StemConn2015 Poster Sessions
Full Abstract Booklet
Poster Map on last page
Posters are located in Ballroom AB
Poster viewing times:
10:15 - 11:00 am (presenters of odd-numbered posters
will be at their posters during this time)
12:00 - 1:00 pm (lunch break; note presenters of posters
may not be present)
3:00 - 3:45 pm (presenters of even-numbered posters
will be at their posters during this time)
Poster #1
A Move Towards Therapies
Administration of embryonic stem cell-derived thymic epithelial progenitors expressing
MOG induces antigen-specific tolerance and ameliorates experimental autoimmune
encephalomyelitis
Laijun Lai (1, 2), Min Su (1, 3), Yinhong Song (1), Zhixu He (3), Rong Hu (1), and Debra
Rood (1)
(1) Department of Allied Health Sciences, (2) University of Connecticut Stem Cell
Institute, University of Connecticut, Storrs, CT; (3) Guiyang Medical College, Guizhou,
China
Technical Abstract
Tolerance induction, and thus prevention or treatment of autoimmune disease, is not only
associated with the persistent presence of self-antigen in the thymus, but also relies on a functional
thymus; however, the thymus undergoes profound age-dependent involution. Thymic epithelial
cells (TECs) are the major component of the thymic microenvironment for T cell development.
We have reported that mouse embryonic stem cells (mESCs) can be induced in vitro to generate
thymic epithelial progenitors (TEPs) that further develop into functional TECs in vivo. In this
study, we determined whether mESC-TEPs expressing myelin oligodendrocyte glycoprotein
(MOG) could improve the thymic environment, induce antigen-specific tolerance and render
experimental autoimmune encephalomyelitis (EAE) resistant. Our results show that
transplantation of mESC-TEPs expressing self-antigen myelin MOG in mice results in enhanced
T cell regeneration, long-term expression of MOG in the thymus, prevention of EAE development,
and remission of established EAE. Our findings indicate that transplantation of ESC-TEPs
expressing disease-causative self-antigen(s) may provide an effective approach for the prevention
and treatment of MS and other autoimmune diseases. This project was partly supported by a grant
from the Connecticut Stem Cell Program (12-SCB-UCON-02).
Lay Abstract
Autoimmune disease affects approximately 5% of the population in Western countries and arises
when the immune system actively targets and destroys self-tissues, leading to a range of clinical
synd romes. Multiple sclerosis (MS) is a devastating autoimmune disease of the central nervous
system. It is generally believed that MS is mediated by immune responses against myelin antigens,
followed by neurological impairment. Experimental autoimmune encephalomyelitis (EAE) is the
most commonly used animal model for MS, and is induced by immunization with diseasecausative self-antigens such as myelin oligodendrocyte glycoprotein (MOG). Currently, strategies
to treat autoimmune disease including MS often rely on immunosuppression in a nonspecific
manner. Despite achieving some success, immunosuppressive strategies have difficulty in
sustaining long-term tolerance, and often lead to the development of immune deficiency with a
consequence of a high rate of opportunistic infections. The most logical way to induce self-antigenspecific tolerance in MS would be to use the same mechanisms that the immune system uses to
maintain self-tolerance throughout life. To achieve this tolerance, the thymus, the major organ
implicated in self-tolerance induction, has to be sufficiently functional, and the self-antigen has to
be persistently present in the thymus. However, it is well known that the thymus undergoes agedependent involution, and its functions are seriously compromised in the elderly. Thymic epithelial
progenitors (TEPs) and their derived cells are the major component of the thymic
microenvironment for T cell development. We have demonstrated that transplantation of mouse
embryonic stem cells (ESCs)-derived TEPs expressing MOG can prevent EAE development and
reverse established EAE. Therefore, development of human ESC-derived TEPs expressing MOG
has the potential to prevent MS development and treat patients with MS.
Poster #2
A Move Towards Therapies
The computer-enabled design of novel peptides to mobilize endogenous stem and progenitor
cells for transplant, tissue repair and chemo-sensitization
Joseph Audie(1), David Diller(1), Jon Swanson(2) and Mark Jarosinski (1)
(1) CMDBioscience, 5 Science Park, New Haven, CT 06511, (2) ChemModeling, Suite 101, 500
Huber Park Ct, Weldon Spring, MO 63304
Technical Abstract
The bone marrow (BM) houses populations of endogenous stem and progenitor cells. These
stem/progenitor (SP) cells are anchored in the BM by adhesive protein-protein interactions (PPIs).
Pharmacological strategies aimed at disrupting these PPIs can mobilize SP cells from the BM to
the peripheral circulation where they can be available for tissue repair and transplant. Cancer SP
cells, tethered by various PPIs, can hide in the BM where they are protected from chemotherapy.
Thus, drug induced mobilization of cancer SP cells from the BM could sensitize them to
chemotherapy. Importantly, several PPIs, including CXCR4/SDF-1, are known to anchor SP cells
in the BM. Indeed, the drug Plerixafor is a small molecule CXCR4/SDF-1 antagonist that is used
clinically to mobilize SP cells. Owing to Plerixifor's safety and efficacy liabilities, efforts are
underway to develop improved CXCR4/SDF-1 antagonists. Other PPI mobilization targets (MTs),
however, are also worthy of consideration. Indeed, multiple agents aimed at multiple mobilization
targets (MTs) could revolutionize therapeutic SP cell mobilization. In many ways, peptides
represent promising MT agents due to their good safety profile, ability to block PPIs, the
accessibility of the BM compartment and the acceptability of intravenous (IV) drug infusion in
relevant disease settings. With this in mind, we leveraged our computational peptide drug design
platform (CMDInventus) to design novel CXCR4/SDF-1 peptide antagonists. Three rounds of
computational design resulted in multiple linear and cyclic lead peptides with Plerixifor potencies.
The peptides are novel and small and incorporate chemical "tricks" to stabilize them against
proteolysis. We are expanding our research to include the design of peptide antagonists for other
MTs, including VLA-4 and CD44, with the ultimate goal of developing personalized peptide
"cocktails" to mobilize endogenous SP cells for transplant, tissue repair or chemosensitization.
Lay Abstract
Stem cells are cells that can reproduce themselves and turn into many other types of cells. Thus,
stem cells from healthy patients can help sick patients and stem cells from sick patients can help
repair their damaged organs. Some adult stem cells live in the bone marrow. Drugs can be
developed to release stem cells from the bone marrow so they can enter the blood. Once in the
blood, the stem cells can be used to repair damaged tissue or collected for transplant into sick
patients. Recent research suggests that cancer relapse is due to cancer stem cells. Cancer stem
cells can hide in the bone marrow and protect themselves from chemotherapy. Thus, drugs to
stimulate cancer cells from the bone marrow may expose them to chemotherapy. We have used
sophisticated computer software to design drugs to stimulate stem cells to leave the bone marrow
and enter the blood. Some of our designed drugs appear to be as good as an existing drug called
Plerixafor. We are expanding our research to develop several new drugs that can be combined and
personalized for specific patients to do an even better job releasing healthy stem cells for transplant
and tissue repair and cancer stem cells for killing by chemotherapy.
Poster #3
A Move Towards Therapies
Investigating iPS Cells Derived From Newborn Patients for the Treatment of HyperoxiaInduced Lung Injury
Charles T. Drinnan, PhD (1), Stephanie Vadasz, PhD (1), Todd J. Jensen, MSc (1), and
Christine M. Finck, MD (1,2)
(1) Department of Vascular Biology, University of Connecticut Health Center,
Farmington, CT 06030, (2) Department of Surgery, Connecticut Children’s Medical
Center, Hartford, CT 06106
Technical Abstract
Purpose: In the US, more than 500,000 infants are born prematurely and conventional therapy
may induce hyperoxia induced lung injury. Unfortunately, these under-developed lungs cannot
recover from the early damage, and this condition could benefit from novel regenerative medicine
techniques. An exciting strategy proposes use of stem cells to treat hyperoxia damaged lungs.
Previous studies have had mixed success in animal models utilizing human mesenchymal stem,
cord blood, and amniotic fluid stem cells and their respective conditioned media. Methods:
Fibroblasts from neonatal foreskin (IRB# FINC003364HU) were infected with an excisable
lentiviral vector in order to generate induced pluripotent stem cells (iPScs). Cells were treated with
cre-recombinase in order to generate transgene-free iPScs. These cells were then differentiated
utilizing specific growth factors and a commercially available small airway growth medium. NSG
pregnant mice were allowed to deliver normally, and neonatal mouse pups were exposed to 75%
oxygen environment for 24 hours within 12 hours of birth. Stem cells were then intra-tracheally
injected and allowed to engraft for 24 hours. Controls consisted of hyperoxia alone, normoxia, and
PBS injected. Results and Conclusion: Mice exposed to hyperoxia and PBS injections had
significant lung damage. Injection of iPScs into hyperoxic mice demonstrated improvement in
histology and morphometric measurements. We conclude that differentiated patient-specific
human iPScs are a potential therapeutic option for the treatment of hyperoxia induced lung injury.
Furthermore this option provides an autologous approach to cell therapy, therefore circumventing
the need for immunosuppressive treatment. Further studies will be needed in order to evaluate the
engraftment efficiency and long term effects of administering these cells.
Lay Abstract
According the World Health Organization, 15 million infants are born premature worldwide
including over 500,000 in the United States alone. Typical therapies for premature births are to
place the infant within a neonatial-intensive-care-unit intubated on a ventilator. While improving
the survival rate of premature infants, neonatal ventilation can lead to increased inflammation,
lung permeability, vasculature leakiness, and epithelial cell death. The primary mechanism is the
increased oxygen (hyperoxia) conditions present during ventilation leading to a buildup of free
radicals. Premature infants have a reduced capacity to scavenge these free radicals, and can lead
to long-term consequences such as severe retinal disease, chronic lung disease, and compromised
neurodevelopmental outcomes. Stem cells have been proposed as a treatment platform by
providing a new source of cells to repopulate damaged tissue. Furthermore, studies have shown
that stem cells and their conditioned media can limit inflammation, thus limiting adverse effects
of neonatal ventilation. Studies have been conducted with adult, human mesenchymal stem, cord
blood, and amniotic stem cells, both cell populations and conditioned media, with mixed success.
While allowing for the potential to use autologous cell populations and thus, eliminating the need
for immunosuppressive treatments, plasticity toward pulmonary lineages is limited. The use of
induced pluripotent stem cells (iPScs) can allow for autologous transplantation while providing a
highly potent cell source. This study demonstrates the potential of iPScs derived from newborn
patients for the treatment of hyperoxia induced injury using a standard mouse model. Histological
analyses demonstrate that iPScs limited damage to neonatal mouse pups.
Poster #4
A Move Towards Therapies
Derivation, Differentiation and Characterization of Human Embryonic Stem Cell Derived
GABAergic Interneuron Progenitors
Nickesha C. Anderson (1), Christopher Y. Chen (1), Daniel F. Moakley (1), Katharine W.
Henderson (1), Alex Plocik (2), Brenton Graveley (2), Janice Naegele (1), Laura Grabel
(1)
(1) Wesleyan University, Department of Biology, Middletown CT, (2) University of
Connecticut Health, Department of Genetics and Developmental Biology, Farmington CT
Technical Abstract
The selective loss of GABAergic inhibitory interneurons is characteristic of numerous
neurodegenerative diseases. Absence of these inhibitory subtypes creates an electrical imbalance
in the hippocampal and cortical neural circuits. Our long term goal is to replenish these inhibitory
interneuron subtypes using an embryonic stem cell (ESC) source. During embryonic development,
these inhibitory interneuron progenitors arise from a transient ventral forebrain structure known as
the medial ganglionic eminence (MGE) and are characterized by the expression of Nkx2.1. We
have optimized an adherent monolayer protocol for the generation of Nkx2.1+ neural progenitors
human ESCs using sonic hedgehog treatment. The Nkx2.1+ enriched cell population expresses
elevated levels of MGE markers, including Nkx2.1 and Nkx6.2, based upon qRT-PCR analysis.
Transcriptome analysis using high throughput mRNA sequencing is underway to further
characterize the Nkx2.1+ cell population. To test the differentiation potential of the Nkx2.1+ cells
in vitro, we used co-culture with mouse cortical astrocytes and obtained an enriched population of
interneurons in which 75% of the MAP2+ cells are also GABA+ after 8 weeks. Preliminary studies
examining the fate of human ESC-derived Nkx2.1+ progenitors transplanted into the mouse
hippocampus demonstrate the expression of neuronal markers 3 weeks post-transplant.
Lay Abstract
Epilepsy is a neurodegenerative disease characterized by the loss of neurons responsible for
maintaining proper electrical activity in the brain. Our goal is to use embryonic stem cells to derive
cell types that can replace the lost population of neurons. To ensure the cell population we derive
has all the properties of the lost cell types, we examine their potential to generate the appropriate
neuron type in culture using a variety of approaches. In addition, we transplant the derived neural
progenitors into a mouse model of epilepsy to look at survival, integration and seizure suppression.
Poster #5
A Move Towards Therapies
Embryonic Stem Cell-Derived Neural Progenitor Interactions with the Neurovasculature
Chelsea Lassiter (1), Julian Gal (1),Sandy Becker (1),Laura Grabel (1)
(1) Wesleyan University
Technical Abstract
Before we can safely use embryonic stem cell (ESC)-based cell replacement we must characterize
the behavior of these cells following transplant. Embryonic stem-cell derived neural progenitors
(ESNPs) transplanted to the dentate gyrus region of the hippocampus can differentiate into granule
neurons, repopulating the upper blade lesioned by the injection. These transplants are also richly
vascularized, and surprisingly, the ESNPs appear to migrate great distances from the original site
of injection. We observe that doublecortin (DCX)+ migrating ESNPs are found in close proximity
to endogenous blood vessels, outside of the transplant area. Our preliminary data suggests that
blood vessels and their associated astrocytes provide a source of the chemokine CXCL12, which
promotes ESNP migration in the brain. To test this model, we use organotypic hippocampal slice
culture and find that ESNPs are found closely associated with blood vessels. To directly study the
interaction between ESNPs and endothelial cells and identify a molecular mechanism, we are using
a selective adhesion assay with brain endothelial cells, as an in vitro model. These data raise a
concern for many therapeutic transplantation approaches that cells may migrate away from the
original transplant, and be disruptive at a distant site.
Lay Abstract
We use human embryonic stem cells to study the relationship between blood vessels and neural
progenitors. The vasculature has been shown to play major roles in neural progenitor cell
regulation, both during development and adulthood. We are interested in understanding how neural
progenitors use the vasculature to migrate, since this may lead to unanticipated negative effects on
brain behavior. On the other hand, understanding the signaling that directs neuronal migration may
lead to more successful targeting of cells to areas of neurodegenerative damage.
Poster #6
A Move Towards Therapies
GABAergic Inhibitory Interneuron Progenitor Grafts into the Dentate Gyrus Reverse
Spatial Memory Deficits in a Mouse Model of Temporal Lobe Epilepsy
MA Van Zandt (1), F Harrsch (1), J Gupta (1), JR Naegele (1)
(1) Wesleyan University
Technical Abstract
Temporal lobe epilepsy (TLE) is characterized by spontaneous seizures and cognitive deficits. In
the adult hippocampus, seizures trigger the death of many GABAergic interneurons, leading to
network hyperexcitability. Loss of inhibitory GABAergic inputs also reduces the place and grid
cell network, disrupting precise receptive field properties that shape spatial memories. Our prior
work in a mouse model of TLE showed that GABAergic interneuron grafts reduced seizures and
restored synaptic inhibition onto granule cells (GCs) (Henderson and Gupta et al, 2014). We
hypothesize that restoring GABAergic synaptic input to denervated GCs will also reverse
cognitive deficits. We transplanted embryonic day 13.5 GABAergic progenitors from the medial
ganglionic eminence (MGE) or media (controls) into the dentate gyrus (DG) of mice with TLE
and performed behavioral testing in the Morris Water Maze (MWM), to examine learning and
memory. After receiving transplants, TLE mice with transplants showed significantly fewer errors
during both training and probe trials compared to media controls. Mice with transplants
demonstrated significantly shorter latencies to reach the platform during the learning phase and to
cross the platform area in the probe trial. Additionally, engrafted TLE mice crossed the platform
significantly more times during probe trials compared to media controls. Together, these findings
suggest that transplants of GABAergic progenitors into the dentate gyrus of TLE mice reverse
some memory deficits in TLE. While previous optogenetic and immunohistochemical work
showed that transplanted GABAergic interneurons formed functional synapses onto endogenous
GCs, further work is needed to determine this is the primary mechanism behind restoration of
cognition in TLE mice. This work was supported by NIH NS072879, CURE Epilepsy.org and the
CT Stem Cell Research Program.
Lay Abstract
Temporal lobe epilepsy (TLE), one of the most common forms of epilepsy, is characterized by
spontaneous, recurrent seizures and cognitive deficits. A common hallmark of TLE is dysfunction
of certain inhibitory neurons in a region of the brain called the hippocampus. Loss of these neurons
increases excitatory activity and seizures. As a result, common cognitive deficits in TLE include
diminished spatial and declarative memory. Our previous work in the epileptic hippocampus of
mice with TLE showed that restoring these neurons through transplantation significantly reduced
seizures. To investigate whether transplants can also reverse memory deficits, we grafted
embryonic progenitor cells of this type of neuron into the hippocampus of a mice with TLE. Six
weeks later, we tested engrafted mice and media-injected controls using an assay that tests spatial
memory. Our findings show that TLE mice with transplants exhibit significantly improved
memory compared to media controls. These findings suggest that stem cell transplants are a
promising approach for therapies to regenerate damaged neural circuits in TLE.
Poster #7
A Move Towards Therapies
Humanized Mice Afford Efficient Engraftment and Disease Replication of Myelodysplastic
Syndromes
Ashley Taylor, M.Sc. (1*), Yuanbin Song M.D. (1*), Anthony Rongvaux, Ph.D. (2*),
Nikolai Podoltsev, M.D. (1), Mina Xu M.D. (3), Natalia Neparidze M.D. (4), Richard
Torres M.D. (5) , Lisa M. Barbarotta BSN, MSN, AOCNS, APRN-BC (1), Kunthavai
Balasubramanian, M.Sc. (1), Karin Finberg, M.D. (3), Richard Flavell, Ph.D. (2#),
Stephanie Halene, M.D. (1#)
(1) Section of Hematology, Department of Internal Medicine and Yale Comprehensive
Cancer Center, (2) Department of Immunobiology, (3) Department of Pathology, (4)
Hematology/Oncology, West Haven VA and Yale Comprehensive Cancer Center, (5)
Department of Laboratory Medicine, Yale University School of Medicine; New Haven,
CT, USA, (*) contributed equally, (#) co-corresponding
Technical Abstract
Myelodysplasia is a disorder of the hematopoietic stem cell caused by a large number of genetic
and epigenetic alterations. With the development of novel therapeutics a reliable model to test their
efficacy in correlation with genetic and epigenetic alterations and disease phenotype is essential.
We optimized xenotransplantation into Rag2-/-IL2rγ-/- mice, named MISTRG, that express
human macrophage receptor signal regulatory protein-alpha (SIRPα) to prevent murine
macrophage-mediated immune rejection as well as several human, non-crossreactive cytokines.
We optimized irradiation dose, transplantation route, CD34+ cell number and preparation prior to
injection for MDS engraftment. We performed direct comparison between NOD/scid/IL2rg-/(NSG) and MISTRG mice. Mice were allowed to engraft for >10 weeks and peripheral blood,
bone marrow and spleen were analyzed for engraftment by flow cytometry, histology, and
genomics.
MISTRG mice consistently supported higher engraftment in peripheral blood and bone marrow
than NSG mice for all MDS samples assessed. Over 70% of all MDS samples engrafted in
MISTRG mice, with detection of the abnormal clone via genetic testing. MISTRG mice support
myeloid engraftment with improved terminal differentiation, with improved immunophentypic
concordance between MISTRG mice and the patient’s primary bone marrow. Bone marrow
histology of engrafted MISTRG mice replicates histologic findings in patient bone marrows. The
number of engrafted MISTRG mice per sample ranged from 2-10 mice which may be improved
with optimal bone marrow sample collection. Physiologic expression of essential noncrossreactive human cytokines greatly facilitates long-term engraftment of MDS patient derived
CD34+ HSPCs in the murine immunodeficient host. MISTRG mice engraft lower and higher grade
MDS with replication of the disease geno- and phenotypes.
Lay Abstract
Myelodysplastic Syndrome (MDS) is a disorder of the blood caused by alterations in the blood’s
stem cells. In order to test novel therapeutics, it is essential to have a model that accurately mimics
myelodysplasia in patients. We optimized xenotransplantation (transplantation of human MDS
cells into mice) into genetically altered mice, named MISTRG. The MISTRG mice express human
cytokines that allow the human cells to better engraft into the mouse, which would normally reject
human cells. We optimized the bone marrow transplantation protocol to allow for the best
engraftment of MDS cells into the mice. We also performed direct comparison between previously
available NSG mice and the new and improved MISTRG mice. Mice were allowed to engraft for
>10 weeks after injection of human MDS cells. After at least 10 weeks peripheral blood, bone
marrow and spleen were taken from the mice and analyzed for engraftment by flow cytometry,
histology, and genomics. MISTRG mice consistently supported higher engraftment in peripheral
blood and bone marrow than NSG mice for the majority of MDS samples assessed. Over 70% of
all MDS samples engrafted in MISTRG mice with detection of the same cell mutations the patients
also had via genetic testing. The MISTRG mice provided an environment where the human MDS
cells could mature as they would normally in a human. When the bone marrow was examined, the
abnormal patterns that were seen in patients were also seen in the mice. The number of engrafted
MISTRG mice per sample ranged from 2-10 mice which may be improved with optimal bone
marrow sample collection. By expressing human cytokines in MISTRG mice, we are able to
improve engraftment of patient MDS stem cells into an animal model, allowing us to better study
MDS and test treatments.
Poster #8
A Move Towards Therapies
Müller glia based Co-culture system induces hESC into retinal lineages
Shao-bin Wang(1)(2) Bo Chen (1)
(1) Department of Ophthalmology and visual science, Yale School of Medicine (2)
Department of surgery, Yale School of Medicine
Technical Abstract
Purpose: Müller glia cells are critical for retinogenesis in mammalian retina as they form scaffolds
to maintain the retinal structure, secrete various factors to support the survival of retinal neurons,
maintain the extracellular milieu, and recycle neurotransmitters. This study aims to establish a
Müller glia based co-culture system, and to investigate the ability of Müller glia on promoting
human embryonic stem cells (hESCs) differentiation into retinal neurons, as well as to evaluate
whether Müller glia Co-culture induced retinal neurons integrate into host tissue after ocular
transplantation. Methods: The co-culture system was created using purified Müller glia cells as
feeder cells for embryoid body cells derived from hESCs. The reaggregating spheroids of Cocultured hESCs were identified by specific immunostaining or labelling with AAV-GFP (2/2)
transduction. Finally, hESC-derived retinal cells from the co-culture were transplanted into mouse
retina to examine their integration into the recipient retina. Results: After co-culture with Müller
glia cells, the dissociated hESC-derived embryoid body cells formed reaggregating spheroids on
co-cultured Müller glia layers. Around 35.2%~43.5% cells of the spheroids showed retinal
progenitor properties to express CRX and Pax-6. Around 10.4%~20.7% cells of the spheroids
showed expression of photoreceptor marker (Rhodopsin), and retinal ganglion cells marker (Tuj1
and NeuN). For transplantation test, the AAV-GFP (2/2) labeled hESC-derived retinal cells
survived for at least 4 weeks after transplantation in the normal or diseased recipient retina. A few
of them successfully integrated into recipient retina layers and extended dendritic neuronal
processes. Conclusion: Müller glia based co-culture system is sufficient for inducing hESCs into
retinal lineages. Some hESC-derived retinal cells showed characteristics of retinal neurons and
were viable after transplantation with a few integrating into the recipient retina.
Lay Abstract
Retina degenerative diseases, such as glaucoma and age related macular degeneration, are
characterized by loss of retinal ganglion cells and photoreceptors. They are leading causes of visual
impairment and blindness. Stem cell transplantation is a promising therapeutic strategy to treat the
disease by replacing lost retinal neurons. A major challenge is to efficiently induce human
embryonic stem cells to become transplantation-competent retinal neurons such as photoreceptors
or retinal ganglion cells. Several approaches have been developed to induce embryonic stem cells
or induced pluripotent stem cells into retinal photoreceptors or pigment epithelial cells, however,
these approaches were unable to generate retinal ganglion cells or other types of retinal neurons.
These methods require gene manipulations and/or treatment with various factors, increasing the
difficulty and risk for clinical application. Müller glia cells are a neuronal support cell in the retina
that plays an important part in the development of the retina. Muller cells secrete factors that
regulate retinal differentiation. We established a co-culture system with primary Müller glia and
hESC-derived embryoid body cells. After induction with the co-culture, we can efficiently induce
the differentiation of hESCs into photoreceptor, as well as retinal ganglion cells. After
transplantation into the recipient retina,,some cells integrate into the retina layers with dendritelike neuronal processes. In conclusion, Müller glia based co-culture system enables an efficient
induction of hESCs to differentiate to various retinal neurons, which may be used for cell
transplantation therapy for retinal degenerative diseases.
Poster #9
A Move Towards Therapies
Fate of mesenchymal cells on human decellularised lung matrix
Sumati Sundaram(1), Kevin Boehm(1), Jenna Balestrini(1), Jonas Schwan(1), Michael
Carapezza(1), Caihong Qiu(2),Laura Niklason(1)
(1)Yale University, Department of Biomedical Engineering, New Haven, CT, (2)Yale
Stem Cell Center, New Haven, CT
Technical Abstract
The aim of the current study is to evaluate the utility of human induced pluripotent stem cell
derived
mesenchymal
cells
to
repopulate
decellularized
lung
tissue.
Human iPS cell lines were induced into the mesenchymal lineage via a neural crest intermediate
in a serum-free 2D differentiation format. The mesenchymal identity of derived cells was
confirmed by flow cytometry for positive expression of cell surface marker CD90, CD73, CD105
and negative expression of CD45, CD34. Derived cells were also successfully differentiated into
osteogenic, chondrogenic and adipogenic lineages. Taken together, these tests confirm the
mesenchymal identity of iPS-derived cells. Further, cells were seeded on slices of decellularized
rat lung in various growth media such as SAGM (small airway growth medium), BEGM (bronchial
epithelial growth medium), and DMEM/10% FBS (serum containing medium). In order to
understand the progression of differentiation, reseeded slices were tested at day 1 and day 7 for
various epithelial marker expression. Our preliminary data suggest a mesenchymal to epithelial
differentiation in the presence of the microenvironment of the decellularized lung scaffold.
Histological analyses revealed distinct cellular cuboidal morphologies. Cells were found to express
markers indicative of epithelial phenotypes such as CCSP, Pro-SPC, Keratin14. Epithelial marker
expression was confirmed by PCR, and immunostaining. Further studies are underway to
understand specific anatomical locations that cells bind preferentially. We will also perform PCNA
and Tunel analysis to determine the state of the cells. We will utilize TEM to investigate ultra
structures in the cells such as presence of lamellar bodies in the cells.
Lay Abstract
End-stage lung disease is the fourth leading cause of death in the United States. Lung
transplantation remains the only viable solution in many cases as the inherent potential of the lung
to regenerate in vivo is very limited. However, due to the extreme shortage of donor organs,
extended immunosuppression requirements as well as the costs involved it is an extremely
challenging option. Recent advances in stem cell research and whole organ regeneration provide
unprecedented possibilities for future therapies. Perhaps one of the most important is the
identification of stem or progenitor cells with the capacity for long-term self-renewal and
differentiation. Given their unique properties, hES and hiPS cells show great promise as an
enhanced renewable regenerative cell source. The aim of the current study is to evaluate the utility
of human induced pluripotent stem cell derived mesenchymal cells to repopulate decellularized
lung tissue. Results and conclusions from these studies have the potential to lead to several key
findings. At the very best, they will identify a possible celltype that could possibly form a novel
renewable cell source for repopulating decellularised lungs and thus aid the field of lung tissue
engineering. At the very least, they will help expand the overall knowledge base of lung biology
by providing new insights into the role of MSCs in the context of lung regeneration.
Poster #10
A Move Towards Therapies
OUTCOMES FOLLOWING REVISION ROTATOR CUFF REPAIR WITH
CONCENTRATED BONE MARROW ASPIRATE OBTAINED FROM THE PROXIMAL
HUMERUS
Andreas Voss (1), Mary Beth McCarthy (1), Mark P. Cote (1), Alexander R. Hoberman
(1), Jessica DiVenere (1), Augustus D. Mazzocca (1)
(1) University of Connecticut UConn Musculoskeletal Institute Department of Orthopaedic
Surgery
Technical Abstract
INTRODUCTION:
The purpose of this study was to assess the effectiveness of biologic augmentation of revision
massive rotator cuff repair using concentrated bone marrow aspirate (cBMA) obtained from the
proximal humerus during surgery and using an allograft patch as a scaffold for the BMA.
METHODS:
All patients with large or massive rotator cuff tears as well as patients who had failed a primary
rotator cuff repair were considered eligible. We biologically augmented the patch with both bone
marrow aspirate and autologous concentrated plasma through injected into the patch < 30 min
prior to implantation. cBMA samples in the patch were sent to the laboratory to obtain cell
numbers. All patients followed the same postoperative rehabilitation protocol.
RESULTS:
There were 23 repairs in 22 patients. Mean follow up of 21 ± 13 months. Two patients were lost
to follow up. Of the remaining 21 procedures in 20 patients, there were 12 failures. The average
CTPs were 20,545 ± 23,316. In patients who had not failed, the average CTPs were 13, 294 ± 3985
compared to 26,476 ± 30,564 in those that did not fail.
DISCUSSION:
We have previously demonstrated that bone marrow aspiration from the proximal humerus and
distal femur is a safe, simple, efficient and reproducible procedure1,2. We’ve also found that a
non-fenestrated trocar is preferable for BMA when looking at the CTP preference (numbers of
CFU/cc BMA). In the present study, we did not find a statistical significance in CTPs between
those that had failed and those who were stable.
Lay Abstract
In this study we could not show any significance between the amount of stem cells obtained from
the proximal humerus and a stable or failed revision rotator cuff repair. These results led us to
intensify the research in stem cells to improve healing of the rotator cuff.
Poster #11
A Move Towards Therapies
Autologous Progenitor Cells for Bone Tissue Engineering
Paiyz E. Mikael (1) Deborah Dorcemus (1) Brian Barnes (2) Syam P. Nukavarapu (1)
(1) University of Connecticut (2) Arteriocyte, Cellular Therapies Medical Systems
Technical Abstract
Treating large bone defect is still a significant challenge in orthopedic surgery. Finding a clinically
relevant cell source is only a first step in the right direction. Bone marrow aspirate (BMA) is an
excellent source of mesenchymal stem cells, vascular forming cells (endothelial progenitor cells)
and growth factors such as bone morphogenic proteins (BMPs), vascular endothelial growth factor
(VEGF) and platelet-derived growth factor (PDGF) and is currently used by orthopedic surgeons
to treat fractured bones by directly infusing the bone marrow aspirate onto allografts or autografts.
However, the volumetric amount of BMA required to fill in a large defect does not contain
adequate numbers of progenitor cell population, thus leading to limited bone formation. Here we
propose the direct use of concentrate BMA (cBMA) using an FDA approved and automated system
(Magellan). Flow cytometry and colony forming assay were utilized to establish the superior
quality of cBMA. The aim of this study is to enrich human BMA for bone marrow stromal cell
population and study its potential for enhanced bone regeneration. The long-term goal is to develop
bedside strategies to rapidly translate tissue-engineering technologies/products into the clinic for
patient use
Lay Abstract
In the United States alone about 3 million musculoskeletal procedures are done annually.
If left untreated, large bone defects lead to non-union and eventually loss of function. Large bone
defects are typically caused by trauma, cancer, inflammation or congenital abnormalities.
Currently available treatments include the infusion of bone marrow aspirate (BMA) which is a rich
source of stem cells and growth factors that work in concert to rebuild the damaged bone. However,
the volume of BMA required to fill in a large defect does not contain adequate numbers of stem
cells which leads to very limited bone formation. Here we propose the concentrating a larger
volume of BMA (cBMA) using an FDA approved and automated system, Magellan. Our results
show increased level of mesenchymal stem cells in the concentrated BMA. Other assays further
confirm the potential of the concentrated BMA for bone formation.
Poster #12
A Move Towards Therapies
Implantable Tissue-Engineered Blood Vessels from Human Induced Pluripotent Stem Cells
Liqiong Gui (1), Biraja Dash (1), Lingfeng Qin (1), Liping Zhao (1), Kota Yamamoto (1),
Takuya Hashimoto (1), Hongwei Wu (1), George Tellides (1), Alan Dardik (1), Laura E.
Niklason (1), Yibing Qyang (1)
(1)Yale University School of Medicine
Technical Abstract
Human tissue-engineered blood vessels (TEBVs) have been developed using primary aortic
smooth muscle cells and shown great success in clinical trials. However, due to the batch variation
of donor cells, it remains a challenge to establish products without variable mechanical properties
for clinical treatment. Human induced pluripotent stem cells (hiPSCs) offer the advantage of
providing unlimited number of cells that can be differentiated into comparable vascular smooth
muscle cells (VSMCs) from batch to batch for tissue engineering. In this study, transgene-free
hiPSCs were differentiated into alpha-smooth muscle actin (a-SMA) and calponin positive cells
and then seeded onto biodegradable polyglycolic acid (PGA) scaffold in bioreactors. After 9
weeks, a completely biological blood vessel (2-mm inner diameter) was constructed, which
showed a burst pressure of at least 500 mmHg and suture retention up to 70g. These TEBVs
contained a large amount of collagen matrix but no elastic fibers. Significant amounts of the cells
were stained positive for a-SMA and myosin heavy chain (MHC), indicating more contractile
phenotype. When TEBVs were implanted into nude rats as abdominal aorta interposition grafts,
they remained mechanically intact and patent for up to 2 weeks. Interestingly, there appeared to
be an increase in the percentage of MHC positive human cells in the grafts after implantation. In
addition, rat cells that stained positive for a-SMA and calponin but not MHC were observed at the
adventitial side of the implanted human grafts. These results indicate that hiPSC-derived TEBVs
are implantable and support vascular remodeling. Future studies will further improve their
mechanical strength and investigate their long term in vivo stability and function. This is the first
report of implantable TEBVs based on hiPSCs and establishes the foundation for developing
autologous grafts for clinical intervention in patients with vascular diseases.
Lay Abstract
Every year millions of patients in the United States are diagnosed with cardiovascular diseases and
over a quarter million of them undergo coronary bypass surgery. There is a constant demand for
functional small-diameter (< 6mm) vascular grafts for clinical applications. Due to the shortage of
autologous blood vessels for the procedure, tissue-engineered vascular grafts have been developed
as alternative replacement conduits and some of them are currently being evaluated in humans.
Despite the success in clinical trials, tissue-engineered human vascular grafts generated from
primary vascular smooth muscle cells (VSMCs) are limited by the availability and batch variation
of donor cells. In comparison, human induced pluripotent stem cells (iPSCs) have provided
revolutionarily new possibilities for obtaining unlimited amount of autologous VSMCs for tissue
engineering. In this study, human iPSC-derived VSMCs were cultured on biodegradable polymer
scaffolds in bioreactors under a condition that mimics the in vivo vascular development
environment. After 9 weeks, cells generated large amounts of collagen matrix to form a completely
biological blood vessel that was robust enough for implantation. When the human iPSC-derived
tissue-engineered vessels were implanted into nude rats as abdominal aorta interposition grafts,
they remained mechanically intact and patent for up to 2 weeks and supported vascular remodeling.
This is the first report of implantable tissue-engineered vascular grafts based on human iPSCs and
establishes the foundation for developing autologous tissue-engineered grafts for clinical
intervention in patients with vascular diseases.
Poster #13
A Move Towards Therapies
Tissue Engineered Vascular Rings From HIPSC Derived Smooth Muscle Cells
Biraja Dash (1), Karen Levi (2), Hongwei Wu (1), Marsha Rolle, (2)Yibing Qyang (1)
(1) Dept of Internal Medicine, Yale University, New Haven, CT, (2) Dept of Biomedical
Engineering, Worcester Polytechnic Institute, Worcester, MA
Technical Abstract
For the past few decades, tissue engineered vascular grafts (TEVGs) grown using vascular smooth
muscle cells (VSMCs) have been used as a promising tools for surgical replacement of the affected
vessels in patients with vascular disease as well as model systems to study vascular tissue function
in vitro. There are two approaches to generate TEVGs: 1) by seeding cells within a scaffold and
2) scaffold free approach where cells self-assemble and secret ECM to make TEVGs. However,
the major limitation for generating an autologous tissue engineered construct is the limited
accessibility to patient VSMCs. The over-all goal of this study was to generate tissue engineered
vascular constructs using hiPSC-VSMCs using a facile scaffold free approach. The VSMCs of
95% purity were derived from hiPSC using an embryoid body method. Three-dimensional vascular
rings were engineered by seeding VSMCs around posts of 2 mm diameter in agarose wells and
cultured them for two weeks. The three dimensional rings were then fused to fabricate a TEVG.
The three-dimensional vascular rings showed UTS of around 600-1000 kPa and average thickness
of around 0.7 to 0.9mm. Histological analysis of the constructs showed high cellularization and
collagen deposition. Immunofluorescence studies showed that the VSMCs maintained their
phenotype in culture. The tissue rings are mechanically robust and can be used for vascular tissue
engineering, disease modeling and drug screening. Our method may further serve as a model
system, extendable to other tissue rings based on patient-specific iPSC-derived cells. Thus, this
study describes an exciting platform technology with broad utility for manufacturing cell-based
materials for a variety of biomedical applications.
Lay Abstract
Vascular disease due to dysfunction of vascular smooth muscle cells (VSMCs) is one of the largest
causes of mortality in the developed world. For the past few decades, VSMCs have been used as
to grow tissue engineered grafts. These grafts have been used either as surgical replacement of the
affected vessels in patients with vascular disease or as tool to study disease mechanism. However,
there are few limitations such as i) limited availability of patient specific VSMCs; and ii) lack of
patient specific cell-based, tissue engineered graft that provides a closer approximation of the in
vivo environment for the research of vascular disease and for use as patient specific graft. Thus, it
is critically important to obtain an abundant amount of patient specific VSMCs and to establish a
robust, three dimensional tissue-engineered construct for disease intervention and also for studying
the pathogenesis of vascular disease. Thus, the primary objective of this study was to establish
tissue-engineered vascular grafts (TEVGs) using VSMCs derived from virus free human induced
pluripotent stem cells (hiPSCs). hiPSCs can be derived from a person’s own somatic cells by
forced gene expression. hiPSCs can self-renew and differentiate into virtually every cell type in
the human body, including VSMCs. We established a protocol to generate large amount of VSMCs
and a unique approach to engineer the vascular grafts which can be used for studying disease
mechanism as well as grafts for patients.
Poster #14
A Move Towards Therapies
ISL1 CARDIOVASCULAR PROGENITOR CELLS: A NEW SOURCE FOR
CARDIOMYOCYTES AND ENDOTHELIAL CELLS IN CARDIAC REPAIR
Oscar Bartulos (1), Zhen Wu Zhuang (1), Yan Huang (1), Nicole Mikush (1), Carol Suh
(1), Alda Bregasi (1), Lin Wang (1), William Chang (1), Diane S. Krause (1), Lawrence
H. Young (1), Jordan S. Pober (1), Yibing Qyang (1)
(1) Yale University
Technical Abstract
Acute myocardial infarction (MI) leads to patient’s death in up to 25% of the cases within one year
of hospital discharge. Angiotensin converting enzyme (ACE) inhibitors and β-adrenergic blockers
are effective treatments for MI but they are unable of fully reversing adverse ventricular
remodeling. In the last 15 years, cell therapy has emerged as an important tool for heart
regeneration. Despite the achievements in this field, there are significant pitfalls that require
special attention. In most of the cases, implanted cells did not survive well in long-term
studies,neither could differentiate to working myocardium in vivo. When differentiated
cardiomyocytes (CMs) were used, although long-term survival is achieved, arrhythmias are
unavoidable.
In the present study we used ISL1 cardiovascular progenitor cells (ISL1-CPCs) delivered in 3D
structures to treat MI in a mouse model. ISL1-CPCs are cells present in the second heart field with
potential to give rise to CMs, smooth muscle cells (SMCs) and endothelial cells (ECs) during
embryonic development. Mouse embryonic stem cell (mESC)-derived ISL1-CPCs differentiate in
vitro to CMs, SMCs and ECs mimicking their differentiation properties during development.
When implanted into infarcted mice undergoing permanent ligation of left coronary artery, ISL1CPCs survived efficiently for up to 5 weeks. Moreover, ISL1-CPCs in mice with MI differentiated
into CMs and ECs, last ones incorporated in host’s blood vessels. Echocardiography analysis
showed that ISL1-CPC-treated mice had enhanced left ventricular contractile function with respect
to animals without treatment or treated with vehicle control. No arrhythmic processes were
detected in ISL1-CPC-treated mice. Recently, we have generated transgene-free human ESCderived ISL1-CPCs, capable of differentiating to CMs and ECs and readily available for in vivo
studies. Overall our results show that ISL1-CPCs could be a valuable approach to be considered
for cell therapy after MI.
Lay Abstract
Cardiovascular diseases remain the leading cause of mortality worldwide and heart failure after
myocardial infarction (MI) is the most common one. MI is the result of deprivation of oxygen and
nutrients caused by the blockage of a coronary artery and produces cell death, affecting specially
cardiomyocytes (CMs). When CMs die after MI, they are replaced by cells that produce fibrotic
tissue to maintain the structure of the heart, affecting the global heart contraction. Scientists have
been trying for decades to replace the injured area with new CMs, capable of recovering the
contractile function. Cell therapy emerged more than a decade ago as an important tool to be
considered. However, previous cell therapy studies faced many important challenges. When
different cell types were implanted in MI hearts the vast majority of them did not survive too long
after implantation. Another problem was that most of the cell types cannot differentiate to CMs in
infarcted hearts. In this study we used for the first time a cell type that forms the embryonic heart:
ISLET 1 cardiovascular progenitor cells (ISL1-CPCs). ISL1-CPCs have the potential to form the
3 main lineages present in the heart: CMs, smooth muscle cells (SMCs) and endothelial cells
(ECs). We obtained ISL1-CPCs in the laboratory from embryonic stem cells (ESCs) that contain
the same differentiation potential that the ones present in the embryo. Using mouse ESC-ISL1CPCs we have seen that these cells can survive in infarcted mice for at least 5 weeks (last time
point analyzed). Moreover, ISL1-CPCs implanted in mice with MI improved the heart contractile
function when compared to control mice with MI. ISL1-CPCs differentiated in vivo to CMs and
ECs. These ECs were fully integrated in the host blood vessels. Additionally, ISL1-CPCs reduced
the fibrotic area and increased the number of blood vessels in MI mice. Recently, we have
developed a method to obtain human ESC-ISL1-CPCs readily available for pre-clinical studies.
Poster #15
A Move Towards Therapies
Senescent fibroblasts promote vasculogenesis in vitro in a fibrin gel
Yang Xiao[1], Jing Zhou[1], Laura Niklason[1,2], and Rong Fan[1,3]
(1)Department of Biomedical Engineering, Yale University, CT 06520, USA.
(2)Department of Anesthesiology, Yale University, CT 06520, USA.
(3)Yale Comprehensive Cancer Center, CT 06520, USA.
Technical Abstract
How to fabricate a microvascular network to support the tissue growth is crucial to tissue
engineering. The major challenge in bottom-up synthesis of large-scale neotissues for
transplantation is revascularization. Herein, we report on a generic route to generate
endothelialized microvessel networks in a fibrin gel system with the aid of senescent fibroblasts.
The microvessels rapidly develop in the vicinity of senescent fibroblasts within 6 days and
potentially can be retrieved as a free-standing bio-compatible transplant towards regenerative
medicine.
Senescent fibroblasts (FB), induced by bleomycin treatment, secrete significantly higher levels of
VEGF, IL 6, and IL8, compared to normal human lung fibroblasts. The sustained elevated level of
VEGF promotes proliferation of HUVECs towards vasculogenesis in fibrin gel (2 mg/ml
fibrinogen). Premature endothelial sprouts after a 24-hour co-culture with senescent fibroblasts.
High quality interconnected capillary-like (20~80 μm in diameter) microvascular structures fully
develop in 4-6 days. We process the 2-D images to compute the total length and the number of
branches, and find that HUVECs and HUVEC+FB groups do not give rise to a lot of capillary-like
micro vessels. Instead, most of them are cell aggregates or sheets. But the HUVEC+senescent FBs
group does give capillary like micro-vessels. Further investigation would confirm whether 3-D
lumen-like EC sprouts are present in the HUVECs only and HUVEC+FB group at early stages
(day 2-4). Compared to HUVECs cultured alone in fibrin gel, senescent fibroblasts significantly
accelerate the vasculogenesis process and improve the quality of the microvessels with increased
length, branches, and more uniform capillary diameters. In addition, senescent fibroblasts do not
proliferate, so they donʼt take much space as other mural cells such as non-senescent fibroblasts
and mesenchymal stem cells, and thus maintain the high degree of vascular integrity.
Lay Abstract
We developed a novel engineering approach to generate micro blood vessels mimicking human
capillaries in vitro within 4~6 days. We grow the micro-vessels in a biocompatible jelly-like
hydrogel with drug-treated fibroblasts, a type of vessel supporting cells. These fibroblasts would
secrete a sustained elevated level of vessel promoting proteins and accelerate the time period for
vessel formation. This stack of microvessel-containing hydrogel could be used as a transplant
encapsulating tissue cells such as liver cells and islets. In addition, the system could potentially be
used as a platform for drug screening, toxicity test, and even individualized therapy.
Poster #16
A Move Towards Therapies
Culture model of the human retina: effects of scaffold and co-culture
Deepti Singh(1), Peter Zhao(1), Shaomin Peng(1), Shao-Bin Wang(1), Bo Chen(1), Ron
A. Adelman(1), and Lawrence J Rizzolo(1)
(1)Yale University
Technical Abstract
To restore vision, stem cell therapies for age-related macular degeneration must address the loss
of both photoreceptors and the retinal pigment epithelium. To explore the effects of each tissue
layer on the other’s maturation, we co-cultured human embryonic stem cell derived retinal
progenitor cells (hESC-RPC) with retinal pigment epithelium (hESC-RPE). Cultures were derived
from the H9 human embryonic stem cell line using published protocols. To form a multilayered,
retina-like structure hESC-RPC were seeded on scaffolds of electrospun polycaprolactone (PCL),
gelatin/chondritin sulphate/hyaluronic acid (GCH) or polyethylene. hESC-RPE were seeded on
laminin-coated Transwell (polyethylene) filters. In the co-culture group, RPC cultures were placed
on top of the RPE cultures. In the control group, RPC and RPE cultured separately. After 2-4
weeks, co-cultured tissues were compared to controls using RT-PCR and immunofluorescence.
The transepithelial electrical resistance (TER) of the culture was monitored to assess the integrity
and function of the RPE. RPC invaded the PCL scaffold only to a depth of ~ 20 µm regardless of
whether it was coated with Matrigel or hyaluronic acid. In contrast the RPC populated the entire
thickness of the ~60 µm GCH scaffold. Co-culture promoted the maturation of both the RPC and
RPE. Compared to controls, co-cultured RPC expressed higher levels of the photoreceptor genes
Crx, M/L-opsin, and rhodopsin. Cells expressing photoreceptor markers were restricted to the
surface layer opposing the RPE. A RT-PCR array for monitoring maturation of the RPE showed
that co-cultured RPE was more mature than RPE maintained in SFM-1. Co-cultured RPE
maintained a high TER, whereas the TER decreased in control RPE that was maintained in retinal
differentiation medium. These studies indicate that co-culture increases the maturation of both
cultured RPC and RPE. Tests of retinal functions and the effects of putative pharmaceutical agents
is ongoing.
Lay Abstract
Age-related retinal degeneration is the leading cause of visual impairment in developed nations.
The retina is the light-sensitive portion of the eye. Clinical trials are already in progress to that use
stem cell-derived retinal cells to replace damaged cells. This study addresses two problems that
face this promising therapy. 1) Animal studies show this therapy would be most effective when it
is performed early, before most patients would consent to risky surgery. 2) Medical therapies to
augment and even replace surgery. The goal is to engineer a three-dimensional culture model of
the retina that could be used for transplantation and to develop pharmaceutical therapies. The
methods are well established for differentiating stem cells into retinal pigment epithelium (a retinal
support cell), and retinal progenitor cells (precursors for various retinal nerve cells).
Bioengineering techniques were used to manufacture several types of scaffolds to hold the retinal
progenitors. These were cultured together with retinal pigment epithelium. We developed a
scaffold that allowed retinal progenitors to flat sheets that were many cell layers thick as would be
found in the retina. Co-culture promoted the expression of retinal and epithelial genes. Ongoing
studies are examining whether retinal functions develop in the culture model and whether this
maturation process can be furthered using potential pharmaceutical agents.
Poster #17
A Move Towards Therapies
Mitochondrial functional characterizations of Parkinson’s disease associated mutations in
iPSC derived dopamine neural progenitors and neurons
Carina S. Peritore (1), Serene Keilani (1), Sashi Nadanaciva (1), Keith Haskell (1), William
Blake (1), Rosalind Norkett (2), Kelly Bales (1), Sandra Engle (1), Yvonne Will (1)
(1) Pfizer, Inc. (2) UCL, London, UK
Technical Abstract
Parkinson's disease (PD) associated mutations impair mitochondrial function and increase the
vulnerability of induced pluripotent stem cell (iPSC)-derived neural cells from patients to
oxidative stress, mitochondrial DNA damage, compromised oxidative phosphorylation and
mitochondrial dynamics. In order to better understand these deficits, we generated stem cell
derived dopaminergic neural progenitors (SCD dNPCs) and neurons (SCD DN) from induced
pluripotent stem cells (iPSCs) carrying PD associated mutations (LRRK2 G2019S, SNCA
triplication, PARK2 deletions, and GBA N370S). We characterized PD SCD dNPCs and DNs to
verify that they were patterned toward the dopamine neuron fate. Preliminary results indicate that
when
treated
with
the
respiratory
chain
uncoupler
carbonylcyanide
ptriflouromethoxyphenylhydrazone (FCCP), SCD dNPCs from all four PD lines showed increased
sensitivity compared to wild-type controls. Basal oxygen consumption rates (OCRs) were lower
for two SCD dNPCs (PARK2 del and GBA N370S). We hypothesize that once the cells are
functionally closer to their vulnerable cell types in the PD brain (SCD DNs), we will observe
exacerbated decreases in mitochondrial respiration compared to wild-type controls and altered
mitochondrial motility in neuronal processes. Studies using human neurons from iPSCs with
familial PD mutations highlight opportunities to characterize disease pathways and to screen for
new therapeutic agents.
Lay Abstract
Parkinson's disease (PD) associated mutations impair mitochondrial function. In order to better
understand these deficits, we used stem cells to generate progenitors for dopaminergic-type
neurons (SCD dNPCs) from patients carrying PD associated mutations. We characterized SCD
dNPCs to verify that they were patterned toward the dopamine neuron fate. Preliminary results
indicate that SCD dNPCs from PD lines were more sensitive to mitochondrial toxins than normal
patients. Studies using human neurons from iPSCs with familial PD mutations highlight
opportunities to characterize disease pathways and to screen for new therapeutic agents.
Poster #18
Patient-Derived Disease Models
Induced pluripotent stem cell models of 15q duplication syndrome
Noelle Germain (1) and Stormy Chamberlain (1)
(1) Department of Genetics and Genome Sciences, University of Connecticut Health
Center
Technical Abstract
15q duplication syndrome (Dup15q), is a neurodevelopmental disorder resulting from duplication
of the q11-q13 region of chromosome 15. This syndrome is associated with intellectual disability,
seizures, and autism spectrum disorders. Dup15q accounts for an estimated 3% of all autism cases.
Individuals carrying 2 extra maternal copies of chromosome 15q11-q13 on an isodicentric
chromosome (idic(15)) are more severely affected than those carrying 1 extra copy as an interstitial
duplication. Individuals with duplications of paternal chromosome 15q11-q13 are unaffected. This
link to parent-of-origin of the duplication suggests a role for several 15q11-q13 imprinted genes,
including the ubiquitin ligase E3A (UBE3A) gene, in the disease mechanism. Our goal is to
develop iPSC models of Dup15q to investigate the underlying cellular and molecular mechanisms
of
the
disorder.
We generated iPSCs from five individuals carrying different copy number variations of 15q11q13 and differentiated these iPSCs into mature neurons, a cell type biologically relevant to the
dup15q neuronal abnormalities. Methylation analysis at the imprinted SNRPN promoter in each
iPSC line revealed maintenance of imprinting following cellular reprogramming and confirms
duplication of maternal or paternal chromosome 15q11-q13. Gene expression analysis shows that
while transcript levels for 15q genes largely corresponds with gene copy number in iPSCs,
expression levels for several genes no longer reflects their copy number following neural
differentiation. This data suggests possible disruption of transcription regulatory elements as a
result of chromosomal rearrangements. We compared global gene expression patterns in idic15,
Angelman Syndrome, and normal iPSC-derived neurons, using mRNA-sequencing, and identified
aberrant expression levels of several genes involved in neuronal development and function.
Lay Abstract
Many neurodevelopmental disorders, including autism, are believed to result from disruption in
neuronal function. Copy number variants (CNVs) of several genes known to affect neuronal
function have been linked to various forms of autism, including Fragile X, Tuberous Sclerosis, and
15q11-q13 Duplication Syndrome (Dup15q). However, the specific contributions of these autism
candidate genes to the autism phenotype is not well understood. Investigation of disease
mechanisms in syndromic autism spectrum disorders (ASD) may provide insight into idiopathic
forms of autism and other neurodevelopmental disorders. In depth study of the molecular
mechanisms underlying these disorders requires access to live neurons which can be manipulated
in vitro. IPSC technology allows us to develop in vitro models of a variety of neural diseases which
I believe will be an invaluable resource for uncovering the gene functions and cellular pathways
involved. Ultimately, these tools will aid in the identification of novel therapeutic targets. IPSCs
are pluripotent cell lines, derived from patient samples, such as blood, skin cells, or umbilical cord
blood cells, which can be pushed to mature into any of the cells types in the human body, including
functional neurons. We generated IPSCs from seven individuals carrying different copy number
variations of 15q11-q13 and differentiated these iPSCs into mature neurons, a cell type
biologically relevant to the dup15q neuronal abnormalities. We have thoroughly characterized
these cells and used them to investigate chromosome 15-specific as well as global changes in gene
expression that may be functionally relevant to the disease phenotypes.
Poster #19
Patient-Derived Disease Models
Restoring Angelman Syndrome Neurons to Normal Phenotype by Unsilencing Paternal
UBE3A
Carissa L. Sirois (1), Noelle Germain (2), Stormy Chamberlain (2)
(1) Department of Neuroscience, University of Connecticut Health Center, Farmington,
CT
(2) Department of Genetics and Genome Sciences, University of Connecticut Health
Center, Farmington, CT
Technical Abstract
Genomic imprinting is an epigenetic phenomenon that results in differential expression of genes
depending on a chromosome’s parent of origin (maternal or paternal). In neurons, UBE3A, which
encodes for an E3 ubiquitin ligase, is expressed from the maternal copy of chromosome 15 and is
silenced on the paternal copy. This is due to the extension of a long non-coding RNA (lncRNA)
transcript that is transcribed in the opposite direction of UBE3A, thus acting as an antisense
transcript (UBE3A-ATS). Loss of expression of the maternal copy of UBE3A results in the
disorder known as Angelman Syndrome (AS). AS is characterized by severe seizures, ataxia,
absent speech, learning disability, happy demeanor, and characteristic “puppet-like” arm
movements. Most of these symptoms appear during early childhood and persist through adulthood.
Currently, there is no treatment for AS. Induced pluripotent-stem cell (iPSC)-derived neurons
made from AS patient fibroblasts provide a unique opportunity to test potential therapeutics in
human neurons. We have established an iPSC cell line from an AS patient with a point mutation
in UBE3A (F603S). We are using the CRISPR-Cas9 genome editing system to correct this AScausing mutation in the iPSCs, thus creating an isogenic control iPSC line., Upon establishment
of this genetically corrected iPSC line, we will differentiate both corrected and uncorrected cells
into neurons. Using mRNAseq, we will then establish a molecular phenotype for the AS cells by
comparing the transcriptomes of the corrected and uncorrected cells. This phenotype will comprise
a list of transcripts differentially expressed in the AS neurons. After establishing our molecular AS
phenotype, we will then test the ability of a drug to restore UBE3A expression from the imprinted
(silent) paternal allele and to correct our AS phenotype. Success of this drug in correcting an AS
phenotype in vitro will lead to further investigation of this drug as a potential AS therapy.
Lay Abstract
Angelman Syndrome (AS) is a neurodevelopmental disorder characterized by severe seizures,
walking and gait problems, absent speech, learning disability, happy demeanor, and characteristic
“puppet-like” arm movements. AS is caused by the loss of expression from the maternal copy of
the UBE3A gene. Individuals with AS have an intact paternal copy of UBE3A that is silenced.
Drugs that can unsilence the paternal copy of UBE3A have been identified, and are predicted to
provide a potential “gene” therapy for AS patients.. We have previously generated patient-specific
stem cells (iPSCs) from an AS patient with a mutation in the maternal copy of UBE3A. We plan
to use genome editing tools to correct the mutation in AS stem cells to produce cells that are
genetically identical to the AS patient, but do not have AS. We will use these cells to make AS
and normal neurons to identify differences between them. Once we have identified these
differences, we will treat the AS neurons with a drug that unsilences paternal UBE3A to determine
whether it can correct the differences between AS and normal neurons. Success of this drug in our
cultured human neurons will lead to future investigation of this drug as a potential therapy for
individuals with AS.
Poster #20
Patient-Derived Disease Models
Synaptic pathophysiology in stem cell-derived neurons from dup15q autism and Angelman
syndrome patients
James J. Fink (1), Tiwanna M. Robinson (1), Kaitlyn A. Bolduc (1), and Eric S. Levine (1)
(1) University of Connecticut Health Center
Technical Abstract
Individuals with a maternal deletion of chromosome 15q11-q13 suffer from Angelman syndrome
(AS), while those with a duplication of the same region suffer from a form of autism known as
15q duplication syndrome (dup15q), which is one of the most frequent chromosomal abnormalities
associated with autism. Both of these syndromes are neurodevelopmental disorders that often
present with common features, including intellectual disability, impairments in language, and
seizures. The gene believed to be responsible for these phenotypes encodes the ubiquitin ligase
UBE3A, but other genes may also contribute. In both syndromes, alterations in synaptic signaling
and plasticity appear to play a critical role in the disease phenotype.
In this study, we are using electrophysiological approaches and calcium imaging to examine
synaptic activity and plasticity of iPSC-derived neurons from AS, dup15q, and control subjects.
Low levels of spontaneous excitatory synaptic activity were seen after six weeks in vitro in both
patient-derived and control neurons. After twelve weeks, control neurons displayed a dramatic
increase in the frequency of synaptic events, which was significantly greater than the frequency in
both AS-derived neurons and dup15q-drived neurons, suggesting a genotypic difference in
synapse number and/or release probability. These iPSC-derived neurons also show spontaneous
action potential firing and synchronous activity, which can be enhanced by NMDA receptor
activation and increased intracellular cAMP levels. We are currently exploring potential
differences in these processes in AS and dup15q-derived neurons using single cell recordings and
multi-cell calcium imaging. Overall, these approaches may prove useful for identifying novel
targets for drug discovery and for screening potential therapeutics aimed at reversing the seizures,
movement disorders, and language and cognitive impairments in Angelman syndrome and autism.
Lay Abstract
Angelman syndrome (AS) and 15q duplication syndrome (Dup15q) are related brain disorders that
share a few common features including intellectual disability, seizures, and impairments in speech
and language. Previous work suggests that the signaling that occurs between neurons, the principal
cell type of the brain may be disrupted in both of these disorders. Although much is known about
the genes that are disrupted in these syndromes, how these genes cause disruptions in neuron
signaling is unknown. In this study we are using patient-derived induced-pluripotent stem cells
(iPSC) to produce live human neurons from AS, Dup15q, and control subjects. We are using these
patient-specific neurons to understand how neuron signaling is disrupted in AS and Dup15q
syndrome. We have observed significant differences in the maturation of neuron function in these
cells. Overall, these approaches may prove useful for identifying novel targets for drug discovery
and for screening potential therapeutics aimed at reversing the seizures, movement disorders,
speech/language, and cognitive impairments in Angelman and Dup15q syndromes.
Poster #21
Patient-Derived Disease Models
Alternative splicing factors, RBFOX1 and RBFOX2, are dispensable in iPSCs and iPSCderived neurons and do not contribute to neural-specific paternal UBE3A silencing.
Ivy P-F. Chen (1), Michael O. Duff (1), Brenton R. Graveley (1), Stormy J. Chamberlain
(1)
(1) University of Connecticut Health Center, Farmington, USA
Technical Abstract
Angelman syndrome (AS) is a neurodevelopmental disorder characterized by microcephaly,
seizures, ataxia, and severe cognitive disability. Patients often display a characteristic happy affect
and are non-verbal. AS affects ~1/20,000 live births with no cure available. This disorder is caused
by the loss of function of maternal UBE3A, which is located in an imprinted domain on
chromosome 15q11-q13. Because of tissue-specific genomic imprinting, UBE3A is expressed
biallelically in non-neuronal cells, while it is only expressed from the maternal allele in neurons.
In all individuals with AS, the paternal UBE3A allele is intact and functional, although
epigenetically silenced. We are interested in UBE3A gene regulation during neural development
because reactivation of paternal UBE3A is a potential therapeutic strategy for treating AS.
We hypothesized that splicing changes during neural differentiation would result in neuronspecific processing of UBE3A-ATS, and thus lead to the silencing of paternal UBE3A. By
performing cross-linking immunoprecipitation on AS iPSCs and iPSC-derived neurons, we found
that two of the RBFOX family of alternative splicing proteins, RBFOX1 and RBFOX2, bind
extensively to the paternally-expressed UBE3A-ATS in neurons. However, by knocking out
RBFOX1 and/or RBFOX2 using CRISPR/Cas9 lentiviruses, we demonstrated that their absences
do not affect gene expression at the UBE3A-ATS and UBE3A loci. Contrary to a previous report,
RBFOX2 knockout iPSC lines are viable and we did not observe any gross defects in RBFOX1
and RBFOX2 knockout iPSC lines other than previously reported splicing changes. Moreover,
despite the fact that both proteins play a role in regulating splicing of several important neuronal
genes, we did not observe significant perturbation during in vitro neural differentiation. Our results
suggest that RBFOX1 and RBFOX2 are dispensable in iPSCs and iPSC-derived neurons and do
not contribute to neural-specific paternal UBE3A silencing.
Lay Abstract
Angelman syndrome (AS) is a brain development disorder caused by deletion or mutation of the
mother’s copy of the UBE3A gene. Normally, humans inherit two copies of UBE3A, one from
father and one from mother. The copy inherited from father does not make the UBE3A protein in
the brain, so the mother’s copy of the gene is the only source of UBE3A in this organ. Therefore,
when the mother’s copy of UBE3A is deleted or mutated in AS patients, the father’s copy cannot
compensate, and the patient has no UBE3A in their brain, causing AS. Nonetheless, every AS
patient still has the intact, but non-functional copy of UBE3A that they inherited from their father.
Activation of the non-functional paternal copy of UBE3A is a promising therapeutic approach to
treating AS because it would provide the UBE3A protein to the brains of AS patients. We have
been investigating the role of alternative splicing, the process by which different pieces of genes
are glued together, in the regulation of the paternal copy of UBE3A. Our results suggest that
although two proteins involved in alternative splicing, RBFOX1 and RBFOX2, bind next to the
UBE3A gene, they do not play a role in regulating the activity of the paternal copy of UBE3A,
and thus cannot be used to develop a potential therapy.
Poster #22
Patient-Derived Disease Models
Patient-derived iPS Cells from Primary Progressive Multiple Sclerosis Reveal Defect in
Myelin Injury Response
Alexandra M. Nicaise (1), Erin Banda (1), Kristen Russomanno (1), Kasey M. Johnson (1),
Wanda Castro-Borrero (2), Rosa M. Guzzo (3, 4), Albert C. Lo (5, 6), Stephen J. Crocker
(1, 4)
(1) Department of Neuroscience, (2) Neurology, and (3) Orthopedic Surgery, and (4) Stem
Cell Institute, University of Connecticut School of Medicine, Farmington, CT;(5) Mandell
Center for Multiple Sclerosis, Hartford, CT; (6) Department of Neurology and Department
of Epidemiology, Brown University, Providence, RI; Providence Veterans Affairs Medical
Center, Providence, RI
Technical Abstract
Multiple sclerosis (MS) is characterized by chronic demyelination of the central nervous system.
Most MS patients experience episodes of clinical worsening followed by recovery (called
relapsing-remitting, or RRMS), while patients with primary progressive multiple sclerosis (PPMS)
develop a disease course without relapses and persistent chronic demyelination. Because PPMS is
refractory to current immunomodulatory therapies for RRMS, regenerative therapies offer promise
to treat PPMS, but will depend on key endogenous regenerative capacity of the PPMS brain to
promote remyelination. Here we tested the regenerative potential of iPSC lines developed from
PPMS patients in an established cuprizone mouse model of CNS demyelination. Blood taken from
PPMS patients, and either their spouse or blood relative, was reprogrammed into iPSCs. Our
previous work using human embryonic stem cell-derived neural progenitor cells (NPCs)
determined that these cells, when administered intravenously into cuprizone-fed mice migrated
into demyelinated CNS lesions and effectively reversed demyelination. To determine whether
NPCs from PPMS patients had equivalent capacity to promote brain repair we injected PPMS
NPCs or control NPCs into mice fed cuprizone. We found NPCs from control iPSC lines provided
robust protection and CNS remyelination, while PPMS cases evoked minimal protection or
remyelination in the actively demyelinating brain. Analysis of NPC cell fates within the affected
area of the corpus callosum in treated mice, we determined that PPMS NPCs tended to differentiate
towards astrocytes, while control NPCs differentiated towards an oligodendrocyte cell fate.
Additionally, conditioned media from control NPCs support oligodendrocyte differentiation but
conditioned media from PPMS NPCs did not. These data provide the first direct evidence that
there is an inherent defect in PPMS to promote CNS myelination which may contribute to the
chronic nature of this disease.
Lay Abstract
Multiple sclerosis (MS) is the most common cause of chronic neurological disability affecting
people 18-40 years of age. Clinically, MS manifests in early adulthood as different subtypes with
the most common type exhibiting bouts of exacerbations (or relapses) followed by remission, with
varying degrees of recovery. The most severe form of MS is called primary progressive MS
(PPMS). PPMS is distinct from other forms of MS as patients progressively accrue disability
without remission. PPMS progresses rapidly and significantly reduces life expectancy. Disability
in MS is a result of the loss of myelin, a specialized tissue made by a cell called the
oligodendrocyte. All current treatments for relapsing forms of MS, which target the immune
system, do not benefit PPMS patients. Therefore, promoting repair of brain damage in PPMS is
needed to halt PPMS disease progression, and offers the potential to restore neurologic deficits.
Oligodendrocyte progenitor cells (OPCs) are the cells within the brain that can mature to become
new oligodendrocytes. OPCs are also found within demyelinated lesions in MS; however
remyelination in MS is incomplete. Understanding what limits OPC replacement in MS will guide
future reparative strategies for PPMS. Toward this goal, we developed lines of patient-specific
induced pluripotent stem (iPS) cells generated from PPMS patients, and their spouse or bloodrelatives. When we tested these cells in an animal model of active demyelination, we found that
control cells (non-disease cells) effectively prevented the myelin damage whereas cells from
PPMS did not. These data provide the first direct evidence that there is an inherent myelinating
defect in PPMS which may contribute to the chronic nature of this disease. Using these cells from
PPMS patients we may start to investigate why remyelination fails in these patients and develop
new strategies to promote brain repair to reverse the course of this chronic disease.
Poster #23
Patient-Derived Disease Models
Interstitial Triplication of 15q11-q13: An Assessment of Methylation Status and Gene
Expression
Alexandra Goetjen (1), Noelle Germain (1), Stormy Chamberlain (1)
(1) University of Connecticut Health Center
Technical Abstract
The majority of genes in the human genome are biallelically-expressed, but this is not true for a
number of genes that are located at the 15q11-q13 locus, which are regulated by a process called
genomic imprinting. Genomic imprinting is a phenomenon in which genes are expressed in a
parent-of-origin specific manner. Imprinted genes are functionally haploid. The master regulator
of gene expression from this imprinted region is a differentially methylated region known as the
Prader-Willi imprinting center (PWS-IC). A number of genes are selectively expressed from the
paternal allele, whereas one gene (UBE3A) is maternally-expressed in neuronal cells. There are
three clinical syndromes that result from duplication or deletion of these mono-allelically
expressed genes: Prader-Willi syndrome, Angelman syndrome, and 15q duplication syndrome.
Children with 15q duplication syndrome present with cognitive dysfunction, speech/language
disorders, and autism. Duplication of the 15q11-q13 locus may occur as an interstitial duplication
or as an isodicentric chromosome 15 (idic(15)) that contain two or three copies of the maternal
locus, respectively. The focus of this project was to characterize gene expression and methylation
status of the 15q11-q13 locus in a patient with a maternal interstitial triplication. Induced
pluripotent stem cells (iPSCs) and iPSC-derived neurons were used as the model system.
Methylation at the PWS-IC, was shown to be 75%, consistent with the individual’s cells having
three maternal copies and one paternal copy of the 15q11-q13 locus. There was a positive
correlation between the level of gene expression and copy number in iPSCs generated from the
patient with the maternal interstitial triplication. We observe minor differences in gene expression
between interstitial triplication and idic(15) iPSCs or neurons, suggesting that different structural
presentations of the three maternal copies of 15q11-q13 may be regulated similarly.
Lay Abstract
This project focuses on 15q duplication syndrome, in which children present with cognitive
dysfunction, speech/language disorders, and autism. It is caused by extra copies of a small piece
of chromosome 15. An increased number of maternal copies of this region correlate with a greater
severity of the clinical presentation. However, it is not known which genes in this genetic region
are responsible for the disorder. We made induced pluripotent stem cells (iPSCs) from three
patients who inherited 3 maternal copies of this region of chromosome 15 and converted these
cells into brain cells. Two of the patients carried the extra copies on a separate, extra chromosome,
while one patient carried the extra copies on the maternal copy of chromosome 15. We compared
gene expression between iPSCs and brain cells from these three patients. For most genes, there
appears to be a direct link between the level of expression of genes from this region and the number
of maternal copies of the region. Furthermore, minor differences were observed between the
different structural arrangements of the extra copies in brain cells, suggesting that these different
presentations may be regulated similarly.
Poster #24
Patient-Derived Disease Models
Studying Systemic Lupus Erythematosus (SLE) using human pluripotent stem cells
Yangfei-Xiang (1), Shin Min (2), In-Hyun Park (I), InSoo Kang (2), and Joseph Craft (3).
(1) Dept. of Genetics, Stem Cell Center, Yale University (2) Dept. of Internal Medicine,
Yale University (3) Dept. of Immunobiology, Stem Cell Center, Yale University
Technical Abstract
Systemic lupus erythematosus (SLE or lupus) is a chronic autoimmune inflammatory disease that
affects multiple organs including the skin, joints, kidneys and blood cells. Although the exact cause
of SLE is unknown, both genetic and environmental factors play a role in breaking immune
tolerance to self-antigens and inciting inflammation. Patients with SLE have increased morbidity
and mortality compared to the general population. However, the current treatments are not curative
and carry considerable levels of adverse effects such as infections by suppressing the immune
system globally. Lupus patients have increased levels of inflammatory molecules in the affected
tissues and/or or cells. The increased production of such molecules from immune cells can be
driven by alterations in the genes involved in their production. Thus, these genetic alterations can
be a good therapeutic target in correcting dysregulated inflammation in lupus. In the current
proposal, we have been addressing this critical question by editing a lupus risk genetic variant of
interferon regulatory factor 5 (IRF5), a transcription factor which drives the production of
inflammatory molecules, into the non-risk genetic variant in induced pluripotent stem cells (iPSCs)
from lupus patients and testing whether this editing corrects inflammation in a humanized lupus
mouse model. By utilizing iPSCs, gene editing and humanized mice, the results of our study would
open a new paradigm in treating lupus.
Lay Abstract
Systemic lupus erythematosus (SLE or lupus) is a chronic autoimmune inflammatory disease that
results from both genetic and environmental factors. SLE affects multiple organs including the
skin, joints, kidneys and blood cells. The exact cause of SLE is still unknown and SLE is disease
with high morbidity and mortality. The current treatments are not curative and carry considerable
levels of adverse effects. Lupus patients have increased levels of inflammatory molecules in the
affected tissues and/or or cells. Here, we propose to study SLE using human pluripotent stem cells,
especially induced pluripotent stem cells (iPSCs) derived from patients. iPSCs are generated by
overexpressing four proteins regulating protein expression in pluripotent stem cells. iPSCs carry
the same genetic materials as donor cells. From iPSCs from SLE patients, we will correct a variant
in gene well known for SLE, interferon regulatory factor 5 (IRF5). The iPSC lines of pathogenic
and corrected IRF5 will be differentiated into blood cell type called monocytes which is critical in
SLE development. We will study the role of IRF variant in monocytes using cellular and molecular
tools. Our results will be an important step to understand and to find therapeutics for SLE.
Poster #25
Patient-Derived Disease Models
Restoring mitochondrial morphology rescues axonal defects in patient-derived neurons of
hereditary spastic paraplegias
Kyle R. Denton (1), Chongchong Xu (1), Craig Blackstone (2), and Xue-Jun Li (1,3)
(1) Department of Neuroscience, The University of Connecticut Health Center,
Farmington, CT 06032, USA, (2) Cell Biology Section, Neurogenetics Branch, National
Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD
20892, USA, (3) The Stem Cell Institute, The University of Connecticut Health Center,
Farmington, CT 06032, USA.
Technical Abstract
Hereditary spastic paraplegias (HSPs) comprise a large and diverse group of inherited
neurodegenerative disorders (SPG1-71). The hallmark of all HSP subtypes is a length-dependent
distal axonopathy, resulting in prominent lower limb spasticity, which is primarily caused by the
degeneration of cortical-spinal motor neurons. Two autosomal recessive forms of HSP, SPG15
and SPG48, are associated with thinning of the corpus callosum, cognitive impairment, and
juvenile parkinsonism. These two forms are caused by loss-of-function mutations to the ZFYVE26
and AP5Z1 genes, which encode spastizin and the adaptor protein complex 5 zeta 1 (AP5Z1)
protein respectively. Little is known about the function of these two proteins, and the mechanisms
by which the loss of these two proteins results in axonal defects are unknown. Here, we generated
induced pluripotent stem cell (iPSC) from SPG15 and SPG48 patients and differentiated these
iPSC lines to telencephalic glutamatergic and midbrain dopaminergic (mDA) neurons.
Interestingly, neurite number, length, and branching were significantly reduced in SPG15 and
SPG48 telencephalic glutamatergic neurons and mDA neurons, suggesting that spastizin and
AP5Z1 play a role in neurite development of affected neurons. Next, the morphology of
mitochondria was analyzed, because spastizin partially colocalizes to mitochondria. This revealed
a significant reduction in the length and density of mitochondria within neurites of SPG15 and
SPG48 telencephalic glutamatergic neurons and mDA neurons. Mitochondrial membrane potential
was reduced in SPG15 neurons, and there was an increase in apoptosis in both SPG15 and SPG48
cells. Treatment with a mitochondrial fission inhibitor, mdivi-1, rescued mitochondrial
morphology, neurite outgrowth, and levels of apoptosis in SPG15 neurons. These results link
mitochondrial fission/fusion alterations to SPG15 and SPG48, and identify mitochondria as a
potential target for therapeutics in the future.
Lay Abstract
A large group of inherited neurodegenerative disorders, known collectively as hereditary spastic
paraplegias (HSPs), are a significant source of long term disability. All HSP patients suffer from
the degeneration of nerves needed for voluntary movement of the legs, however some severe forms
of HSP present with other symptoms, including intellectual disability and early onset
parkinsonism. Here, we have studied two forms of HSP associated with parkinsonism, called
SPG15 and SPG48, using stem cell-derived neurons. A number of cellular abnormalities were
observed in patient-derived neurons, including smaller, less complex shape, increased cell death,
and dysfunctional mitochondria. As mitochondria are the main source of energy for cells, we tested
whether drugs that targeted mitochondria would be beneficial to SPG15 and SPG48 neurons.
Following treatment with a drug that improved the shape of mitochondria, the health of the neurons
was improved. This study found that therapeutics targeting mitochondria may be beneficial for
SPG15 and SPG48 patients in the future.
Poster #26
Patient-Derived Disease Models
Dendritic Spine Morphogenesis in Neurons derived from Neurotypical and Dup15q iPSCs
Olena O. Marchenko(1), Leslie M. Loew(1), Stormy J. Chamberlain(2)
(1)Department of Genetics and Developmental Biology and Uconn Stem Cell Institute,
University of Connecticut Health Center, Farmington, CT 06030-6403 (2)Richard D.
Berlin Center for Cell Analysis and Modeling, University of Connecticut Health Center,
Farmington, CT 06030-6403
Technical Abstract
Dendritic spines are small mushroom-like membranous structures that grow on neuron dendrites
and receive signals from nearby axons. Increased hippocampal spine density and abnormal spine
shapes have been associated with many neurodevelopmental disorders, as shown in animal models
and postmortem human brain studies. While symptoms of Dup15q Syndrome are wellcharacterized, there is no neuronal phenotype associated with them. This knowledge is required
for development of novel clinical therapies and diagnostics that target debilitating symptoms of
Dup15q.
Our goal is to investigate actin network changes that make up spine formation, in neurons obtained
by in vitro differentiation of dup15q patient stem cells, and compare them with network changes
of the neurotypical neurons. We compared filopodia motility and density observed in human
neuron culture derived from neurotypical and dup15q iPSC lines and found an increased filopodia
density in dup15q lines. We measured, and compared spine shape and density in dup15q and
neurotypical neurons. To analyze experimental results we have developed a mathematical model
that can predict actin network growth and stabilization in dendritic spines and used it to determine
factors that promote spine shape formation and reproduce Dup15q spine shape. The characterized
Dup15q spine phenotype can be used for drug testing platforms and early intervention strategies
that target upstream regulators of dendritic spinogenesis.
Lay Abstract
Dendritic spines are small mushroom-shaped structures present on neurons that are involved in
communication between neurons. Studies of dendritic spine shapes in human cadaver brains and
in animals showed association of abnormal shapes with cognitive and motor disorders. Dup15q
syndrome is a genetic disorder in which affected individuals have intellectual disability, motor
skills deficits, seizures, and autism. Symptoms of Dup15q syndrome are well documented, but
dendritic spine formation and shape has not been studied. This knowledge may be helpful for the
development of novel clinical therapies and diagnostics that target the debilitating symptoms of
Dup15q syndrome.
We compared spine shapes and density in control and Dup15q neurons derived from human stem
cells and found altered spine density and shape in Dup15q neurons. Dendritic spines are filled with
actin, a protein that links with itself to form complex networks. To identify factors important for
the altered spine formation in Dup15q neurons, we used a mathematical model that generates
different spine shapes by calculating how the actin network grows and shrinks. This will help us
identify new ways in which dendritic spine shape and density can be manipulated and how spines
grow in general. This information can be used for drug testing platforms and to identify potential
therapeutics that can regulate dendritic spine formation.
Poster #27
Patient-Derived Disease Models
Novel discovery of muscle lim-protein gene as a common modifier in hypertrophic
cardiomyopathy using patient induced pluripotent stem cells
Y. Ren* (1), T. Yi* (1), D. Jacoby (1), I-Ping Chen(2),Y. Qyang(1) et al.
(1) Yale School of Medicine Cardiology (2) University of Connecticut Health Center
* Contributed equally
Technical Abstract
Hypertrophic cardiomyopathy (HCM) is the most common inheritable heart disease in the world.
While the individual mutations causing HCM continue to be studied actively and there are likely
to be additive effects at the organ and organism levels, very little is known regarding possible
interactions between multiple HCM-causing mutations at the cellular and tissue levels. We are
using a family case to study how different genes may interact with each other and cause severe
symptoms. In this family, the asymptomatic father is heterozygous for the cardiac myosin MYH7
(R723C) gene, who has mild hypertrophy. The asymptomatic mother is heterozygous for the
muscle lim protein (MLP) (W4R) gene, a common genetic allele appearing in 1 out 100
Caucasians. Their child, who has an abnormally large heart, is heterozygous for both mutations.
In this study, we derived and characterized patient-specific induced pluripotent stem cells (iPSCs)
from this double mutation family and differentiate them into cardiomyocytes.. We measured the
cell area of the derived cardiomyocytes from the patient family iPSCs. The results showed the
father and mother have relatively normal cardiomyocytes size while the boy has an unusually
larger cell size.. Whole cell calcium transient measurements showed that the boy and the father
have significantly lower systolic calcium while the mother is relatively normal. The infection of
MLP lentivirus in the double mutant cardiomyocytes showed that over-expressed wild type MLP
partially rescued the double mutant systolic calcium. Our results suggested that the MLP gene is a
common disease modifier instead of the disease causative gene. In this case, MYH7 is the diseaseinitiating mutation to cause hypertrophy in the double mutant while the MLP mutation exacerbates
the phenotype. Thus, our hiPSC approach has revealed a novel hypertrophic cardiomyopathy
disease mechanism and will set the stage for developing novel therapies to treat this deadly disease.
Lay Abstract
Enlarged heart, or hypertrophic cardiomyopathy (HCM), is the most common inheritable heart
disease in the world. While the individual mutations that cause HCM continue to be studied, it is
possible that multiple mutations together can cause a more severe disease. Very little is known
about what happens when an individual has multiple different HCM-causing mutations. We are
using a family case to study how mutations in two different genes interact with each other to
influence heart function. In this family, the father has mild HCM but has no symptoms. He has
DNA change in one copy of his MYH7 gene. The mother also has no symptoms. She is has a
DNA change in one copy of her MLP gene that occurs in 1 out 100 Caucasians. This change does
not cause disease by itself. Their child, who has an abnormally large heart, is has both DNA
changes. In this study, we derived and characterized patient-specific induced pluripotent stem cells
(iPSCs) the father, mother, and child from this family and converted them into heart cells known
as cardiomyocytes. Our primary data showed that the cardiomyocytes from the child, which have
both DNA changes, are larger and can store less calcium. Furthermore, we found that in this
family, the DNA change in the MYH7 gene is likely causing HCM and the DNA change in the
MLP gene makes it worse. The interactions between these two genes have never been described
before, and thus, our hiPSC approach has revealed a new heart disease mechanism. Understanding
this mechanism will help in the development of specific therapies to treat this deadly disease.
Poster #28
Patient-Derived Disease Models
Modeling Human Chondrodysplasias with Induced Pluripotent Stem Cells
1
Renee Wasko, 1,2,*Sara Patterson, Ph.D., 3Dan Cohn, Ph.D., 4Arthur Hand, D.D.S., and
1,2,5
Caroline Dealy, PhD; 1Department of Reconstructive Sciences, 4Department of
Orthopaedic Surgery and Center for Regenerative Medicine and Skeletal Development,
and 4Department of Craniofacial Sciences, University of Connecticut Health Center;
3
Department of Molecular, Cell and Developmental Biology, University of California Los
Angeles, CA; and 5Chondrogenics, Inc., Farmington, CT. *Current: Jackson Laboratories
Technical Abstract
Chondrodysplasias are a group of rare, heritable disorders of cartilage that affect children and
adults. Chondrodysplasias disrupt the growth and homeostasis of the cartilaginous skeleton, and
cause severe disability and even death. Disease features include extreme short stature (dwarfism);
skeletal malformation and premature osteoarthritis. Although each disorder is itself very rare, as a
group Chondrodysplasias are a considerable health concern, occurring in 1/4000
births. Development of treatments for Chondrodysplasias requires better understanding of disease
mechanisms to identify druggable targets, and development of systems in which to test potential
treatments. However, these efforts are hampered by lack of available tissue from patients with
which to study the disease, and the unsuitability of available animal models, which do not always
reproduce the same genetic or phenotypic characteristics. The goal of this project is to develop an
in vitro model of human Chondrodysplasias using patient-derived induced pluripotent stem cells
(iPSCs). We have previously established a rapid and efficient method for directing the
differentiation of pluripotent human cells including iPSCs into the chondrogenic lineage. Here, we
have used this method to direct the chondrogenic differentiation of iPSCs derived from the
fibroblasts of a patient with Spondyloepiphyseal dysplasia congenita (SEDC), a Chondrodysplasia
caused by mutations in collagen type II, a major component of cartilage matrix. We have identified
a cell-stress response in SEDC-iPSC-derived chondrocytes that offers a novel target for potential
patient treatments. We found that cell stress responses are inappropriately activated in SEDCiPSC-derived chondrocytes, as confirmed by molecular profiling and by ultrastructural
examination via Transmission Electron Microscopy. These responses can explain the phenotypic
changes seen in SEDC patients, including the growth retardation of the cartilaginous skeleton that
leads to extreme short stature, as well as the premature degradation of the articular cartilage of the
joints that causes early-onset osteoarthritis. Our SEDC-iPSC model could be used to test drugs
for SEDC that target these cell-stress responses, and may also be useful in modeling more than
100 other equally rare, disabling, and currently untreatable Chondrodysplasias.
Lay Abstract
Chondrodysplasias are a group of rare, heritable disorders of cartilage that affect children and
adults, and cause extreme short stature (dwarfism), skeletal malformation and premature
osteoarthritis. Development of treatments for these disabling disorders requires reliable models for
disease study and drug testing. In this project, we have established a model for one of the most
severe Chondrodysplasias using patient-derived induced pluripotent stem cells (iPSCs). Using this
model, we have identified a cell-stress response in the diseased iPSC-derived cartilage cells that
offers a novel target for potential patient treatments. Our iPSC-based Chondrodysplasia model has
exciting utility for mechanistic study and drug testing for more than 100 other rare, disabling, and
currently untreatable Chondrodysplasias.
Poster #29
Resources and Techniques
Generation of Induced Pluripotent Stem Cell (iPS) Lines from Human Dendritic Cells
Arvind Chhabra
University of Connecticut Health Center
Technical Abstract
Human induced pluripotent stem cells (iPS) represent a unique source to create donor specific cells
of choice. Among the approaches available to reprogram human somatic cells includes integrating
recombinant viral vector mediated methods as well as non-integrating virus based approaches,
such as episomal vectors. While integrating virus based approaches have higher iPS generation
efficiencies, non-integrating virus based approaches are highly desirable for translational usage of
the iPS lines generated, due to clinical safety concerns associated with the integrating virus based
approaches. Sendai virus is a RNA virus that has recently been shown to effectively program the
transduced somatic cells, without integrating in the genome. We here report the generation of
human induced pluripotent stem cell (iPS) lines from human dendritic cells (DC), by Sendai virus
based approaches. We here show data from three iPS lines derived from the DC of two different
individuals. We show that these iPS lines exhibit pluripotent phenotype, and can effectively
yielded embryoid bodies (EB) that could generate hematopoietic stem cells (HSC), characterized
by FACS and colony forming unit (CFU) assays.
Lay Abstract
Human induced pluripotent stem cells (iPS) can be used to create donor specific cells of choice.
Towards generating iPS lines from human subjects by methods that do not involve integrating
viruses, we here show successful derivation of three iPS lines from peripheral blood derived
dendritic cells (DC) of two different healthy individuals, by the Sendai Virus based approach. The
iPS lines exhibit pluripotency, proof that they are stem cells, and can be used to derive
hematopoietic stem cells (HSC). These lines will be good source to develop donor specific cell
lineages and personalized therapeutics.
Poster #30
Resources and Techniques
Next Generation High-throughput Sequencing at Genomics Core of Yale Stem Cell Center
Mei Zhong and Haifan Lin
Stem Cell Center and Department of Cell Biology, Yale University School of Medicine,
New Haven, CT 06520
Technical Abstract
Yale Stem Cell Center (YSCC) Genomics Core is one of the five core facilities at the YSCC. The
mission of the Genomics Core is to offer a high level of expertise in next generation sequencing
technology to support stem cell research at Yale and across the State of Connecticut. The Genomics
Core is a hub for collaborations and a training site for stem cell researchers who wish to extend
their research using genomic approaches. Using the Illumina HiSeq 2000 platform, the Genomics
Core offers standard services including DNA sequencing (DNA-Seq), transcriptome analysis
(RNA-Seq), small RNA discovery (smRNA-Seq), gene regulation & epigenetic analysis (ChIPSeq) and cost effective multiplex sequencing (multiple samples in one lane). As part of technical
development, the Genomics Core also offers collaborative research on projects to explore the
sequencing technology to go beyond the Illumina protocol limitation tailoring to the project needs
of stem cell researchers. In 2014, the genomics core had purchased a C1 Single-Cell Auto Prep
System from Fluidigm Corporation, which extends our mRNA sequencing services down to single
cell resolution.
Lay Abstract
Yale Stem Cell Center (YSCC) Genomics Core is one of the five core facilities at the YSCC. The
mission of the Genomics Core is to offer a high level of expertise in next generation sequencing
technology to support stem cell research at Yale and across the State of Connecticut. In addition
to providing deep sequencing services, the Genomics Core trains investigators and collaborates
with investigators to develop new technologies to support their research using genomic
approaches. Using the Illumina HiSeq 2000 platform, the Genomics Core offers standard services
including DNA sequencing (DNA-Seq), transcriptome analysis (RNA-Seq), small RNA discovery
(smRNA-Seq), gene regulation & epigenetic analysis (ChIP-Seq) and cost effective multiplex
sequencing (multiple samples in one lane). The Genomics Core also offers consulting services to
investigators who are looking to explore the sequencing technology to go beyond the Illumina
protocol limitations by tailoring to the project needs of the stem cell researchers. In 2014, the
Genomics Core purchased a C1 Single-Cell Auto Prep System from Fluidigm Corporation to
extend our mRNA sequencing services down to single cell resolution.
Poster #31
Resources and Techniques
Modular Cell Type Database
James Lindsay (1)Ion Mandoiu, Craig Nelson (2)
(1) Smpl Bio LLC(2) University of Connecticut
Technical Abstract
The definition of a cell type is contentious at best, therefore Smpl Bio has adopted a customizable
and modular strategy toward aggregating single cell, cell line and bulk tissue expression profiles
into unique cell types. This database will not only serve as a repository for single cell data, but by
accepting cell line and bulk mixtures (coupled with deconvolution) will allow it to become a
unified source to cell type definitions. The Cell Type DB is composed of 3 distinct layers; the raw
expression layer, an organization layer and finally an aggregation to cell type layer.
The raw gene expression layer accepts 3 types of data, single cell gene expression, cell line gene
expression and finally bulk (heterogeneous mixtures) gene expression. The organization layer’s
primary purpose is to stratify each individual gene expression profile into clusters corresponding
to cell type, then converted into the canonical expression profiles. The procedure for this is
different for each data type; single cell will be processed using the clustering and visualization
methodology, cell line data is highly homogeneous and require minimal preprocessing, while bulk
data will be computationally deconvolved into its constituent cell types. The final aggregation
layer will consist of the computed cell type expression profiles. A query can be constructed to
extract desired cell type profiles. This module access layer enables the database to support
multiple,
well
defined
strategies
for
defining
a
cell
type.
We have developed a new guided-deconvolution algorithms to address discern the composition of
heterogeneous mixtures leveraging single cell data. With these algorithms the user can accurately
reconstruct the population structure of the original sample, including accurate estimates of cell
type frequencies, the presence of rare that may be missing from the single cell analysis.
Lay Abstract
This work presents a modular cell type database which aggregates multiple sources of gene
expression profiles and cell type definitions. The database can be used to aide in the matching of
gene expression profiles to known cell types and the identification and storage of novel types. It
leverages single cell, cell line and heterogeneous gene expression data types.
Poster #32
Resources and Techniques
Genome editing service of the UConn-Wesleyan Stem Cell Core
Christopher Stoddard (1) and Marc Lalande (1)
(1)University of Connecticut Health Center
Technical Abstract
Precise gene editing in hESC/iPS cells provides a powerful tool for the study of disease models in
vitro. The ability to edit the human genome has been hampered by the low levels of homologous
recombination in human cells as opposed to mouse stem cells. The ability to make precise genome
breaks has allowed us to induce homologous recombination in human cell lines. The use of
TALENS and CRISPRS allow us to precise cause a double stranded break in the genome that
induce the machinery for homologous recombination. The use of these genome cutters in along
with carefully designed targeting vectors allows for the precise modification of the genome. The
ability to achieve efficient homologous recombination in hESC and iPS cells enables researchers
to generate specific mutations in wildtype cells or to genetically correct mutations in patientspecific cells. These isogenic cell lines with and without specific genetic changes are powerful
tools to study human development and disease. Gene editing can also be used to generate reporter
cell lines that can be used for lineage tracing, identification of specific cell sub-populations, or
drug screening. The gene editing services provided by the UConn-Wesleyan Stem Cell Core and
Gene Targeting and Transgenic Facility (GTTF) provides TALEN and CRISPR constructs as well
as targeting vectors for these studies. We also offer full start-to-finish gene editing services for
nearly any cell line. We have demonstrated the ability to create/verify and use these tools to edit
multiple cell lines in both murine and human cells. As a result, we are excited to provide these
services to the research community through the UConn-Wesleyan Stem Cell Core and the GTTF.
Lay Abstract
The gene editing services provided by the UConn-Wesleyan Stem Cell Core and the Gene
Targeting and Transgenic Facility (GTTF) provides TALEN and CRISPR –based tools that
enable precise gene editing in human pluripotent stem cells. These tools can be used to create
mutations in normal cell lines, or to correct mutations in patient-specific cell lines. The ability to
compare mutated and normal cell lines that are otherwise genetically identical is a powerful tool
for scientific research. In addition to offering validated DNA vectors for researchers to use, our
core facilities also provide full gene editing services. These vectors and services can be used in
most mouse and human cell types and are available to the entire research community.
Poster #33
Disease Models and Mechanisms
Cloning and Variation of Ground State Intestinal Stem Cells
Xia Wang (1), Yusuke Yamamoto (1), Lane H. Wilson (1), Gang Ning (1), Yue Hong (1),
Benoit Chevalier (1), Frank McKeon (2), Wa Xian (1)
(1) The Jackson Laboratory (2) Genome Institute of Singapore
Technical Abstract
Stem cells of the gastrointestinal tract, pancreas, liver, and other columnar epithelia collectively
resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly
clonogenic, “ground state” stem cells of the human intestine and colon. We show that derived
stem cell pedigrees sustain limited copy number and sequence variation despite extensive serial
passaging and display exquisitely precise, cell-autonomous commitment to epithelial
differentiation consistent with their origins along the intestinal tract. This developmentally
patterned and epigenetically maintained commitment of stem cells likely enforces the functional
specificity of the adult intestinal tract. Using clonally-derived colonic epithelia, we show that
toxins A or B of the enteric pathogen C. difficile recapitulate the salient features of
pseudomembranous colitis. The stability of the epigenetic commitment programs of these stem
cells, coupled with their unlimited replicative expansion and maintained clonogenicity, suggests
certain advantages for their use in disease modeling and regenerative medicine.
Lay Abstract
Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) face formidable barriers to
clinical use, including risk of teratoma, complicated and inefficient differentiation strategies, and
limited regenerative capacity of the cells ultimately produced. Stem cells intrinsic to regenerative
tissues largely bypass many of these issues. Green and colleagues developed methods for cloning
epidermal stem cells that can be engrafted. These methods have been successfully applied to
cornea, thymus, and airway tissues. However, it is difficult to clone these types of stem cells from
columnar epithelial tissues, such as stomach and intestine, in a manner that maintains their stem
cell state. The present study reports the cloning and propagation of “ground state” human intestinal
stem cells (ISCGS). This technology offers insights into the molecular and functional features of
intestinal stem cells and their utility for disease modeling and regenerative medicine.
Poster #34
Disease Models and Mechanisms
p63+Krt5+ distal airway stem cells are essential for lung regeneration
Yusuke Yamamoto (1), Wei Zuo (2), Ting Zhang (2), Daniel Wu (2), ShouPing Guan (2),
Audrey-Ann Liew (2), Xia Wang (1), SiewJoo Lim (2), Wa Xian (1) and Frank McKeon
(2)
(1) The Jackson Laboratory for Genomic Medicine (2) Genome Institute of Singapore, ASTAR
Technical Abstract
Lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis involve the
progressive and inexorable destruction of oxygen exchange surfaces and airways, and have
emerged as a leading cause of death worldwide. Mitigating therapies, aside from impractical organ
transplantation, remain limited and the possibility of regenerative medicine has lacked empirical
support. However, it is clinically known that patients who survive sudden, massive loss of lung
tissue from necrotizing pneumonia or acute respiratory distress syndrome often recover full
pulmonary function within six months. Correspondingly, we recently demonstrated lung
regeneration in mice following H1N1 influenza virus infection, and linked distal airway stem cells
expressing Trp63 (p63) and keratin 5, called DASC p63/Krt5, to this process.Here we show that
pre-existing, intrinsically committed DASC p63/Krt5 undergo a proliferative expansion in
response to influenza-induced lung damage, and assemble into nascent alveoli at sites of interstitial
lung inflammation. We also show that the selective ablation of DASC p63/Krt5 in vivo prevents
this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we
demonstrate that single DASC p63/Krt5-derived pedigrees differentiate to type I and type II
pneumocytes as well as bronchiolar secretory cells following transplantation to infected lung and
also minimize the structural consequences of endogenous stem cell loss on this process. The ability
to propagate these cells in culture while maintaining their intrinsic lineage commitment suggests
their potential in stem cell-based therapies for acute and chronic lung diseases.
Lay Abstract
The 1918 ‘‘Spanish’’ influenza pandemic killed more than 600,000 people in the United States
and an estimated 40 million individuals worldwide. Infections by this H1N1 influenza A strain is
thought to induce acute respiratory distress syndrome (ARDS) marked by a rapid onset of
pneumonia, lung damage and associated lack of oxygen, as well as massive inflammation. In vitro
models of lung are necessary to study devastating diseases such as H1N1 influenza. In this study,
we highlight the remarkable regenerative capacity of the lung following large-scale, severe lung
damage, and demonstrate the function of a pre-existing population of lung stem cells in this
process. In addition, we demonstrate that upon transplantation, single cell- derived lung stem cells
contribute to multiple cell types in the lung, and thus may be useful for lung regeneration, as well.
Poster #35
Disease Models and Mechanisms
Human iPSC Modeling of Autism-associated Mutations in the Gene CHD8
Wenzhong Liu (1), Cynthia A. Zerillo (1), Abha R. Gupta (1)
(1)Yale School of Medicine
Technical Abstract
Our goal is to create cellular models of autism spectrum disorders (ASDs) to help elucidate its
pathophysiology and serve as a platform for drug screening. ASDs are defined by persistent
deficits in social communication and social interaction and restricted, repetitive patterns of
behavior, interests, or activities. These syndromes are common in the population, with a
prevalence of approximately 1.5%. There is no known cure and treatments are limited since the
pathophysiology is still largely unclear. However, high-throughput sequencing approaches have
provided substantial insight into the genomic architecture of ASDs, which are quite genetically
heterogeneous. For example, whole-exome sequencing data demonstrate an over-representation
of heterozygous de novo truncating mutations in brain-expressed genes in affected individuals.
Several ASD genes have been identified based on the clustering of these mutations in the same
gene in unrelated individuals, providing significant evidence for association. Among those with
the strongest evidence is CHD8, which encodes a DNA helicase that regulates transcription by
remodeling chromatin structure, binds beta-catenin, and is involved in the Wnt signaling pathway,
which is important in early development. It is now critical to perform the in vitro and in vivo
studies to determine how these mutations disrupt cellular and molecular processes to advance our
understanding of the pathophysiology of ASD. We are studying human induced pluripotent stem
cells (iPSCs) and subsequently derived neural progenitor cells (NPCs) and neural cells which are
stably knocked down (KD) for CHD8 by genome editing and are comparing these with lines
derived from ASD patients with truncating CHD8 mutations. Preliminary phenotyping has
revealed that CHD8 KD and ASD patient NPCs do not organize into typical rosette formations,
suggesting that CHD8 mutations disrupt neural cell adhesion during brain development.
Lay Abstract
The genetic analysis of autism spectrum disorders (ASDs) has identified a number of genes
strongly associated with ASD, especially CHD8, which is involved in regulating how other genes
are activated. To advance our understanding of how ASD develops, we need to study how
mutations in these genes disrupt the structure and function of brain cells. One promising method
is to study the cells of patients carrying high-risk mutations. Blood cells are usually readily
available from patients, but they do not accurately represent brain cells. Stem cell technology can
transform patient blood cells to an immature form called induced pluripotent stem cells (iPSCs),
which can in turn be transformed into brain cells. Furthermore, genome editing technology can
introduce and correct genetic mutations in cell lines. Combining these two techniques, we are
creating cellular models of ASD to determine if mutations found in patients are necessary and
sufficient to cause dysfunction. These results will shed light on how mutations which have been
strongly associated with ASD actually cause the disorder and point to potential targets for
treatment.
Poster #36
Disease Models and Mechanisms
Understanding the impact of location and heterogeneity towards malignancy
Cristiana Pineda (1), Markus Woelfel (1), Valentina Greco (1)
(1) Yale University, Genetics, New Haven, CT
Technical Abstract
Stem cells and their environment, the so-called niches, are critical components that sustain not
only proper tissue homeostasis, but also diseased states such as cancer. One challenge to
understanding the initiating events towards malignancy is the inability to follow the same cells
over time in an intact animal. Specifically, this roadblock hinders the ability to understand both
the role of specific cells and how their location contributes to their growth, whether that growth be
normal or cancerous. To overcome this, we have established a novel live imaging approach to
track cellular behaviors in normal, healthy skin and capture the emergence of the cellular behaviors
that lead to malignant cutaneous Squamous Cell Carcinoma (cSCC) in an intact, live mouse.
Through these approaches we have investigated stem cell behaviors/choices and determined how
these impact stem cell fate within normal tissue during homeostasis. For example, our lab has
shown that location dictates the fate and behaviors of the hair follicle stem cells during skin
regeneration (Rompolas et al. Nature 2013). Through a combination of genetic and viral
approaches we are currently studying tumor initiation within a cSCC model using the approach of
simultaneous Hras activation and TGFβ loss of function. We utilize inducible fluorescent markers
to perform in vivo lineage tracing and track the behaviors of these mutant cells including:
proliferation, migration, and death. We will then compare these behaviors to the wild-type system
and analyze how the specific identity and location of mutant clones within the skin affects these
behaviors and the subsequent development of cSCC.
Lay Abstract
In order to comprehensively understand cancer, it is critical to gain an understanding of how
tumorigenesis is initiated. In order to accomplish this, we are utilizing a live-imaging approach to
track skin stem cells as they develop into a skin tumor, cutaneous Squamous Cell Carcinoma
(cSCC). This cSCC will be generated through manipulation of two signaling pathways established
to give rise to this type of tumor. Fluorescent tags will be utilized to label these mutant cells so
that they can be tracked over time during live-imaging revisits. The proposed experiments will
provide important clues towards the role of stem cells in the initiation and progression of skin
tumors. Furthermore, topical treatment of these tumors will also enable us to study mechanisms
that occur during tumor resolution.
Poster #37
Disease Models and Mechanisms
Exploring the origins of fibrotic and adipogenic infiltrates in muscular dystrophy.
Arpita A. Biswas (1), Kevin Zheng (1) and David J. Goldhamer (1).
(1) University of Connecticut
Technical Abstract
Muscular dystrophies refer to a set of more than 30 genetic diseases characterized by progressive
degeneration of skeletal muscle leading to a loss of motor control. While the underlying causes
and pathomechanisms vary depending on the type of dystrophy, the loss of muscle mass typically
is accompanied by an accumulation of fibrotic tissue and fat, which disrupts muscle architecture
and function. Rostrocaudal muscular dystrophy (rmd) is a recently developed mouse model in
which the hindlimb musculature shows progressive and severe dystrophic changes during early
adulthood. We used lineage-tracing strategies to investigate the cells-of-origin of fatty and
connective tissue infiltrates in rmd mice. Our data indicate that muscle-resident satellite stem cells
do not adopt non-myogenic fates in this dystrophic environment, consistent with their stable
commitment to the myogenic program. Interestingly, an interstitial, multipotent mesenchymal
progenitor marked by the expression of the receptor tyrosine kinase, Tie2, contributed to muscle
fibrosis. Of note, adipogenic infiltrates, which are pronounced in rmd muscle by 4 months of age,
were not derived from these progenitors, indicating that at least two distinct cell types contribute
to non-myogenic infiltrates in this model. Experiments are underway to determine the source of
intramuscular fat in rmd mice.
Lay Abstract
Muscular dystrophies refer to a set of more than 30 genetic diseases defined typically by skeletal
muscle weakness accompanied by a loss of muscle mass and simultaneous buildup of connective
and fatty tissue. Massive research efforts continue to shed light on the defects in multiple muscle
proteins that cause the muscle wasting. Our studies, however, lay emphasis on identifying the
source of abnormal non-muscle tissue that invades into muscle and compromises motor function.
To do this we use a mouse model of muscular dystrophy that displays all classical disease
symptoms called the rostrocaudal muscular dystrophy (rmd) model. Genetic tools enable us to
permanently label distinct cell populations within these mice, right from birth and through all
stages of development. This technology allows us to precisely track a group of cells and establish
whether they contribute to intramuscular fat or connective tissue. Our data indicate that adult
muscle stem cells that can form new fibers upon injury to normal muscle are not the source of fat
or fibrotic tissue in diseased tissue. Interestingly, a stem cell that is closely associated with muscle
vasculature contributed to muscle fibrosis. Of note, fatty infiltrates, which are pronounced in rmd
muscle by 4 months of age, were not derived from these stem cells. Experiments are underway to
determine the source of intramuscular fat in rmd mice.
Poster #38
Disease Models and Mechanisms
MyoD or Myf5 is Required for Satellite Cell Myogenic Commitment
Nicholas Legendre (1), Masakazu Yamamoto(1), Alexander Lawton(1), Samantha
Cummins(1), Shoko Yamamoto(1) and David J. Goldhamer(1)
(1) University of Connecticut, Storrs
Technical Abstract
The functions of the muscle regulatory factors (MRFs), MyoD and Myf5, in postnatal myogenesis
have not been fully examined due to perinatal lethality of double-null mice. Mutations in either
MyoD or Myf5 result in relatively minor defects, although it is unclear whether this reflects
functional overlap between these MRFs, as in the embryo, or regulation of postnatal myogenesis
by alternative pathways. We developed a MyoD conditional knockout allele, MyoDCKO, and
analyzed satellite cell (SC) fate from mice having different gene doses of MyoD and Myf5 using
the satellite cell-specific Cre deleter Pax7CreER. Double-null mice have severe regeneration
defects after injury; satellite cells fail to differentiate into myofibers, and some SCs undergo a fate
change to adipocytes. In order to investigate issues of cell autonomy, and recombination
efficiency, we use a culture system of low-passaged primary mouse double knock-out (dKO) SCs
isolated via FACS. In early culture time points, dKO satellite cells are morphologically identical
to wild type SCs and express SC markers Pax7 and Desmin. By day 7 dKO SCs undergo a dramatic
morphological change. This progressively continues so that by day 28, the majority of dKO SCs
have lost Pax7 and Desmin expression, and all dKO SCs completely fail to differentiate into
multinucleated, Myosin heavy chain-positive (MyHC) myotubes. This indicates that MyoD and
Myf5 are required for SC differentiation. However, SCs lacking MyoD and Myf5 maintain some
myogenic traits. Although freshly isolated dKO SCs do not form fat, a minority of dKO cells
become adipocytes when given low serum medium, suggesting heterogeneity in the dKO SC pool.
Transcriptional profiling experiments are ongoing in order to determine the roles of MyoD and
Myf5 in SC programming and the genetic regulation of adult myogenesis.
Lay Abstract
Satellite cells (SCs) are adult muscle stem cells that are indispensable for muscle regeneration. The
roles of Myf5 and MyoD, two important genes in muscle development and regeneration, have been
investigated individually with respect to adult muscle regeneration. However, due to neonatal
lethality of mice lacking both MyoD and Myf5, it has not been possible to study the roles of both
genes simultaneously. Here we use an inducible system to delete MyoD in SCs that are already
lacking Myf5, and label the now double-null SCs in adult mice. We then trace the cells and analyze
them both in the mouse and in cell culture. Mice with double-null SCs show major regeneration
defects after injury and an impaired ability to form muscle in culture. Additionally, a minority of
the double-null SCs can become fat cells.
Poster #39
Disease Models and Mechanisms
An in vitro model of Prader-Willi syndrome via genome editing of induced pluripotent stem
cells (iPSCs).
Elodie Mathieux (1), Heather Glatt-Deeley (1), Christopher Stoddard (1), Kristen MartinsTaylor (1) and Marc Lalande (1).
(1) Department of Genetics and Genome Sciences, Stem Cell Institute and Institute for
Systems Genomics University of Connecticut, Farmington, CT, USA.
Technical Abstract
Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, followed by hyperphagia,
obesity and distinctive behavioral problems. PWS is a disorder of genomic imprinting that is
caused by the absence of a normal paternal contribution to chromosome 15q11-q13. Recently the
PWS critical region (PWSCR) has been narrowed to an ~91kb region encompassing several noncoding RNAs including a cluster of box C/D snoRNAs (SNORD116). In order to gain an
understanding of how the loss of the SNORD116 cluster contributes to PWS phenotypic
abnormalities, we developed an in vitro model of PWS by generating isogenic pairs of human
iPSCs that differ exclusively at the SNORD116 cluster. To construct these isogenic lines, we
electroporated a pair of custom-designed CRISPRs into a normal iPSC line, along with a targeting
construct. We isolated the targeted iPSCs by drug selection and PCR to confirm the insertion of
the targeting construct and then excised the drug selectable marker. We confirmed the loss of
expression of the SNORD116 cluster in the isogenic deletion line by qRT-PCR and RNA
fluorescence hybridization. We are currently differentiating isogenic normal and SNORD116
deletion lines into neural precursor cells and neurons in order to identify novel cellular and
molecular targets of the SNORD116 regulation. We are also using our in vitro model of PWS to
activate the silent maternal copies of SNORD116 by knockout of a Zinc Finger protein 274
(ZNF274) containing chromatin-silencing complex with the objective of developing a therapeutic
intervention for the disease.
Lay Abstract
Prader-Willi syndrome (PWS) is a genetic disorder characterized by poor muscle tone at birth,
followed by an extreme drive to eat, obesity, and behavioral problems. PWS is caused by the
absence of the father’s copy of chromosome 15q11-q13 and occurs in one out of every 15,000
births. Recently the genetic region responsible for PWS has been narrowed to a smaller region that
contains a cluster of non-protein coding RNAs called SNORD116. In order to gain an
understanding of how the loss of the SNORD116 cluster causes the symptoms of PWS, we used
genome engineering technologies to remove the paternal copy of the SNORD116 cluster from
normal induced pluripotent stem cells (iPSCs). This allows us to study cells with and without a
SNORD116 deletion that are otherwise genetically identical to one another. We confirmed the loss
of expression of the SNORD116 cluster in the engineered line. We are currently converting the
SNORD116 deletion and control iPSC lines into neurons in order to determine how loss of
SNORD116 causes PWS. We are also using our SNORD116 deletion iPSCs as well as other PWS
iPSCs that we previously developed from patient samples to activate the silent maternal copies of
SNORD116 with the objective of developing a therapeutic intervention for PWS.
Poster #40
Disease Models and Mechanisms
GABAergic progenitor transplants suppress seizures in a mouse model of TLE by forming
synapses onto newborn granule cells in the dentate gyrus
J. Gupta (1), E. Paquette (1), J. Radell (1), A. Fine (1), F. Harrsch (1), M. Van Zandt (1),
B. Luikart (2), G.B. Aaron (1), J.R. Naegele (1)
(1) Department of Biology, Wesleyan University, Middletown, CT; (2) Department of
Physiology and Neurobiology, Geisel School of Medicine at Dartmouth, Lebanon, NH
Technical Abstract
Temporal lobe epilepsy (TLE), associated with intractable seizures, is the most common form of
epilepsy in adults. A major feature of TLE is loss of GABAergic interneurons in the hippocampus,
decreasing inhibition onto granule cells (GC) in dentate gyrus (DG). Increased excitation is also a
result of morphological changes in adult-generated GCs, including: abnormal migration and
retention of basal dendrites. The dysmorphic GCs have been proposed to form a hyperexcitable
hub that promotes seizures. To determine whether stem cell grafts into this hub would regenerate
inhibitory circuits and control seizures, we transplanted E13.5 medial ganglionic eminence
(MGE)-derived GABAergic progenitors into the DG of mice with pilocarpine-induced TLE. We
demonstrated that grafts containing Channelrhodopsin (ChR2)-expressing GABAergic neurons
could be activated by light in brain slices to induce inhibitory currents in GCs, confirming that
transplants formed functional inhibitory synapses. We also showed by electroencephalography
(EEG) that stem cell grafts of GABAergic interneurons in the hippocampus could suppress
spontaneous seizures (Henderson, Gupta, et al. 2014). In the present work we investigated the
mechanisms responsible for seizure suppression, by examining whether grafted MGE cells form
inhibitory synapses onto adult-generated GCs born after mice develop TLE. We transplanted MGE
cells into the DG of TLE mice after labeling adult-generated GCs with retrovirus. High resolution
confocal imaging and morphometric analyses were conducted to determine whether MGE cell
grafts formed synapses onto GCs. We found extensive synaptic contacts between the grafts and
adult-generated GCs, with an average of more than 150 synapses onto each GC. Most of these
synaptic contacts were on secondary and tertiary dendrites. Taken together, these studies suggest
that adult-generated GCs are hyperinnervated by GABAergic interneuron grafts, providing a
plausible mechanism for seizure suppression
Lay Abstract
Epilepsy is a neurological disorder characterized by occurrence of spontaneous seizures and affects
more than 1% of the global population. Temporal lobe epilepsy (TLE) is the most common type
of epilepsy that affects adults. TLE is characterized by increased excitation resulting from loss of
inhibitory neurons in the hippocampus and rewiring of excitatory granule cells that are born in the
adult brain after the development of epileptic seizures. About one-third of the TLE patients are
resistant to treatment with anti-epileptic drugs, requiring surgical removal of the hippocampus and
regions of the temporal lobes for good seizure control. In some cases, this approach is not possible.
We are investigating novel stem cell-based therapies for TLE and have shown that transplantation
of inhibitory neurons into the hippocampus can suppress seizures in mouse models of TLE.
However, the mechanisms for seizure suppression are not well understood. We hypothesize that
the transplanted cells suppress seizures by wiring up with the hyperexcitable granule cells. To test
this hypothesis, we selectively labeled populations of newborn granule cells born after the onset
of TLE and visualized by confocal microscopy the synaptic connections formed onto these cells
by inhibitory interneuron transplants. Our preliminary studies suggest that transplanted cells form
dense inhibitory connections onto hyperexcitable granule cells, suggesting that the transplants
regenerate inhibitory neural circuitry that controls seizures.
Poster #41
Disease Models and Mechanisms
Mechanisms Driving the Development of Adipose Tissue and Metabolic Disease
Brandon Holtrup, Christopher Church, Elise Jeffery, Laura Colman, Matthew Rodeheffer.
(1) Yale University, New Haven, CT
Technical Abstract
A significant increase in the incidence of obesity, defined as the excess accumulation of white
adipose tissue (WAT), has been observed over the last few decades. Currently 70% of adults and
over 30% of children in the United States are either overweight or obese. Obesity puts individuals
at higher risk of developing several comorbidities such as cardiovascular disease, type-II diabetes,
and hypertension. However, our understanding of how increased WAT mass is linked to these
metabolic diseases is limited. During normal development, an adipocyte number ‘set point’ is
established by early adulthood. While it is known that adolescence is a critical time period for the
establishment of adipocyte number in humans, adipocyte precursor (AP) proliferation and
adipogenesis have never been fully characterized during this time period. Here I characterize for
the first time the activation of AP cells during adolescent development in mice. We show that
transient high fat diet feeding during adolescence leads to defective heart function and altered
glucose metabolism in adulthood. We have recently shown that the molecular mechanisms that
regulate developmental adipogenesis are distinct from those seen in obesogenic adipogenesis, yet
the mechanisms that drive developmental adipogenesis and adipocyte number maintenance in vivo
are not known. As insulin signaling is known to play a crucial role in metabolism and is required
for adipogenesis in vitro, we chose to examine insulin signaling in AP cells in vivo. To this end, I
used PDGFrα-cre mice in combination with InsR flox/flox mice to knockout insulin signaling in
AP cells. As expected, InsR flox/flox; PDGFrα-cre mice display metabolic dysfunction and defects
in adipogenesis that are consistent with a role for insulin in regulating the establishment of normal
adipocyte number.
Lay Abstract
A significant increase in the incidence of obesity, defined as the excess accumulation of white
adipose tissue (WAT), has been observed over the last few decades. Currently 70% of adults and
over 30% of children in the United States are either overweight or obese. Obesity puts individuals
at higher risk of developing several comorbidities such as cardiovascular disease, type-II diabetes,
and hypertension. However, our understanding of how increased fat is linked to these metabolic
diseases is limited. During normal development, the number of fat cells in your body is determined
by early adulthood. While it is known that adolescence is a critical time period for the proper
development of fat in humans, this process has never been fully characterized. Here, I characterize
for the first time the activation of fat stem cells during adolescent development in mice. We show
that short-term high-fat diet feeding during adolescence leads to reduced heart function and
diabetes in adulthood. Additionally we look at the genes that may be involved in turning fat stem
cells into mature fat cells.
Poster #42
Disease Models and Mechanisms
Transcriptional regulation in pluripotent stem cells by methyl CpG-binding protein 2
(MeCP2)
Kun-yong Kim (1),Yoshiaki Tanaka (1),Mei Zhong (1), Xinghua Pan (1), Sherman Morton
Weissman (1), In-hyun Park (1).
(1)Yale Stem Cell Center, Yale School of Medicine (New Haven, CT 06520, USA)
Technical Abstract
Rett syndrome was formerly classified as a pervasive developmental disorder, together with the
autism spectrum disorders. Initial development is normal and onset occurs between 6 and 18
months of age. Recent studies, demonstrate that neurological deficits resulting from mutation of
MeCP2 can be reversed upon restoration of gene function. These studies are quite exciting and
provide hope for restoring neuronal function in patients. However, the strategy in humans will
require providing the critical factors that function downstream of MeCP2 and the identification of
the molecular mechanisms underlying Rett syndrome phenotypes. To pick out the candidates and
molecular mechanism that can be therapeutically targeted, here we performed the comparative
analysis of global gene expression with hESCs and normal iPSCs and Rett patient derived iPSCs
using massively parallel RNA sequencing (RNA-seq). We found several functions of MeCP2 in
pluripotent stem cells. First, results showed that gene sets involved in neuronal development or
function are differentially expressed between Rett-iPSCs and control iPSCs. And the comparative
analysis among Rett patients has different type of MeCP2 mutation revealed that distinct genes are
affected by different mutations in MeCP2, although the expression of MeCP2 is relatively lower
in pluripotent stem cells compared with neurons. Second, Rett-iPSCs showed the up-regulation of
mitochondria-related genes whose expression is also altered in other neuronal diseases.
Furthermore, genes involved in splicing and ubiquitin-mediated proteolysis are also highly
enriched in Rett-iPSCs. Third, we found that MeCP2 is also important in inactivation of X-linked
genes. Together, our data highlight the MeCP2 regulatory pathway in undifferentiated pluripotent
cells. These data provide novel insights into MeCP2 functions in the undifferentiated stage and
implicate the importance of dissecting the function of MeCP2 in diverse cell types for Rett
syndrome.
Lay Abstract
Rett syndrome is one of the most prevalent female mental disorders and exhibits clinical symptoms
that include severe mental disabilities, an absence of speech, stereotypic hand movements,
encephalopathy, and respiratory dysfunction. Mutations on MeCP2 gene are known as a major
cause of syndrome. MeCP2 is highly expressed in neuronal cells and regulates gene expression as
a transcription regulator as well as through long-range chromatin interaction. The functions of
putative MeCP2 genes have been probed using mouse models as well as human postmortem brains.
However, generating Rett syndrome mouse models is not always feasible, and both normal and
human Rett syndrome postmortem brains are not readily available for study. To overcome these
difficulties, we generated derived human induced pluripotent stem cells (iPSCs) from Rett patient
using reprogramming method and compared the global gene level with normal pluripotent stem
cells. We found that MeCP2 regulates genes encoding neuronal developmental and mitochondrial
membrane genes. In addition, MeCP2 also can regulates gene set related with X chromosome
inactivation. These studies suggest that fundamental cellular physiology is affected by mutations
in MeCP2 from early development, and that a therapeutic approach targeting to unique forms of
mutant MeCP2 is needed.
Poster #43
Disease Models and Mechanisms
Stem cell models for autophagy in the retinal pigment epithelium
Maneesh Vij(1), Katherine Davis(1), Tina Xia(1), Ron A. Adleman(1), Tim Blenkstuff(2),
Sally Temple(2) and Lawrence J Rizzolo(1)
(1)Yale University, (2)Neural Stem Cell Institute
Technical Abstract
Age-related macular degeneration is the leading cause of visual impairment in developed
countries. Diminished autophagy by retinal pigment epithelium (RPE) may contribute to the
pathophysiology, but stimulators of autophagy show little clinical benefit. To explore autophagy
across the lifespan, we compared RPE from various sources: 16-week human fetuses (hfRPE),
cadaveric eyes (adRPE), human embryonic stem cells (hESC-RPE), and iPSC. The iPSC were
derived from fibroblasts (fibro-iPSC-RPE) or adult RPE (RPE-iPSC-RPE). We examined resting
cultures and cultures challenged with photoreceptor outer-segments (POS). Gene expression was
monitored using quantitative RT-PCR arrays designed for autophagy and RPE maturity.
Autophagic flux was monitored by measuring the conversion of LC3-1 to LC3-II. Autophagic flux
was reduced in 90-year-old adRPE relative to RPE isolated from younger individuals. Although
RPE markers were similar, the expression of autophagy genes varied among cultures. Autophagy
gene expression in RPE-iPS-RPE more closely resembled adRPE than hfRPE or hESC-RPE. As
expected, rapamycin stimulated RPE autophagy. However, spautin-1 inhibited autophagy only
partially, which suggests the presence of a non-canonical autophagy pathway that was absent in
90-year-old RPE. Continuous feeding of POS up-regulated a subset autophagy genes in hfRPE,
but had little effect on gene-expression in hESC-RPE. In RPE-iPS-RPE, POS resulted in the
accumulation of autofluorescent granules, undigested POS associated with AMD. In contrast,
fibro-iPSC-RPE up-regulated apoptosis genes and a broad range of autophagy genes. Even if
marker genes are similar, stem cell-derived RPE vary in the expression of autophagy-related genes
and in response to a phagocytic challenge. The data suggest that RPE uses a non-canonical pathway
for digesting phagosomes that is not regulated by rapamycin or spautin-1. Further studies may
reveal pharmaceutical targets that would be effective for treating AMD.
Lay Abstract
Age-related macular degeneration is the leading cause of visual impairment in developed
countries and may be related to process known as autophagocytosis. Autophagocytois is an
intracellular process that removes and recycles damaged or aged organelles. The disease begins
in the Retinal Pigment Epithelium (RPE), a layer of cells that supports the light-sensitive cells of
the retina known as photoreceptors. The photoreceptors are elongated cells that shed their tips
every day. The RPE engulfs the shed tips and digests them by a process call phagocytosis. With
age, undigested material accumulates, which leads to disease. The relationship between
phagocytosis and autophagocytosis is unclear, but it is known that stimulation of
autophagocytosis is not an effective treatment for the disease. To study these processes we
compared various culture models of RPE that were derived from various sources, including: 16week human fetuses, cadaveric eyes, human embryonic stem cells, and induced pluripotent cells.
Each RPE model expressed typically monitored marker genes, but the expression of autophagy
genes was variable. As a collection, these RPE models revealed different aspects of phagocytosis
and how the process differs from autophagocytosis. Hopefully, this approach will identify drugs
that target the phagocytic pathway that is relevant for treating AMD.
Poster #44
Disease Models and Mechanisms
Establishing a human surrogate model to study PAX7 mutations in etiology of cleft lip/palate
Alan W. Leung (1), Andrew Xiao (1), Martin I Garcia-Castro (2)
(1) Yale University (2) University of California Riverside
Technical Abstract
Over 4000 babies are born with a cleft lip with or without a cleft palate (CLP) each year in the
United States. CLP is also amongst the most common malformations worldwide with a frequency
of 1 of every 700 births. CLP arises from environmental and/or genetic effects on neural crest
cells, and a number of genome-wide association analyses have identified PAX7 and other genes
as major risk alleles for CLP. Pax7 mutations (coding or non-coding) are proposed to modify the
function of the protein. Here we aim to construct a human surrogate model to analyze the outcomes
of PAX7 mutations in neural crest development, and their derivatives associated with lip and palate
development to improve our understanding and capacity to better diagnose and treat CLP. To this
end we have successfully devised a fast and efficient model of human neural crest (hNC) cell
development based on human ES (hES) cells that mimics endogenous neural crest development.
These hNC are induced from hES cells by canonical WNT signaling in 5 days and we found that
these hNC arise independently from neural and mesodermal tissues, in agreement with our own
studies in early chick embryos. FGF and BMP pathways also play a complex role in controlling
human NC development. In order to assess the role of PAX7 mutations in NC, lip and palate
development we will mimic human mutations from CLP afflicted individuals in our hES cell
system via CRISPR/Cas9 technologies. Once mutated Pax7-hES lines are established, we will
subject them to hNC and terminal differentiation assays to systematically analyze survival,
proliferation, fate modifications, and interactions. We foresee that results from our ongoing work
will significantly contribute to an increased understanding of the etiology of CLP. The human
surrogate model we are establishing may also allow future high throughput screening of synthetic
compounds that mimic or alter functions of PAX7 or other risk factors that potentially can be
applied to CLP prevention.
Lay Abstract
Twenty-first century will be an age for regenerative and personalized medicine. The
groundbreaking discovery in 1998 of methods to harvest and culture human embryonic stem cells
has altered the whole landscape for biology and medicine. This unique type of human stem cells
has proved to be a potentially useful tool for generating clinically applicable cell types/tissues for
repairs, modeling diseases, and screening drugs. Cells like neurons, liver cells, and heart cells have
been successfully made and been used for these purposes. Our team focuses on finding ways to
produce cell types and tissues in the human face and palate. These cells and tissues share an origin
from a unique part of the developing embryo called the border of the neural plate or what scientists
in our field called the 'neural crest’. Neural crest cells can regenerate themselves in culture dish
and can give rise to bones, cartilages and connective tissues in the face and palate. Malformation
of the neural crest is implicated in cleft lip and cleft palate at birth. We have invented a very
efficient way to generate neural crest cells in the culture dish from human embryonic stem cells.
We will introduce mutations found in patients with cleft lip/palate into these human neural crest
cells. The cells harboring the mutations are expected to have issues in maintaining their identity,
progressing through cell doubling, and/or communicating with one and other. We will scrutinize
these different aspects using various cutting edge tools available in our laboratory. Our ongoing
research will allow us to find out why these mutations cause the cleft defects in babies. The
ultimate goal is to obtain insights that will facilitate the invention of ways to prevent or cure these
birth defects.
Poster #45
Disease Models and Mechanisms
Expression of the FOP-causing BMP receptor, Acvr1(R206H), in a Tie2-positive progenitor
cell population is sufficient to induce heterotopic ossification
Lees-Shepard JB (1), Cummins S(1), Biswas A(1), Cogswell C(1), Yamamoto S(1),
Yamamoto M(1), Goldhamer DJ(1)
(1) University of Connecticut
Technical Abstract
Heterotopic ossification (HO) is a debilitating condition in which ectopic bone forms within the
skeletal muscle and associated soft tissues. A devastating form of HO is manifested in the rare
genetic disorder, fibrodysplasia ossificans progressiva (FOP), where progressive heterotopic bone
formation occurs throughout life, resulting in painful and disabling cumulative immobility. Hyperactivation of bone morphogenic protein (BMP) signaling, within the context of an inflammatory
environment, has emerged as the major factor promoting the onset and progression of HO,
regardless of etiology. We hypothesized that a recently identified progenitor cell population
functions as the major source of osteogenic and chondrogenic cells in response to dysregulated
BMP signaling. To test this hypothesis, we investigated the cellular basis of HO by generating
conditional mouse models in which Cre-mediated recombination of the FOP-causing
Acvr1(R206H) gene mutation (designated Acvr1FOP/+)was driven by specific promoters
allowing us to target satellite cells, endothelium, and Tie2+ progenitors, all of which have been
previously implicated in HO. Our results demonstrated that injury-induced HO was only detected
in Acvr1FOP/+;Tie2-Cre mice. Furthermore, spontaneous HO, which is the major cause of
morbidity in human FOP, has only been observed in Acvr1FOP/+;Tie2-Cre mice. In both injury
induced and spontaneous HO, fluorescent lineage tracing confirmed that the Acvr1(R206H)
mutation functions cell autonomously in Tie2+ progenitors and that wild type cells did not
significantly contribute to HO. These data strongly indicate that expression of FOP-causing
Acvr1(R206H) mutation within a Tie2-positive cell population, but not satellite cells or
endothelium, is sufficient to induce HO. This study represents the first physiological test to
determine cell responsible for HO, and will facilitate development of novel therapeutic treatments
to mitigate or prevent this debilitating condition.
Lay Abstract
A distinct but poorly understood population of adult stems cells is ultimately responsible for a
debilitating condition in which bone inappropriately forms within the skeletal muscle and
associated soft tissues. This condition, called heterotopic ossification, is a serious and common
complication of combat‐related injury and surgical interventions such as knee and hip replacement.
The most severe example of heterotopic ossification is manifest in the rare genetic disorder
fibrodysplasia ossificans progressiva, in which inappropriate bone formation occurs progressively
throughout life, resulting in devastating effects on health, quality of life, and life expectancy. While
the central role of a stem cell population as the causative agent in this condition is firmly
established, the identity of the offending cell type remains unclear, and little is known of the
mechanisms that direct these stem cells to initiate bone formation. Our foundational studies have
discovered the identity of the offending adult stem cells, will provide knowledge of key regulatory
signals that control their behavior, and define the relationship of soft tissue trauma to activation of
the bone forming program in this adult stem cell population. Understanding the cellular basis of
this condition represents a major breakthrough, as the ability to target therapies to specific cell
populations is of primary importance to minimize collateral effects. Additionally, the present
mouse models will be valuable tools for future studies that focus on drug discovery and testing
treatment modalities.
Poster #46
Disease Models and Mechanisms
The use of human embryonic stem cells to elucidate pro-apoptotic signaling in glioblastoma.
Erin M. Boisvert (1), Michael Michaud (1), Joseph A. Madri (1), Samuel G. Katz (1)
(1) Yale University
Technical Abstract
Glioblastoma Multiforme (GBM) is the most common primary malignant tumor of the central
nervous system with a dismal median survival of 15 months following diagnosis. Underlying their
highly malignant phenotype is a small subset of cells with increased proliferative capacity, and
high resistance to irradiation and chemotherapy induced apoptosis. A similarly proliferative cell
type, the neural stem cell (NSC) is posited as the cell of origin for GBM. Normally, NSCs have a
low apoptotic threshold set point in order to quickly remove highly proliferative cells that
accumulate damage. BAX and BAK are pro-apoptotic proteins that once activated permeabilize
the outer mitochondrial membrane and irreversibly implement apoptosis. BAK null mice with
conditional deletion of BAX in Nestin-positive cells demonstrate profound accumulations of Sox2
positive neural progenitor cells. These cells do not mature and the mice are more prone to highly
aggressive brain tumors. Compared to WT, NestinCreBaxfl/flBak-/- neurospheres retain increased
Sox2 and GFAP expression under neuronal promoting cell culture conditions. With the use of
embryonic stem cells, we are evaluating the role of these critical genes in human neuronal stem
cells. Recently, we adapted a human cerebral organoid method to provide us with a model system
that more accurately represents the complexities of the human brain. These cerebral organoids
display an appropriate neuronal differentiation pattern, and an exceptionally high tendency to
become forebrain neurons., We are using CRISPR CAS9n to disrupt Bax and Bak as well as other
genes commonly mutated in glioblastoma in this cerebral organoid model. This system will
provide a unique way to evaluate the efficacy of therapies. We have generated extracts from local
plants, which selectively kill BAX/BAK double knockout cells more than WT cells. We are
excited to evaluate the effect of these extracts as well as other potential therapeutics in the cerebral
organoid model.
Lay Abstract
Glioblastoma Multiforme (GBM) is the most common and deadliest form of malignant primary
brain tumors, having a median survival of only 15 months, and accounting for more than 13,000
deaths in the United States annually. Despite our best efforts with radiation, chemotherapy, and
surgery, GBMs manage to avoid cell death. Programmed cell death, however, has the potential to
prevent GBMs. Programmed cell death is a fundamental biological pathway regulated by a family
of proteins that are frequently impaired in GBM. At the crux of this pathway’s ability to kill are
two proteins named BAX and BAK, which deliver the final blow by poking holes in the
mitochondria, the cell’s powerhouse. In our new mouse model that eliminates BAX and BAK from
the brain, we find a massive increase in neural stem cells, the presumptive cell of origin for GBM.
Using mouse neural stem cells and human embryonic stem cells, we will explore the hypothesis
that the loss of BAX and BAK permits neural stem cells to undergo misguided differentiation and
transform into GBM. Human embryonic stem cells are differentiated into human neural stem cells,
which we have recently used to develop a three dimensional model human brain. Using these
models, I will explore the concept that while loss of BAX and BAK makes cell resistant to
apoptosis, it also makes them vulnerable to other forms of cell death. Applying state-of-the-art
techniques in mouse modeling and human embryonic stem cells, our goal is to both improve our
understanding of how GBM arise and identify novel pathways by which to kill them.
Poster #47
Disease Models and Mechanisms
Balancing Act: Investigating the role of Neuroligin2 in GABAergic synapse development and
stabilization
Maisel, S.M.(1), Shrestha, S.(1), Bumsch, K.(1), Naegele J.R(1).
(1) Wesleyan University
Technical Abstract
Neurodegenerative conditions, traumatic brain injury, and some genetic disorders are
characterized by a loss or alteration of particular neuronal populations. Stem cell transplantation
is a promising approach for regenerative medicine, aiming to stimulate repair in pathologically
altered neural circuits and possibly reverse aberrant phenotypes. Knowledge about the mechanisms
by which these transplanted cells functionally integrate into host circuitry is limited. Our work is
focused on the molecular mechanisms regulating GABAergic interneuron synapse formation in
endogenous circuitry as well as in models of stem cell transplantation. Neuroligin 2 (NLGN2) is a
membrane-bound post-synaptic adhesion molecule present at GABAergic synapses and is thought
to play a critical role in their formation and stabilization. AAV-mediated gene delivery is a
powerful and effective approach for manipulating the expression of NLGN2 in the developing and
adult brain, to further elucidate the role of NLGN2 in GABAergic synapse formation and
stabilization. In this study, we have overexpressed NLGN2 in the cerebral cortex and hippocampus
of postnatal and adult VGAT-ChR2-eYFP mice. Following 3-6 week survival periods, we
compared the extent of GABAergic synapse formation onto cells with normal vs. high levels of
NLGN2-expression. Immunohistochemistry and confocal analyses suggest increased NLGN2
puncta in cells infected with an overexpression vector and increased GABAergic synaptic inputs
onto these neurons. While further studies will be needed to verify that the supernumerary inhibitory
synapses are fully functional, our preliminary findings suggest that viral-mediated NLGN2
delivery is a promising approach for increasing the formation of inhibitory synaptic circuitry in
both developing and adult nervous system. Funding from NIH has supported this work.
Lay Abstract
Neurodegenerative conditions and traumatic brain injury are often characterized by dysfunction or
death within particular brain regions and cell types. Stem cell transplantation is a promising
approach for regenerative medicine, aiming to replace damaged neurons and repair altered neural
circuits. While optimizing the survival and integration of transplanted neurons is one of the central
goals of transplantation therapies for neural repair, little is known about how neurons make proper
connections in the adult nervous system. In this study, we examined the role of one putative protein
called Neuroligin 2 (NLGN2), in neuron connection. NLGN2 is thought to play a vital role in the
formation and stabilization of inhibitory connections in the brain. We used adeno-associated virus
(AAV) to deliver the NLGN2 gene into specific brain regions of developing and adult mice and
studied the effects of overexpressing this protein on the inhibitory connections.
Microscopic analyses in both developing and adult mice showed that neurons
overexpressing NLGN2 protein also had more inhibitory connections. These preliminary findings
suggest that viral-mediated NLGN2 delivery may be a promising approach for increasing the
formation of inhibitory connections in both developing and adult nervous system. Funding from
NIH supports this work.
Poster #48
Disease Models and Mechanisms
Genetic Requirement for Progenitor Cell Responses in a Mammalian Limb Regeneration
Model
Sandra Lopez1, Melanie Fisher1, Nickesha Anderson1*, Tatiana Blanchard1, Ken
Muneoka2, Caroline Dealy1,3
1
Department of Reconstructive Sciences, Center for Regenerative Medicine and Skeletal
Development; 3Department of Orthopaedic Surgery; University of Connecticut Health
Center; and 2Department of Veterinary Medicine and Biomedical Sciences, Texas A&M
University. *Current: Wesleyan University
Technical Abstract
At least 2 million people in the United States live with limb loss due to trauma, birth defects or
disease. Although prosthetics can restore some functionality to patients with limb loss, there is
currently no means to generate a biological replacement for lost human limbs. Some animals, like
salamanders, possess a remarkable ability to spontaneously regenerate lost limbs. Spontaneous
limb regeneration capacity also exists in mammals, including humans, but it is limited to the digit
tip. By understanding the signals that control limb regeneration responses in the mammalian digit
tip, including signals that regulate the behavior of the progenitor cells that give rise to regenerated
digit tissue, we may one day be able to develop biological approaches to treat human limb loss.
The Epidermal Growth Factor Receptor (EGFR) family of tyrosine kinase receptors plays an
important role in cell proliferation, migration, survival and differentiation. EGFR signaling is also
required for differentiation of skeletal progenitors, and for development of cartilage and bone
tissue. In prior studies, we identified a genetic requirement for the EGFR-related receptor, ErbB3,
for spontaneous regeneration of the mouse digit tip following digit tip loss. The goal of this project
is to understand the cellular and molecular mechanisms underlying this requirement. In this study,
we compared the amount of cell proliferation by progenitor cells of the amputated digit stump of
wildtype mouse digits, which undergo successful digit tip regeneration, with the amount of cell
proliferation by progenitor cells of the amputated digit stump of transgenic mice with limb-targeted
genetic loss of ErbB3, in which digit regeneration is impaired. We also used uCT (micro Computer
Tomography) to quantify bone formation by wildtype and ErbB3-deficient amputated digit tips.
We found that cell proliferation and bone formation were both impaired in the ErbB3-deficient
amputated mouse digit tips. Based on these results, we propose that a key function of ErbB3
signaling during mouse digit tip regeneration is to maintain the proliferation of progenitor cells,
including skeletal progenitor cells, which are required to re-form lost digit tip structures. The
identification of molecular cues that regulate limb regeneration responses in mammals offers
potential for future development of therapeutic agents that may one day facilitate limb regeneration
in humans.
Lay Abstract
At least 2 million people in the United States live with limb loss, including individuals who
suffered traumatic wounds in the military that resulted in amputation. Some regenerative capacity
exists in mammals, including rodents and humans, but it is restricted to the digit tip. If we can
identify the molecular cues that control limb regeneration responses, it is conceivable that one day
we could stimulate limb regeneration in humans by providing these signals at the site of
amputation. The goal of this project is to understand the role of certain growth factor signals that
control progenitor cell responses in a mouse digit tip model of limb regeneration.
Poster #49
Exploring the Basics
Indy reduction maintains fly health and homeostasis
Blanka Rogina(1), Ryan P. Rogers(2)
(1) Department of Genetics and Genome Sciences, Institute for Systems Genomics, School
of Medicine, University of Connecticut Health Center, 263 Farmington, CT 06030-6403,
(2) Department of Sciences, Wentworth Institute of Technology, 550 Huntington Ave.,
Boston, MA 02115
Technical Abstract
Indy (I’m not dead yet) encodes the fly homologue of a mammalian transporter of the Krebs cycle
intermediates. Reduced Indy gene activity has beneficial effects on energy balance in mice, worms
and flies, and worm and fly longevity. In flies, longevity extension is not associated with negative
effects on fertility, mobility or metabolic rate. Others and we show that Indy reduction extends
longevity by mechanism similar to calorie restriction (CR). Some of the hallmarks of these changes
are altered intermediate nutrient metabolism and increased mitochondrial biogenesis. These
changes have been found in fly heads, thoraces and the midguts. The observed changes in midgut
energy metabolism, specifically decreased production of free radials, results in preservation of
intestinal stem cell (ISC) homeostasis, which is characterized by a decrease in age-associated
accumulation of ISCs. Our studies show a direct connection between changes in energy
metabolism, caused by the Indy mutation and preservation of ISC homeostasis and midgut
integrity. The data suggest that Indy reduction preserves homeostasis in tissues that contribute
extended health and longevity.
Lay Abstract
Indy (I’m not dead yet) gene transports important nutrients necessary for energy production in
cells. Reduced Indy gene activity has beneficial effects on energy balance in mice, worms and
flies. In worm and fly Indy reduction extends longevity by mechanism similar to calorie restriction
(CR). CR is the most efficient manipulation that promotes organismal health and extends lifespan
in nearly all species. Preservation of intestinal stem cell (ISC) homeostasis has become an
important factor in extending longevity due to a central role for ISCs in maintaining normal midgut
functions such as food absorption and protection from microorganisms and toxins. INDY reduction
in fly midgut changes midgut energy metabolism, specifically decreased production of damaging
free radials. Our studies show a direct connection between changes in energy metabolism, caused
by the Indy reduction and preservation of ISC homeostasis and midgut integrity in flies. The data
suggest that Indy reduction preserves homeostasis in tissues that contribute extended health and
longevity. The similar effects of INDY reduction on metabolism in flies, worms, and mice suggest
an evolutionary conserved and universal role of INDY in metabolism. These findings suggest that
INDY could be potentially used as a drug target for preservation of the ISC homeostasis in
mammals.
Poster #50
Exploring the Basics
Wnt signaling promotes the induction of Sox10+ neural crest precursors from dissociated
human embryonic stem cells
Barbara Murdoch (1), Martín García–Castro (2)
(1) Eastern Connecticut State University, (2) Yale University
Technical Abstract
Neural crest cells are fascinating as they can produce a wide array of cell types throughout the
body, including craniofacial bone and cartilage, melanocytes, blood vessels around the heart and
neurons and glia of the peripheral nervous system. During development, neural crest precursors
arise at the border of the neural plate in response to extrinsic signals that include FGF, BMP and
WNT pathways. How these various signaling pathways converge to induce neural crest cells is
largely unknown. Here we evaluate the signaling requirements for the production of neural crest
precursors using defined feeder-free conditions, with Sox10 as a key marker. In humans, Sox10
labels early premigratory and migratory neural crest cells and more recently Sox10 was shown, as
a single transcription factor, to produce “induced neural crest cells” from human fibroblasts. We
hypothesized that the manipulation of multiple signaling pathways would best enrich for Sox10+
neural crest precursors from human embryonic stem cells, when tested using low density singlecell cultures. We found that Sox10+ cells were enriched with a combination of FGF and noggin,
which acted via Wnt signaling. Indeed, Wnt signaling alone was sufficient to enrich for Sox10+
cells and for cells co-expressing other early neural crest markers. These Sox10+ precursors could
be differentiated into several neural crest cell types, including peripheral neurons and glia,
pigmented cells and myofibroblasts and demonstrated traits associated with neural crest stem cells.
Our study provides insights regarding the signaling cascades that coax the production of neural
crest precursors from pluripotent human embryonic stem cells.
Lay Abstract
With an aging population there is great interest in regenerative medicine –the ability to use cellular
therapies to treat a variety of damaged tissues. My research focuses on two areas of investigation
that are key to regenerative medicine: i) identify which cells are capable of producing replacement
cells and ii) determine how to coax those cells to make the desired replacement cell types without
making an abundance of unwanted cells. My research focuses on how to replace cells lost in the
nervous system, like in the brain, after damage, injury or disease.
Poster #51
Exploring the Basics
Epigenetic control of iPS cell chondrogenic differentiation
Rosa M. Guzzo (1),Hicham Drissi (1)
(1) UConn Health, Department of Orthopaedic Surgery, Stem Cell Institute
Technical Abstract
The regulation of cartilage genes is key to our understanding of joint cartilage maintenance, disease
progression, and the development of therapeutic strategies for osteoarthritis (OA). Recent studies
suggest that epigenetic changes are key drivers of articular cartilage degeneration during
osteoarthritis (OA). Thus, identifying the epigenetic mechanisms that govern chondrogenic
differentiation may uncover critical processes underlying articular chondrocyte (AC)
dysregulation in disease. We generated novel tissue-specific human induced pluripotent stem (iPS)
cell model systems to study the mechanisms controlling chondrogenic lineage commitment and
differentiation. Our previous studies determined that iPS cells derived from articular chondrocytes
(AC) displayed increased propensity to form cartilage matrix and higher expression of cartilagespecific genes when compared to iPS cells derived from either cord blood (CB) or skin fibroblasts
(SF). This finding suggests that there is a retained epigenetic memory in iPS cells. To gain insight
into the epigenetic mechanisms that govern iPS cell chondrogenesis, we analyzed the expressions
of ~170 genes encoding regulators of heterochromatin, chromatin remodeling, histone
modifications, and DNA methylation. Quantitative analyses identified significant changes in
epigenetic modifier gene profiles at distinct transitions from pluripotency to the chondrogenic
lineage. The up-regulation of cartilage genes during differentiation was concomitant with
regulated expressions of components of the histone methylation pathway. In current studies, we
are targeting key histone methylation enzymes in iPS cell-derived progenitors to reveal the
epigenetic mechanisms that control chondrogenic differentiation. An improved understanding of
these mechanisms may provide a basis for innovative approaches to alter or reset the chondrogenic
differentiation propensity of stem cells or the phenotypic stability in chondrocytes.
Lay Abstract
Osteoarthritis (OA), a leading cause of disability in the United States, results from progressive
degeneration of joint cartilage tissue. Since cartilage has limited to no capacity for regeneration,
the irreversible joint damage leads to chronic pain and loss of mobility. Despite the high prevalence
of OA, current treatments are ineffective and surgical joint replacement is often the final outcome.
Thus, there is an overwhelming need to develop effective new strategies to repair joint cartilage.
This research will use novel human stem cells to identify the critical mechanisms that control
expression of genes important for normal cartilage. It is expected that results from this work will
provide new information and a deeper understanding of how cartilage genes are regulated, which
will be essential for developing new therapeutic strategies to treat this common disease.
Poster #52
Exploring the Basics
Genome-wide analysis of a FGF/ERK regulated transcriptional networks in temporal and
spatial control of neural stem cells
Qiuxia Guo and James Li
Department of Genetics and Genome Sciences, University of Connecticut School of
Medicine, 400 Farmington Avenue, Farmington, CT 06030-6403
Technical Abstract
Self-renewal of neural stem cells (NSC) is important for growth and homeostasis of mammalian
brains. Significant progress has been made in our understanding of the maintenance and
heterogeneity of NSC. However, one critical knowledge gap is how the potency of NSC is
temporospatially regulated leading to NSC heterogeneity and differential growth of different
structures of the brain. The midbrain provides an excellent experimental paradigm to study NSC
development because the embryonic midbrain displays a pronounced developmental gradient and
expands along the rostral-to-caudal direction. Although the precise mechanism remains unknown,
graded FGF signaling activities are important for the developmental gradient in the midbrain. We
have recently discovered that an FGF-ERK signaling pathway regulated by protein phosphatase
Shp2 is essential for maintaining NSC and thereby unidirectional expansion of the midbrain. To
gain insights into the temporospatial control of NSC development, we performed RNA-SEQ to
examine that the transcriptome of midbrain cells at different positions along the rostrocaudal axis
and at different embryonic stages from both wildtype and Shp2-deficient mouse embryos. We also
measured high-order gene expression patterns in NSC derived from human embryonic stem cells
during the early phases of FGF signaling. These unbiased and genome-wide analyses have
provided novel insights into how FGF/ERK signaling pathway controls the temporospatial
development of NSC.
Lay Abstract
The extracellular signal regulated kinase (ERK) signaling cascade plays critical roles in brain
development, learning, memory, and cognition. Mutations of molecular components of this
signaling pathway cause developmental syndromes in humans that are associated with impaired
cognitive function and autism. Genetic studies in animal models have demonstrated that ERK
signaling is important in regulation of neural stem cells in both embryonic and adult brains. Using
unbiased and genome-wide transcriptional profiling, we have made new discoveries on how
different ERK signaling activities control development of neural stem cells over time.
Poster #53
Exploring the Basics
Cell polarity and neurogenesis in embryonic stem cell-derived neural rosettes
Grabel L (1), Banda E (1,2), McKinsey A (1), Germain N (1,3), Carter J (1), Anderson N
C (1).
(1) Biology Department, Wesleyan University, Middletown CT, (2) Department of
Neuroscience, University of Connecticut Health, Farmington CT, (3) Department of
Genetics and Genome Sciences, University of Connecticut Health, Farmington, CT
Technical Abstract
Embryonic stem cells (ESCs) undergoing neural differentiation form radial arrays of neural stem
cells, termed neural rosettes. These structures manifest many of the properties associated with
embryonic and adult neurogenesis, including cell polarization, interkinetic nuclear migration
(INM), and a gradient of neuronal differentiation. We now identify novel rosette structural features
that serve to localize key regulators of neurogenesis. Cells within neural rosettes have specialized
basal as well as apical surfaces, based on localization of the extracellular matrix receptor β1
integrin. Apical processes of cells in mature rosettes terminate at the lumen, where adherens
junctions are apparent. Primary cilia are randomly distributed in immature rosettes and tightly
associated with the neural stem cell’s apical domain as rosettes mature. Components of two
signaling pathways known to regulate neurogenesis in vivo and in rosettes, Hedgehog and Notch,
are apically localized, with the Hedgehog effector Smoothened (Smo) associated with primary
cilia and the Notch pathway γ-secretase subunit Presenilin 2 associated with the adherens junction.
Increased neuron production upon treatment with the Notch inhibitor DAPT suggests a major role
for Notch signaling in maintaining the neural stem cell state, as previously described. A less robust
outcome was observed with manipulation of Hedgehog levels, though consistent with a role in
neural stem cell survival or proliferation. Inhibition of both pathways resulted in an additive effect.
These data support a model whereby cells extending a process to the rosette lumen maintain neural
stem cell identity whereas release from this association, either through asymmetric cell division or
apical abscission, promotes neuronal differentiation.
Lay Abstract
Embryonic stem cells differentiating into neurons in culture self assemble into radial structures
called neural rosettes. Rosettes provide a model for studying many aspects of neuron production
in the embryonic brain. We show here that components of pathways known to regulate production
of neurons are localized to the internal compartment of the rosette. These data validate the use of
embryonic stem cells to model human development and help in the design of protocols that
maximize production of neural progenitors for use in transplantation therapies.
Poster #54
Exploring the Basics
Human Somatic Cell Reprogramming Involves Human-Specific Alternative Splicing and
Allele Specific Gene Expression
Yoshiaki Tanaka (1,6), Eriona Hysolli (1,6), Juan Su (1,3), Yangfei Xiang (1), Kun-Yong
Kim (1), Mei Zhong (2), Yumei Li (1,4), Ghia Euskirchen (5), Michael P. Snyder (5),
Xinghua Pan (1), Sherman Morton Weissman (1) and In-Hyun Park (1)
(1) Department of Genetics, (2) Department of Cell Biology, Yale Stem Cell Center, Yale
School of Medicine, New Haven, CT 06520, USA, (3) Department of Cell Biology, the
Second Military Medical University, Shanghai 200433, China, (4) Department of
Dermatology, Jiangsu University Affiliated Hospital, PR China, (5) Department of
Genetics, Stanford University, Stanford, CA 94305-5120, (6) Equal contribution
Technical Abstract
In course of reprogramming to the induced pluripotent stem cells (iPSCs), somatic cells undergo
multiple transcriptional and epigenetic changes. Alternative splicing (AS) is one of the
transcriptional events to acquire cell type-specific properties and facilitate functional diversity in
genes. Allele specific gene expression (ASE) is a gene regulatory system to increase gene
variations by distinct epigenetic modifications between two alleles and known to occur in preimplantation embryo and gene imprinting. In addressing the dynamics of both AS and ASE during
reprogramming, we performed RNA-seq on cells defined by a combination of multiple cellular
surface markers. We identified over 50 novel spliced forms of genes uniquely expressed at
progressive stages of reprogramming. More than half of these pluripotent-specific forms show
human specificity, which are not detectable in mouse embryonic and epiblast stem cells. In
particular, overexpression of a human-specific pluripotent spliced form of CCNE1 (pCCNE1), but
not ubiquitously expressed CCNE1 (uCCNE1), dramatically enhances the reprogramming. In
addition, SNP expression analysis uncovers that monoallelic gene expression is induced in the
intermediate stages of reprogramming. Biallelic expression is recovered, when the reprogramming
is complete. Overall, our transcriptome data provide unique opportunities to gain new insight into
human iPSC reprogramming.
Lay Abstract
Human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) are not identical to
mouse ESC and iPSC. To investigate the specificity in hESC and hiPSC, we searched alternative
splicing and allele specific gene expression. Alternative splicing (AS) is one of the transcriptional
events to acquire cell type-specific properties and facilitate functional diversity in genes by
changing exon usage. Allele specific gene expression (ASE) is a gene regulatory system to increase
gene variations by distinct epigenetic modifications between two alleles and known to occur in
pre-implantation embryo and gene imprinting. In this study, we found human-specific splicing is
important to gain pluripotency and monoallelic gene expression is induced during iPSC
reprogramming. Our result helps to understand the key difference between human and mouse
ESC/iPSC.
Poster #55
Exploring the Basics
Mechanisms of adipocyte repopulation during involution
Rachel Zwick (1), Valerie Horsley (1)
(1) Yale University, Department of Molecular, Cellular, and Developmental Biology
Technical Abstract
The structure and function of the adult mammary gland relies on the interaction between branched
epithelial ducts and the surrounding stroma. Adipocytes - the major component of the mouse
mammary stroma - exhibit dynamic changes throughout development, especially when they
regress during lactation and repopulate during involution. The abundance of adipocytes in stroma
and the timing of their repopulation suggest a functional role for this cell type in mammary gland
remodeling after lactation, but the cellular and molecular mechanisms that drive their reappearance
are not well understood. Here, I have analyzed whether adipogenesis (proliferation and
specification of adipocyte lineage cells) or hypertrophy of mature adipocytes are involved in
adipocyte repopulation. Both adipocyte number and size increase during involution, suggesting
that adipose tissue increases via both hyperplasia and hypertrophy. I have identified an adipocyte
precursor cell population in the mouse mammary stroma that becomes activated during involution
and will use genetic lineage tracing to determine whether this population differentiates to
regenerate mature adipocytes. My data also demonstrates that epithelial transdifferentiation does
not account for adipocyte repopulation during involution, as had been previously suggested.
Understanding the mechanisms of adipocyte repopulation will enable me to evaluate whether
adipocytes produce signals required for the behavior of epithelial or other cell types during
involution.
Lay Abstract
The mammary gland undergoes a massive cellular restructuring to end lactation and prepare for
future rounds of breastfeeding, which is critical to providing nutrition for infants and protecting
them from disease. In this project, I use genetic mouse models to determine the role of adipocyte
(fat) stem cells in facilitating this reconstructive process, which is largely unknown but of interest
given that a huge portion of mouse and human mammary glands are filled with adipocytes, and
the important role of this cell type in regenerating skin and muscle tissue after injury. Information
revealed in my research is broadly relevant for understanding how adult stem cells maintain
healthy tissues and respond to injury, and holds specific therapeutic potential for problems with
lactation and breast reconstruction after mastectomy.
Poster #56
Exploring the Basics
Spatial induction of cell death by the niche limits the hair follicle stem cell pool
Samara Brown (1), Kailin R Mesa (1), Panteleimon Rompolas (1), Giovanni Zito (8),
Peggy Myung (1,2), Thomas Y Sun (1), David Gonzalez (5), Krastan Blagoev (6,7), Ann
Haberman(5), Valentina Greco(1,2,3,4)
(1) Yale University, Genetics, New Haven, CT, (2) Yale University, Dermatology, New
Haven, CT, (3) Yale University, Stem Cell Center, New Haven, CT, (4) Yale University,
Cancer Center, New Haven, CT, (5) Yale University, Immunobiology, New Haven, CT,
(6) National Science Foundation, Physics of Living Systems, Arlington, VA, (7)
MGH/HST, AA Martinos Center for Biomedical Imaging, Boston, MA, (8) University of
Palermo, Biopathology, Palermo, Italy
Technical Abstract
Tissue regeneration is achieved through a balance of cell production and elimination. A major
focus of study has been to understand how stem cells are essential for this process. However, the
cellular and molecular mechanisms that regulate the selection and preservation of stem cells in a
tissue remain unclear. Using the hair follicle, we show by intravital microscopy in live mice that
the stem cell compartment is selected through spatially restricted cell death along the basal
epithelium. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes
to clear dying epithelial cells. Through cellular and genetic ablation we show that epithelial cell
death requires TGFβ activation through crosstalk with the mesenchymal niche. Finally, we show
that when regression is blocked, stem cell activity is expanded across the basal epithelium. This
study identifies the cellular and molecular mechanisms that contribute to the extrinsic regulation
of stem cell selection in a regenerating tissue.
Lay Abstract
Most tissues in our bodies undergo constant cellular turnover. This process requires a dynamic
balance between cell production and elimination. Stem cells have been shown in many of these
tissues to be the major source of new cells. However, despite the tremendous advances made, it
still remains unclear how stem cell behavior and activity are regulated in vivo. Furthermore, we
lack basic understanding for the mechanisms that coordinate niche/stem cell interactions to
maintain normal tissue homeostasis. Using a novel imaging technique established in our lab, we
are able to investigate these fundamental processes in live mice using the skin as a model system,
with the goal of understanding how tissues successfully orchestrate tissue regeneration throughout
the lifetime of an organism.
Poster #57
Exploring the Basics
The Role of Surfactant Protein C in Lung Repair and Regeneration
Huiyan Jin (1), Ping-Xia Zhang (1), Rachel Tobin (1), Jooeun Lee (2), Barbara Driscoll
(2), Emanuela Bruscia (1) and Diane Krause(1)
(1) Department of Lab Medicine, Yale School of Medicine, New Haven, CT, (2)
Developmental Biology & Regenerative Medicine Program, Children’s Hospital Los
Angeles
Technical Abstract
Surfactant Protein C (SPC) is an important component of pulmonary surfactant that is critical for
reducing surface tension while breathing. It is secreted by Type 2 pneumocytes (T2), which are
facultative stem cells in the lung alveoli. Using a unique murine model that contains an inducible
suicide gene on the SPC promoter (SPC-TK) as well as double transgenic mice that both have the
SPC-TK transgene and are knocked out for endogenous SPC gene expression (herein referred to
as SPC-TK, SPC-KO mice), I induced T2 cell specific lung damage by ganciclovir (GCV)
administration on the SPC+/+ and SPC-KO background. I found that SPC-TK, SPC-KO mice are
far more susceptible to T2 cell depletion injury than SPC-TK, SPC+/+ control mice after depletion
of T2 cells with 50mg/kg GCV for 3 days. After GCV-induced depletion, SPC-TK, SPC-KO mice
have significantly greater weight loss, and higher mortality. Further analysis revealed significantly
more severe lung damage in the SPC-TK, SPC-KO mice, including increased T2 cell death by
apoptosis assessed by TUNEL staining and increased inflammation. The bronchoalveolar lavage
(BAL) from SPC-TK, SPC-KO mice contained more neutrophils and lymphocytes than SPC-TK,
SPC+/+ mice; more protein in the BAL, which indicates leakage at the alveolar-capillary barrier;
and significantly higher levels of pro-inflammatory cytokines (GCSF, CXCL1, IL6). This may be
due to lack of an anti-inflammatory response on the SPC-KO background. Lack of SPC thus
resulted in delayed resolution of inflammation. When I administered of anti-inflammatory drug
(ibuprofen) and anti-apoptotic drug (Z-VAD), it prolonged the survival of SPC-TK, SPC-KO mice.
Our study showed that in addition to its established biophysical role in reducing surface tension,
SPC might also play an important role in T2 cells self-renewal and is essential for the antiinflammatory response that is required for tissue repair.
Lay Abstract
It is known that surfactant protein C (SPC) is an important protein in the lungs that is critical for
reducing surface tension while breathing. SPC is made and secreted by Type 2 lung cells (T2). In
our recent study using a unique inducible mouse model, I induced T2 cell specific lung damage in
the wild type and SPC deficient mice. Our major finding is that SPC deficient mice are far more
susceptible to injury than wild type mice. In SPC deficient mice, they have significantly greater
weight loss, and higher mortality after challenge. Further analysis revealed more severe lung
damage in the SPC deficient mice, including increased T2 cell death and increased inflammation.
After injury, there were more inflammatory cells and protein in the lung fluid removed from SPC
deficient mice, which indicates inappropriate leakage in the lung. In addition, the lungs of SPC
deficient mice had more disrupted lung structure. Lack of SPC thus resulted in delayed healing of
injury. Our study showed that in addition to its established biophysical role in reducing surface
tension, SPC might also play an important role in lung cell self-renewal and is essential for the
anti-inflammatory response that is required for tissue repair. Further studies will focus on how we
can use SPC as a therapy to gain beneficial effect for lung repair and regeneration after injury.
Poster #58
Exploring the Basics
New Evidence for Piwi Association with Specific Sites in the Drosophila Genome
Nils Neuenkirchen (1), Na Liu (1), Mei Zhong (1), Haifan Lin (1)
(1) Yale Stem Cell Center and Department of Cell Biology, Yale University School of
Medicine, New Haven, CT 06520
Technical Abstract
How epigenetic factors without DNA binding ability are recruited to specific sites in the genome
represents a key question in epigenetic programming. Drosophila Piwi is one of the three members
of the PIWI family that bind to short non-coding RNAs of 24-31 nt in length (piRNA). Piwi
localizes to the nucleus and regulates a wide range of processes including germline stem cell
maintenance and self-renewal, transposon repression and epigenetic regulation. The interaction of
Piwi with specific sites in the genome is thought to be guided by its associated piRNAs. While a
recent study from our lab identified Piwi to interact with chromatin using ChIP (Chromatin
Immunoprecipitation), the experimental challenges that accompany this approach make it difficult
to repeat these findings in different labs. The specific Piwi binding sites in the genome remain to
be determined. It is unclear why Piwi does not produce a robust ChIP signal. We suspect that this
might reflect certain peculiar aspects of Piwi-piRNA interaction with chromatin that render this
interaction prone to disruption during the ChIP procedure. Here we present an independent
approach to study the interaction of Piwi with chromatin using DamID-seq (DNA adenine
methylation ID and deep sequencing). Comparing the random methylation of N6 in the adenine of
GATC sites by Dam (DNA adenine methyltransferase) and the Piwi-guided methylation by the
Dam-Piwi fusion protein, our approach covers 78.45% of the genomic GATC sites. We find that
Piwi-specific methylation occurs predominantly in euchromatic regions, indicating specific
association of Piwi with these regions. Heterochromatic regions are targeted less frequently, likely
resulting from their inaccessible chromatin structure. Furthermore, we show that the number of
piRNA complementary sites in close proximity to methylated GATC sites is significantly enriched
for Piwi-specific methylation. These findings support the association of Piwi with chromatin.
Lay Abstract
PIWI proteins play an important role in the germline of worms, flies, mice, humans and plants,
and can lead to infertility if the proteins contain mutations that disrupt their function. Even though
these organisms look very different, they have a lot in common on the molecular level. Fruit flies,
for example, share 75% of the genes that cause diseases in humans. Consequently, we can learn
about human genetics by studying fruit fly genetics. In fruit flies, the Piwi protein is one of three
members of the PIWI protein family that bind to short non-coding RNAs (piRNAs). Piwi is mainly
present in the nucleus and ensures that germline stem cells are maintained. It helps in repressing
transposable elements that could have detrimental effects on the integrity of DNA, and regulates
the control of gene function (epigenetics). While a recent study from our lab showed that the Piwi
protein binds to DNA, other labs were not able to reproduce these findings. Consequently, we
decided to use an independent approach to address this controversial question using a technique
called DamID (DNA adenine methylation identification). In this approach, a bacterial enzyme that
is able to leave chemical marks (methylation) on DNA molecules is joined to Piwi. To study
whether Piwi is able to bind to DNA, we genetically modified flies to generate a Piwi protein that
can leave methyl marks on DNA close to its binding site. Subsequently, these marks were read out
by Next Generation Sequencing (NGS). We find that Piwi binds to specific regions in the genome.
DNA regions with increased methylation marks also contained an elevated number of nearby DNA
sequences that are complementary to piRNA molecules. We consequently reason that the Piwi
protein in fruit fly is able to bind to DNA and identifies specific DNA regions by its associated
piRNA molecule. These findings in fruit flies could help to better understand the role of human
PIWI proteins in germline stem cells and their effect on fertility.
Poster #59
Exploring the Basics
PIWI-piRNA pathway function in the stem cells of “immortal” hydra
Celina E. Juliano(1), Adrian Reich(2), Na Liu(1), Jessica Götzfried1, Mei Zhong(1), Selen
Uman(1), Robert A. Reenan(2), Gary M. Wessel(2), Robert E. Steele(3), and Haifan Lin(1)
(1)Yale Stem Cell Center and Department of Cell Biology, Yale University School of
Medicine, New Haven, CT 06520 (2)Department of Molecular Biology, Cell Biology, and
Biochemistry, Brown University, Providence, RI 02912, (3)Department of Biological
Chemistry and the Developmental Biology Center, University of California, Irvine, CA
92697-1700
Technical Abstract
Adult stem cells maintain tissue homeostasis and a decline in stem cell function contributes to
disease as animals age. Interestingly, Hydra is extraordinarily long-lived and the adult stem cells
show no signs of senescence. We aim to understand the molecular mechanisms that support this
remarkable stem cell longevity. The three cell lineages in Hydra are each supported by a distinct
stem cell type: two strictly somatic and one germline-competent. We find that the PIWI-piRNA
pathway is operating in all three stem cell types and we hypothesize that this may contribute to
Hydra longevity. PIWI proteins and their bound small RNAs (piRNAs) are known for maintaining
genomic stability in the germline, most notably by repressing transposon expression. PIWI has
conserved expression in somatic stem cells, but function in these cases has not been well explored.
In Hydra we find that the PIWI-piRNA pathway is essential in the somatic lineages. We
demonstrate that PIWI proteins in Hydra are strictly cytoplasmic and thus we are focusing on
identifying post-transcriptional targets of the pathway. Our data show that transposon targeting is
a conserved feature of the pathway in Hydra, but notably this function is most prevalent in the
germline-competent stem cells. We are currently exploring the function of the pathway in
regulating somatic stem cell fate through post-transcriptional gene regulation and its potential
function in promoting somatic longevity (Funding Source: NIH 1K01AG044435-01A1).
Lay Abstract
Stem cells are required to repair damaged tissue; as stem cells age they lose this ability thus causing
the decline in health associated with aging. This proposal investigates the molecular mechanisms
controlling the stem cells of Hydra, an animal with negligible aging. Understanding how Hydra
stem cells retain longevity will give us a better understanding of why human stem cells age and
shed light on possible therapies for extending stem cell longevity.
Poster #60
Exploring the Basics
The Dynamics of Connexin Genes in the Context of Spontaneous Activity in Developing
Human Neurons.
Pallavi V. Limaye*, Michele L. McGovern*, Mandakini B. Singh*, Katerina D.
Oikonomou, Glenn S. Belinsky,Srdjan D. Antic (* have made equal contributions)
University of Connecticut Health, Department Of Neuroscience, Farmington, CT 06030
Technical Abstract
Early in development, before the final stages of synapse formation and maturation, nerve cells
exhibit electrical activity in the virtual absence of sensory inputs. Electrical activity drives gene
expression, differentiation and maturation of individual neurons as well as neuronal networks. This
project studied physiological and genetic determinants of spontaneous electrical activity in
developing human neurons. Human stem cells were directed towards neuronal differentiation over
a period of 21 days. Human cells were grouped into three time bins, namely, the stem cell,
neuroepithelial rosettes and the young neurons. Using multi-site calcium imaging technique,
spontaneous calcium transients were detected in populations of cells. This spontaneous activity
was then challenged with drugs that block connexin channels (octanol or gadolinium). The
expression of connexin (Cx) genes Cx26, Cx36, Cx45, Cx47 and pannexin-1 (panx1) was analyzed
by quantitative polymerase chain reaction (qPCR), on the same biological samples where
physiological recordings were performed. In addition, the expression of neuronal marker PAX6 as
well as neuronal genes VGLUT1, GAD1, schizophrenia gene ERBB4, glial lineage marker BLBP
and housekeeping genes beta actin and HPRT1 was also analyzed. Correlation between specific
connexin gene expression levels and parameters of spontaneous activity was used to determine the
relations between individual Cx proteins and spontaneous activity during early phases of neuronal
differentiation in humans.
Lay Abstract
Early during the development of the brain, when contacts between nerve cells have not yet been
completed, individual nerve cells produce electrical activity, without the application of any
external stimulus. This spontaneous electrical activity, in the form of transient depolarizations and
firing of nerve impulses, drives gene expression, differentiation and maturation of both individual
neurons and neuronal networks. Based on experiments performed in rodents, some forms of
communication between cells in developing rodent brains are achieved by membrane pores and
channels formed by structural proteins called connexins. In this study, our aim was to investigate
the role of connexins in the human model of neuronal differentiation. More specifically, we
analyzed the expression of connexin genes and their relation to spontaneous activity during the
earliest phases of neuronal differentiation in humans, which correspond to the first and second
trimester of human gestation. To this aim, human stem cells were directed towards neuronal
differentiation over a period of 21 days. Multi-site calcium imaging technique was applied to detect
populations of cells with spontaneous calcium transients. This spontaneous activity was then
challenged with drugs that block connexin channels. The expression of various connexin genes
was analyzed quantitatively on the same biological samples where physiological recordings were
performed. Correlation between specific connexin gene expression levels and parameters of
spontaneous activity was used to determine the relationship between individual connexin proteins
and spontaneous activity during early phases of neuronal differentiation in humans.
Poster #61
Exploring the Basics
Edges of human embryonic stem cell colonies display distinct mechanical properties and
differentiation potential
Kathryn A. Rosowski (1), Aaron F. Mertz (1), Samuel Norcross (1), Eric R. Dufresne (1),
Valerie Horsley (1)
(1) Yale University
Technical Abstract
Human embryonic stem cells (hESCs) provide an in vitro system to model the processes that
control the earliest stages of cell fate specification. In order to understand the mechanisms that
guide these cell fate decisions, we closely examined the differentiation process in adherent
colonies of hESCs. Live imaging of the differentiation process reveals that cells on the outer edge
of the undifferentiated colony begin to differentiate first and remain on the perimeter of the colony
to eventually form a band of differentiated cells. Strikingly, this band is of constant width in all
colonies, independent of their size. Cells at the edge of undifferentiated colonies show distinct
actin organization, greater myosin activity and stronger traction forces compared to cells in the
interior of the colony. Notably, by increasing the number of cells at the edge of colonies through
plating small colonies, we can increase differentiation efficiency. Our results suggest that human
developmental decisions are influenced by cellular environments dictated by colony geometry.
Lay Abstract
Human embryonic stem cells have great potential for regenerative medicine, due to their ability to
generate all cell types of the body. Current understanding of these cells allows for the specification
of certain cell types, yet these procedures are often inefficient at generating large amounts of fully
functioning cells. To achieve better differentiation protocols, it is important to understand all
factors which regulate pluripotency and cell fate choices in these cells. We find that spatial
organization of cells within a colony impacts differentiation and mechanical environment,
suggesting that these factors could be manipulated to increase differentiation protocol
effectiveness.
Poster #62
Exploring the Basics
Wnt5a Treatment of Embryonic Stem Cell Progenitors Promotes Cartilage Repair in a Rat
Chondral Defect Model
Jason D. Gibson (1), Farhang Alaee (1), David N. Paglia (1), Ryu Yoshida (1), Thomas M.
DeBerardino (1), Rosa M. Guzzo (1), and Hicham Drissi (1)
(1) UConn Health Center, Dept of Orthopaedic Surgery, UConn Stem Cell Institute
Technical Abstract
The poor capacity of native cartilage tissue to self-repair remains a significant clinical challenge.
Identifying an appropriate source of progenitor cells as well as the necessary signals to control
their differentiation into maturationally-arrested chondrocytes is prerequisite for an effective cellbased strategy to repair articular cartilage defects. Multipotent progenitors derived from human
embryonic stem cells (hESC) have been reported to mediate cartilage regeneration, however little
evidence supports the ability of these cells to adopt an articular-like cartilage phenotype. Based on
developmental genetic evidence demonstrating the chondrogenic potential of the canonical BMP2 and the anti-hypertrophic effects of the non-canonical Wnt5a, we hypothesized that Wnt5a may
induce the maturational arrest of BMP-2 mediated chondrocyte differentiation. Moreover, we
hypothesized that the sequential treatment of progenitors with BMP-2 followed by Wnt5a may
promote articular cartilage repair in vivo. The progenitors derived from both the human H9 and
CT-2 hESC lines display multipotent mesenchymal properties. These progenitors were
differentiated into chondrocytes in high density cell pellets, and we observed that treatment with
BMP-2 induced their terminal differentiation, whereas Wnt5a prevented their hypertrophic
maturation. Furthermore, cultures sequentially treated with BMP-2 followed by Wnt5a exhibited
abrogation of chondrocyte hypertrophy and enhanced expression of permanent cartilage matrix
markers. Moreover, our in vivo correlate of these studies demonstrated that this sequential pretreatment of the H9-derived progenitor cell pellets resulted in cartilage regeneration upon
implantation in a rat chondral defect model. The data collectively indicate that Wnt5a can restrict
the differentiation of hESC-derived progenitor cells into maturationally-arrested chondrocytes, a
feature of permanent cartilage and a rate-limiting factor for joint cartilage regeneration.
Lay Abstract
The poor capacity of native cartilage tissue to self-repair remains a significant clinical challenge.
Identifying an appropriate source of progenitor cells as well as the necessary signals to control
their differentiation into functional, permanent cartilage is prerequisite for an effective cell-based
strategy to repair cartilage defects in the joint. Human embryonic stem cells (hESC) have been
used to better understand cartilage biology and to produce cartilage progenitor cells. These
progenitor cells have been reported to mediate cartilage repair, however these cells have not been
reported to regenerate a functional type of permanent joint cartilage. Based on genetic evidence
demonstrating the developmental functions of two factors, BMP-2 and Wnt5a, we hypothesized
that BMP-2 is needed to induce cartilage cell differentiation and Wnt5a is needed to stop the
maturation of cartilage cells for promoting a functional, permanent cartilage tissue. Moreover, we
hypothesized that the sequential treatment of progenitor cells with BMP-2 followed by Wnt5a may
promote cartilage repair in an animal model. The progenitor cells derived from both the H9 and
CT-2 hESC lines behave similar to cartilage stem cells. These progenitors were converted into
cartilage cells, and we observed that treatment with BMP-2 induced their differentiation into
mature cartilage cells, whereas Wnt5a prevented this cartilage cell maturation. Furthermore,
cultures sequentially treated with BMP-2 followed by Wnt5a showed better expression of
functional permanent cartilage markers. Moreover, when implanted into rats with cartilage defects,
we found that the sequential pre-treatment of the H9-hESC derived progenitors were able to
regenerate cartilage tissue. The data collectively indicate that Wnt5a can restrict the differentiation
of hESC-derived progenitor cells into cartilage that is more similar to the permanent cartilage of
the joint. This is an important finding for joint cartilage regeneration.
Poster #63
Exploring the Basics
The RNA exosome in human embryonic stem cell function: beyond the RNA degradation
machinery.
Cedric Belair (1), Sandra Wolin (1)
(1) Yale University School of Medicine Cell Biology Department
Technical Abstract
The unique abilities of human embryonic stem cells (hESCs) to self-new and to differentiate into
all three germ layers are linked to their “open chromatin” status, which allows high levels of
transcription and contributes to pluripotency by keeping many tissue-specific genes in permissive
transcriptional states. As a consequence, ES cells express many RNAs at higher levels than
differentiated cells, including potentially harmful RNAs such as endogenous retrotransposons and
mRNAs that encode proteins promoting differentiation. Although the proteasome and RNA
interference pathway have been implicated in controlling this pervasive transcription, the role of
RNA surveillance pathways in degrading transcripts that escape these and other silencing
pathways is largely unknown.
The RNA exosome, a multiprotein nuclease complex implicated in RNA surveillance, is
an attractive candidate to recognize and degrade RNAs that are harmful or whose products could
jeopardize ES cell function. To assess its role, we generated human ES cell lines expressing
doxycycline-inducible shRNAs directed against the exosome or its catalytic subunits.
Interestingly, we found that the exosome contributes to the ability of ES cells to self-renew and
also limits their differentiation. Our data also suggest that this complex may be involved in the
defense of ES cells against endogenous retrotransposons. By combining whole transcriptome
analysis with in vivo crosslinking of exosome subunits to their target RNAs, we identified several
classes of RNAs which are exosome targets. Our experiments reveal the exosome as a new actor
in regulating the unique properties of self-renewal and pluripotency of human ES cells.
Lay Abstract
In addition to being a model for studying development and disease, human embryonic stem cells
(hESCs) have great therapeutic potential. These cells are able to grow indefinitely in a process
called self-renewal and are also able to differentiate into every cell type of the body, a property
known as pluripotency. These two hallmarks of hESCs are associated with a unique organization
of their DNA that allows the expression of genes implicated in self-renewal and in the maintenance
of pluripotency. As part of this unusual DNA organization, hESCs express many RNA molecules
at much higher levels than differentiated cells. Most of these RNAs are involved in gene expression
but some could be harmful to ESCs, such as RNAs derived from endogenous retrotransposons,
which can cause human disease by mutating DNA. Thus, hESCs need mechanisms to recognize
and degrade these harmful RNAs. Human cells possess several different RNA surveillance
pathways, among which a RNA degradation machine called the RNA exosome is particularly
important. This complex is found in animal and plant cells, although its functions have not yet
been well characterized in human cells.
The aim of this project is to determine the contributions that the RNA exosome makes to
the unique properties of hESCs. By identifying the set of RNAs degraded by the RNA exosome,
we discovered that this complex controls the level of several RNAs involved in the self-renewal
and differentiation of hESCs. We also obtained evidence that the RNA exosome may be important
for defending hESCs against harmful RNAs. Together, these results demonstrate that the RNA
exosome is important for maintaining the unique properties of hESCs. This knowledge may be
helpful in engineering hESCs to form other cell types that can be used to replace defective cells
and tissues.
Poster #64
Exploring the Basics
Inhibition of Retinoic Acid Signaling and Stimulation of Wnt Signaling Allows Efficient
Paraxial Mesoderm Formation from Human Embryonic Stem Cells
Ryan P. Russell (1), Yaling Liu (1), Peter Maye (1)
(1) University of Connecticut Health Center
Technical Abstract
A comprehensive understanding of how to direct embryonic stem cells into mature, functional
skeletal cell types remains essential in the realm of orthopaedic translational medicine. Our work
has focused on a stepwise, embryonic differentiation program to derive cells of the axial skeletal
lineage via paraxial mesoderm and sclerotome intermediates for therapeutic purposes. To assess
paraxial mesoderm formation from human ESCs, we created a Tbx6 reporter line, a gene essential
for paraxial mesoderm induction. Here we report that retinoic acid pathway inhibition combined
with Wnt pathway activation substantially augments paraxial mesoderm formation from human
ESCs. Wnt pathway stimulation in ESCs with Wnt3a and CHIR99021 for 4 days resulted in
nominal TBX6 reporter expression in comparison to untreated cells; however the retinoic acid
receptor antagonist AGN193109 along with Wnt3a and CHIR99021 resulted in a strong increase
in TBX6 reporter expression confirmed by FACS analysis, 26.9% TBX6+ to <1% without
AGN193109. Addition of the BMP antagonist Noggin resulted in a more limited TBX6 reporter
expression pattern (9.9% TBX6+) compared to Wnt3a, CHIR99021 and AGN193109. FACS
sorting at day 4 of differentiation revealed 22.8% of the total cell population was TBX6+. RTPCR for the paraxial mesoderm markers MEOX1 and Mesogenin in the TBX6+ population
showed an 18- and 30-fold increase over the negative population, respectively. A 26-fold
difference in TBX6 expression between the positive and negative sorted populations confirmed
the accuracy of the reporter construct in matching endogenous gene expression. Therefore, Wnt
pathway stimulation combined with retinoic acid inhibition strongly promotes ESC differentiation
into paraxial mesoderm. The ability to isolate a uniform population from this primary
differentiation stage will allow for more effective generation of sclerotome populations and
ultimately lead to generation of skeletal progenitors for therapeutic purposes.
Lay Abstract
Non-union, or failure of bone to properly heal, occurs in roughly 1.6 million fractures per year in
the US, at an estimated cost of $15 million annually. Current bone graft strategies to help bridge
the gap may result in significant and lasting side effects in addition to the added financial losses
for the patient and healthcare system. The goal of our research is to develop a cell therapy approach
to regenerate bone tissue without the need for administration of high-dose growth factors, which
can cause bone repair to go haywire. Our strategy utilizes human embryonic stem cells (hESCs),
which are capable of forming all cell types within the body, to specifically generate forerunners of
osteoblasts, the cells responsible for creation of new bone. Our method is designed to progressively
differentiate the hESCs to reflect their natural maturation during embryonic development. We have
genetically engineered hESCs to produce fluorescent proteins in order to visualize when genes
important for bone production are turned on. This strategy gives us the ability to have a rapid
diagnostic readout on the effectiveness of our experimental conditions and subsequently sort out
the individual cells of interest, depending on their color. This specialized population of cells should
be specifically geared towards forming new bone, and would ideally help fill the void when
appropriately activated. The research presented here demonstrates the capability of inducing
paraxial mesoderm, an intermediate cell type that forms during development and the first phase in
the progression from stem cell to bone-forming cell. By using genetic tools to study the efficiency
of hESCs in response to certain stimuli, we hope to develop a therapeutic approach that harnesses
the body’s natural bone repair mechanism to help heal otherwise irreparable bone defects.
Poster #65
Exploring the Basics
Modeling human telencephalic development using iPS in vivo and in vitro:deleterious effects
of disrupting cell-to cell contact
Mariangela Amenduni (1), Jessica Mariani (1), Gianfilippo Coppola (1), Anahita Amiri
(1), Flora M Vaccarino (1),
(1) Program in Neurodevelopment and Regeneration, Child Study Center, Yale
University
Technical Abstract
To differentiate iPSCs into forebrain neurons several protocols have been developed, that are based
on two main approaches: an organoid system and a dissociated monolayer preparation. Until now
the organoid-based differentiation has proven to be superior to dissociated preparations, in terms
of regional and layer specification, and a more advanced degree of cellular differentiation.
However culturing the cells as dissociated progenitors (NPCs) has the advantage of expanding the
neural progenitor pool for subsequent neuronal differentiation without having to repeat the entire
differentiation process. Dissociated NPCs cultured as monolayers generated generic neurons that
failed to mature into specific neuronal subtypes. In contrast, progenitors derived from the same
iPSC lines grown in 3D conditions generated both excitatory and inhibitory neuron subtypes
expressing specification markers typical of the cerebral cortex and hippocampus. To evaluate
whether the differentiation potential of dissociated NPCs improved in an in vivo context, we
performed grafting experiments in neonatal mice analyzing them at different time points: 2 months
post-transplantations (mpt), and 6 mpt. Surprisingly the NPCs did not express mature excitatory
and inhibitory markers indicating that they remained more immature than at corresponding ages
in vitro using an organoid preparation.To assess the potential of NPCs dissociated at a later time
point to differentiate and mature in the in vivo context, NPC were kept as 3D aggregates until TD
day19, when they were dissociated and transplanted. Delaying the dissociation of human NPCs
until TD day 19 and extending their survival in vivo until 6 mpt did not dramatically enhance the
differentiation of the cells into specific neuronal subtypes. These results suggest that disruption of
cell to cell contacts has a deleterious effect on cell differentiation, and raise an important question
about the effect of dissociation on human neuronal cell cultures
Lay Abstract
Different methods are available to culture human neurons in vitro: they are mainly based on the
use of a 3D structure method and a dissociated monolayer preparation. Neurons grown in 3D are
much better than neurons that are dissociated and cultured in a monolayer. However the
dissociation is helpful to have a source of neuronal progenitors for more than one experiment
without having to repeat the entire differentiation process. The dissociated cultures are producing
generic neurons that are not helpful to study neurodevelopment and neurodegenerative disorders.
Our aim is to improve this method of culturing neurons to be able to have a source of cell that can
be used for future transplantation experiments.
Poster #66
Exploring the Basics
VEGF-C/VEGFR3 SIGNALING IN MOUSE AND HUMAN NEURAL STEM CELLS
Tae Hyuk Kang (1), Jinah Han (2), June Hee Park (1), Charles-Felix Calvo (4), Anne C.
Eichmann (2), Jean-Leon Thomas (1) (3)
(1) Department of Neurology, Yale University School of Medicine, (2) Department of
Internal Medicine, Yale University School of Medicine, (3) Yale Stem Cell Center, Yale
University, (4) University Pierre et Marie Curie-Paris 6, Centre de Recherche de I'Institut
du Cerveau et de la Moelle Epiniere
Technical Abstract
The restoration of neural stem cells (NSCs) within the two neurogenic niches of the adult brain,
subventricular zone (SVZ) and subgranular zone (SGZ), is a possible strategy for curing various
acute and chronic neurological disorders. However, the molecular mechanisms controlling the
function of NSCs still remain largely unknown. Recently, we demonstrated that NSCs in both SVZ
and SGZ of adult mouse brain are directly activated upon stimulation with vascular endothelial
growth factor C (VEGF-C) via its receptor vascular endothelial growth factor receptor 3
(VEGFR3) expressed on NSCs. RNA-Seq analysis performed using primary SGZ NSCs revealed
that the asymmetric dividing function of NSCs could be activated via VEGF-C/VEGFR3 signaling
pathway, i.e., the proliferating and NSC markers were up- and down-regulated after VEGF-C
treatment, respectively. Signaling network analysis using Reactome Functional Interaction (FI)
network showed that the initial signaling cluster induced by VEGF-C stimulation consisted of
several signaling pathways and it implicated the PI3K-Akt signaling pathway as the convergent
mechanism. To delineate this signaling cascade, we generated human embryonic stem cell (hESC)derived NSCs. The phenotypic similarity of these generated cells to as primary NSCs was
confirmed on finding expression of characteristic NSC markers GFAP, BLBP, as well as VEGFR3
in the hESC-derived NSCs. Stimulation of the hESC-derived NSCs with VEGF-C significantly
induced phosphorylation of Akt (T308 and S473 site) and its down-stream targets, pGSK3-β (S9)
and p70S6K. Moreover, phosphorylation of ERK is significantly up-regulated. Activation of
PI3K-Akt and ERK signaling pathways via VEGF-C/VEGFR3 are expected to induce cell cycle
progression and survival of these quiescent NSCs. Taken together, hESC-derived NSCs allowed
us to identify the downstream signaling pathways regulated by VEGF-C/VEGFR3 in human
NSCs.
Lay Abstract
In the project of ‘Vascular growth factor (VEGF) signaling in human neural stem cells’ supported
by the Connecticut (CT) Stem Cell Research Grants Program, the Dr. Jean-Leon Thomas team at
Yale discloses that dormant human neural stem cells (hNSC) can be activated by VEGF-C binding
to its receptor, VEGF receptor 3 (VEGFR3). This study supports previous publications showing
that VEGFR3/VEGF-C signaling can lead to the generation of new neurons from neural stem cells
in the specific parts of the mouse brain. In this hNSC study, hNSCs were produced using the H9
human embryonic stem cell (hESC) line and were characterized by expression of various NSC
markers. To demonstrate the effect of VEGF-C binding to VEGFR3, and the resulting activation
of this receptor, we assessed cell division rate and sequential down-stream pathways of
VEGFR3/VEGF-C signaling after VEGF-C treatment. In response to VEGFR3/VEGF-C
signaling, hNSC division rate was significantly increased. Additionally, the PI3K-AKT and ERK
signaling pathways, which modulate cell cycle progression and survival, were also significantly
increased.
Poster #67
Exploring the Basics
Mismatch repair-dependent damage response in human pluripotent stem cells
Bo Lin (1), Dipika Gupta (1), Christopher D. Heinen (1) *Equal Contributors
(1) Neag Comprehensive Cancer Ctr & Ctr for Molecular Medicine, Uconn Health Center
Technical Abstract
Individuals inheriting mutations in DNA Mismatch repair genes suffer from a colorectal cancer
predisposition disease, Lynch syndrome. Being the only pathway to remove mispaired bases
generated by DNA polymerase, MMR increases DNA replication fidelity and likely prevents
tumorigenesis. In addition, MMR proteins also elicit a cell cycle arrest and apoptosis in response
to DNA damage. Interestingly, MMR-proficient cancers are more sensitive than MMR-deficient
cells to alkylation based chemotherapeutic drugs. Therefore, understanding the basis of this MMRdependent DNA damage response may have implications for tumorigenesis and choice of
chemotherapeutic regimes. From studies in somatic cells, we know that MMR-proficient cells
undergo a permanent G2/M cell cycle arrest after going through two rounds of DNA replication
when exposed to alkylating agents. But, our studies of this pathway in human pluripotent stem cell
(PSC) models, have uncovered a novel and unexpected MMR-dependent DNA damage response.
PSCs express high levels of MMR proteins, have high repair activity and are hypersensitive to
alkylation damage. But in contrast to requiring two rounds of DNA replication, PSCs undergo
apoptosis in the first replication cycle after damage exposure. Furthermore, the activation of
Checkpoint Kinase proteins like CHK1 which help somatic cells cope with DNA damage during
the first S phase, is not observed in PSCs. We observed signs of replication stress like gamma
H2AX and ssDNA formation, followed by p53 activation and rapid apoptosis induction. This
suggests that PSCs when exposed to alkylating agents use a molecular circuitry by which the MMR
pathway generates an overwhelming amount of DNA damage, favoring elimination of damaged
cells. Understanding this response to DNA damage in PSCs will be important if these cells are to
be used therapeutically. Additionally, we may be able to exploit the lessons learned from these
studies for the prevention or treatment of tumors.
Lay Abstract
The most common hereditary disease that predisposes patients to colorectal cancer is Lynch
syndrome (LS) which stems from mutations in the mismatch repair (MMR) genes. MMR proteins
repair errors that accumulate in DNA to maintain genome stability, a function that likely underlies
its role in tumor prevention. However, MMR proteins also elicit a death response in cells that have
been exposed to various DNA damaging agents as a way of preserving their genomic integrity.
Thus, MMR mutations may affect tumorigenesis through multiple mechanisms. Interestingly, cells
with intact MMR systems are more sensitive to agents that damage DNA by alkylation than MMR
deficient cells. The MMR status of the tumors therefore becomes a major consideration in making
therapeutic decisions. MMR-proficient cancer cells when exposed to alkylating agents need to
replicate their DNA twice before they undergo a permanent cell cycle arrest, after which cells
eventually die. Surprisingly, our studies in human Pluripotent stem cells (PSC) show that these
cells are extremely sensitive to damage and die soon after exposure to these drugs. A closer look
indicated that these cells show increased signs of DNA damage and yet do not activate any of the
protective responses that are observed in cancer cells, like activation of checkpoint protein CHK1.
Instead they prefer to die just after one round of DNA replication. We therefore hypothesize that
by switching off their protective mechanisms, PSCs accumulate a large amount of DNA damage
that serves as a point of no return, favoring the rapid elimination of cells exposed to DNA
damaging agents. In doing so, they continuously maintain a pool of stem cells with high genomic
integrity. Understanding this response to DNA damage in PSCs will be important if these cells are
to be used therapeutically. Additionally, we may be able to exploit the lessons learned from these
studies for the prevention or treatment of tumors.
Poster #68
Exploring the Basics
The PIWI/piRNA pathway could maintain genomic integrity independent of its regulation
of transposable elements
Sneha Mani,Na Liu, Mei Zhong, Haifan Lin
(1) Yale University
Technical Abstract
A hallmark of PIWI/piRNA pathway function is its requirement for fertility. The current model
suggests that upregulation of transposons in piwi mutants causes activation of a DNA damage
checkpoint leading to fertility defects. Alternatively, it is possible that piwi mutations directly
affect genomic instability, independent of, or even leading to, increased transposon expression. In
this study, we aim to understand what causes checkpoint activation by studying the developmental
function of Argonaute3 (Ago3), a PIWI family protein. We first examined DNA damage in ago3
mutants by studying the expression and localization of γH2Av, a marker of unrepaired double
strand breaks (DSBs). An upregulation of γH2Av was observed in both germline and somatic cells
despite only ‘germline transposon’ upregulation. To examine genome-wide DNA damage at high
resolution, we devised a method to directly assess DSB formation. Preliminary data suggests that
DNA damage on loss of Ago3 is increased in both gene and transposon regions. Furthermore,
inactivation of the DNA damage checkpoint in ago3 mutants partially rescued oogenesis,
demonstrating an involvement of checkpoint activation in the observed defects. Checkpoint
activation however produced a non-canonical response. Interestingly, transposon levels are
reduced in checkpoint-inactivated ago3 mutants suggesting that transposon upregulation is
downstream of checkpoint activation. Finally, spatial and temporal examination of DNA damage
and transposon mRNA upregulation suggest that these two events might be uncoupled. These
observations represent the first detailed study of the relationship between DNA damage,
transposon silencing, and the PIWI/piRNA pathway and suggest that transposon upregulation may
not be the sole cause of compromised genome integrity in piwi mutants.
Lay Abstract
Our genomes are constantly exposed to insult - from internal sources such as the damaging
byproducts of metabolic processes or external sources such as UV light or chemical mutagens.
Maintaining genomic stability is key to ensuring appropriate function during the lifetime of an
organism. Loss of genomic stability leads to the development of disease such as cancer,
neurodegenerative diseases, immune deficiency, stem cell dysfunction, infertility, and ageing. This
is especially important in the egg and sperm cells (germline cells), where the genome has to be
protected for generational inheritance. The PIWI/piRNA pathway is an evolutionarily conserved
small RNA pathway that participates in the maintenance of genomic integrity and is essential
within the germline. It is commonly thought that this is accomplished through the regulation of
transposons - jumping genes that can cause harmful mutation when inserted into the genome.
However our work demonstrates that this pathway is required for broader roles in the maintenance
of genomic integrity since protection of the genome occurs independent of the regulation of
transposons.
Poster #69
Exploring the Basics
Embryonic stem cells license a high level of dormant origins to protect genome against
replication stress
Xin Quan Ge(1), Jinah Han(2), Ee-Chun Cheng(1), Satoru Yamaguchi(3), Naoko
Shima(3), Jean-Leon Thomas(2) and Haifan Lin(1)
(1) Yale Stem Cell Center and Department of Cell Biology, (2) Yale Cardiovascular
Research Center and Department of Internal Medicine, Yale University School of
Medicine, New Haven, CT 06520, (3) Department of Genetics, Cell Biology and
Development, University of Minnesota, Minneapolis, MN 55455
Technical Abstract
Maintaining genomic integrity during DNA replication is essential for stem cells. DNA replication
origins are licensed by the MCM2-7 complexes, with most of them remaining dormant. Dormant
origins rescue replication fork stalling in S phase and ensure genome integrity. However, it is not
known whether dormant origins exist and play important roles in any stem cell type. Here we show
that embryonic stem cells (ESCs) contain more dormant origins than tissue stem/progenitor cells
such as neural stem/progenitor cells (NSPCs). Partial depletion of dormant origins does not affect
ESC self-renewal but impairs their differentiation, including towards the neural lineage. However,
reduction of dormant origins in NSPCs impairs their self-renewal due to accumulation of DNA
damage and apoptosis. Furthermore, mice with reduced dormant origins show abnormal
neurogenesis and semi-embryonic lethality. Our results reveal that ESCs are equipped with more
dormant origins to better protect against replicative stress than tissue-specific stem/progenitor
cells.
Lay Abstract
For any higher living organism, such as human, life begins with one cell (zygote) formed by sperm
and oocyte. From one cell to an adult, life goes through millions and billions times of cell division.
Before a cell divides, it must make an identical copy of its DNA, so that each daughter cell has a
full complement of chromosomes. Mistakes made during the DNA replication can cause
irreversible genetic modifications that could ultimately lead to cells becoming cancerous.
Embryonic stem cells (ESCs) especially need to make sure that their DNA is accurately duplicated,
otherwise organisms cannot grow. Dormant replication origins (MCM2-7 protein complexes) play
an important role to ensure the accuracy of DNA replication. In addition, we found that embryonic
stem cells (ESCs) employ a higher number of dormant origins than tissue stem cells including
neural stem cells. Without a sufficient number of dormant origins, ESCs cannot grow into different
tissues and organs; mouse cannot develop properly and their brains are smaller. Overall, our
findings have demonstrated a unique mechanism ESCs employ to regulate the accuracy of DNA
replication and to ensure the genome stability. This forms the foundations for fundamental
understanding of stem cells and novel insights into stem cell based therapies.
Poster #70
Resources and Techniques
Next Generation High-throughput Sequencing at Genomics Core of Yale Stem Cell Center
Jason Thomson, Yinghong Ma, Caihong Qiu and Haifan Lin
Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New
Haven, CT 06520
Technical Abstract
The Yale hESC Core, founded with the support of the CT Stem Cell Initiative in 2007, is a key
laboratory in Yale Stem Cell Center to support investigators in Yale and CT for their research on
hESCs. Since then, the Core has fostered Yale hESC research in its state-of-the-art laboratory and
helped the expansion of hESC research from 1 to 36 research laboratories in 23 Yale departments.
We have trained 152 users from Yale and Wesleyan University on hESC culture and
manipulations. The Core distributed 208 plates of hESCs and 25 batches of hESC derivatives to
26 PIs at Yale. In addition, the Core members have strived to develop new cutting-edge
technologies to meet the pressing needs of stem cell researchers throughout the state. In 2010, a
highly efficient transfection of hESC with plasmids and siRNA to modify hESC gene expression
was developed and published in RNA, and we were invited to publish the detailed protocol in
Current Protocol For Stem Cell Biology. In 2012, the Core was expanded to offer services on iPSC
generation using non-integration approaches, characterization of iPSCs generated and training on
generating and culturing iPSCs. Recently, we developed human pluripotent gene knock-out (KO)
technology using CRISPR method and extended the technique to KO microRNAs. This CRISPR
gene editing platform is currently being offered as a service to all stem cell researchers in CT.
Lay Abstract
The Yale hESC Core, founded with the support of the CT Stem Cell Initiative in 2007, is a key
laboratory in Yale Stem Cell Center to support investigators in Yale and CT for their research on
human embryonic stem cells and induced pluripotent cells. Since then, the Core has fostered Yale
hESC research in its state-of-the-art laboratory and helped the expansion of hESC research from
1 to 36 research laboratories in 23 Yale departments. We have trained 152 users from Yale and
Wesleyan University on hESC culture and manipulations. The Core distributed 208 plates of
hESCs and 25 batches of hESC derivatives to 26 PIs at Yale. We have developed new cuttingedge technologies to meet the pressing needs of stem cell researchers throughout the state.
Poster #71
A Move Towards Therapies
Immune modulatory mesenchymal stem/stromal cells derived from human embryonic stem
cells through a trophoblast-like stage
Adam S Lazorchak (1), Li Song (1), Ren-He Xu (1,2), Xiaofang Wang (1)
(1) ImStem Biotechnology, Inc., Farmington, CT
(2) Faculty of Health Sciences, University of Macau, Macau, China
Technical Abstract
We have recently shown that mesenchymal stem/stromal cells derived from human embryonic
stem cells (hESC-MSC) consistently exhibit superior therapeutic efficacy in an animal model of
multiple sclerosis, when compared to human adult tissue derived MSCs. In order to successfully
translate hESC-MSC therapies to the clinic, improved methods to efficiently derive MSCs from
hESCs are required. Here, we present a new method to rapidly differentiate MSCs from hESCs
through a trophoblast-like cell intermediate stage. Through this method, a phenotypically
homogeneous population of MSCs (which we term T-MSC) may be derived from feeder-free
hESC in approximately 3 weeks with a minimum amount of cell culture manipulation. T-MSCs
derived by our method express a phenotype characteristic of MSCs, are capable of multipotent
differentiation to mesenchymal cell lineages and exhibit potent immunomodulatory activity in
vitro and in vivo. We show that T-MSC are capable of inhibiting mitogen dependent lymphocyte
proliferation in vitro and are capable of reducing the disease severity of two distinct mouse
models of inflammatory disease; experimental autoimmune encephalomyelitis and dextransodium sulfate induced colitis. These data demonstrate a simple and fast derivation method to
generate MSCs that possess potent immune-modulatory properties from feeder-free hESCs
which may serve as a novel and ideal candidate for MSC-based immunotherapy.
Lay Abstract
Chronic, auto-inflammatory diseases such as multiple sclerosis (MS) cause lifelong progressive
debilitation in patents and have, thus far, proven challenging to treat with current drug
technologies. Cellular immunotherapy, which involves administering living cells to the patient in
order to arrest or reverse the course of disease, offers the promise of treating or possibly curing
chronic inflammatory diseases. ImStem Biotechnology, Inc. (ImStem) is developing a first-inclass human stem cell derived mesenchymal stem cell (MSC) therapy to satisfy the unmet
medical need for an MS therapeutic that can 1) directly prevent immune mediated inflammation
in the central nervous system of MS patients and 2) promote the healing and repair of a central
nervous system damaged by MS. ImStem has developed a rapid and efficient method to generate
MSCs that possess anti-inflammatory, immune modulating and tissue regenerative properties
from an unlimited, pathogen free source of human embryonic stem cells or reprogramed stem
cells from adult tissue. The MSC immune therapy derived through our proprietary method shows
therapeutic efficacy in animal models of auto-inflammatory disease, including the mouse model
of experimental autoimmune encephalitis (EAE) which is a standard animal model of MS.
StemConn
4/27/2015
Marriott AB
Printed: 4/24/2015 8:35am
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