ARVO 2015 Annual Meeting Abstracts
331 Stem Cells In Development and Disease
Tuesday, May 05, 2015 11:00 AM–12:45 PM
2B/3B Mile High Blrm Paper Session
Program #/Board # Range: 3169–3175
Organizing Section: Retinal Cell Biology
Program Number: 3169
Presentation Time: 11:00 AM–11:15 AM
Generation of retinal tissues from human embryonic stem cells
(hESCs) and induced pluripotent stem cells (hiPSCs) through
self-assisted progenitor cell sorting
Wei Liu, Albert Lowe, Raven Harris, Punita Bhansali, Ales Cvekl.
Ophthalmology and Visual Sciences, Albert Einstein College of Med,
Bronx, NY.
Purpose: This study aims to model human retinal development
by employing human pluripotent stem cells such as human H1
embryonic stem cells (hESCs) and human induced pluripotent stem
cells (hiPSCs).
Methods: H1 hESCs or hiPSCs were cultured on Matrigel-coated
plates in DMEM/F12 medium supplemented with N2 and B27,
0.5% bovine serum albumin, 1mM L-glutamine, 1% penicillinstreptomycin, 100 ng/ml of bFGF (Yao et al., 2006) or in mTeSR
medium. H1 hESCs or hiPSCs were passaged every 5 to 7 days
with laser-enabled analysis and processing (LEAP; Cyntellect),
with Dispase, or ReLeSR™. Retinal progenitor cells (RPCs) were
generated using a modified Matrigel-based method.
Results: In retinal cell differentiation from hESCs or hiPSCs
in vitro, the cell cultures are frequently a mixture of randomly
differentiated many cell types, and thus cell differentiation is in a
disorganized manner in contrast to highly regulated morphogenesis
in vivo. A key concept in organoid culture is to isolate substantial
pure progenitor cells for prolong cultures. Although genetically
engineered fluorescence markers are valuable tools for monitoring
and isolating specific type of cells, identification and characterization
of the intrinsic progenitor cell properties that can be used for cell
isolation are likely to be more relevant to clinical applications.
Here we report the selection and isolation of hPSC-derived retinal
progenitor cells (RPCs) based on their intrinsic properties. These
RPCs were self-sorted into spheres that are readily detectable under
an inverted microscope. Histological and molecular characterizations
of the spheres demonstrate that these cells constituted a polarized
epithelium and highly expressed RPC markers SIX3, RAX, PAX6
and VSX2. Long-term cultures of these RPCs produced laminar
tissues comparable to human retina.
Conclusions: This novel long-term organoid culture system does not
require any genome engineering and generates retina-like laminar
structures comprised of differentiated retinal cells. Ongoing studies
aim to evaluate this system in modeling human retinal development
and disease pathogenesis, drug discovery, and cell replacement
therapy.
Figure 1. Generation of retinal tissues in H1 hESC cultures.
Immunostaining of RECOCERIN (green), OTX2 (red) and nuclear
counterstaining with DAPI (Blue) for a retinal tissue on day 152.
Commercial Relationships: Wei Liu, None; Albert Lowe, None;
Raven Harris, None; Punita Bhansali, None; Ales Cvekl, None
Support: American Health Assistance Foundation M2012044 to
W.L.; NIH grant 1R01EY022645 to W.L.; RPB unrestricted grant;
Joseph Alexander Foundation; NIH grant R01EY012200 and
R01EY014237 to A.C.
Program Number: 3170
Presentation Time: 11:15 AM–11:30 AM
cGMP production of neural retina from hiPSCs
David M. Gamm1, 2, Neehar Bhatia3, Anna Petelinsek2, Jee Min2,
Elizabeth E. Capowski2, Travis Cordie3, Diana Drier3, Connor
Lyons3, Derek Hei3, Joe Phillips2. 1Ophthalmology and Visual
Sciences, Univ of Wisconsin-Madison, Madison, WI; 2Waisman
Center, University of Wisconsin-Madison, Madison, WI; 3Waisman
Biomanufacturing, University of Wisconsin-Madison, Madison, WI.
Purpose: Clinical trials involving human ESC and iPSC-derived
RPE transplantation are well underway. However, in later stages of
most outer retinal degenerative diseases, photoreceptor replacement
alone or in combination with RPE must be considered. The purpose
of this study is to convert an established hiPSC differentiation method
for deriving neural retina (including photoreceptor precursors) to
current Good Manufacturing Practices (cGMP).
Methods: All hiPSC lines investigated were created using an
integration-free reprogramming method. Some hiPSC lines, including
those derived from homozygous HLA “super donors,” were banked
under cGMP conditions. hiPSCs were then thawed and cultured
using multiple cGMP-compliant methods and differentiated to optic
vesicle-like structures (OVs) using our previously established 3-D
protocol. ICC analysis was performed to confirm neural retinal
progenitor cell (iNRPC) and photoreceptor precursor (iPRP) cell
production.
Results: Each hiPSC maintenance platform produced high,
reproducible yields of OVs following retinal differentiation, with a
single 6 well plate of hiPSCs giving rise to up to 400 VSX2+ OVs
containing up to 24 million iNRPCs at early stages of differentiation.
By day 60 of differentiation, >98% of OVs contained iPRPs as
demonstrated by ICC (CRX+/RCVRN+). Cells within the OVs had
>95% viability following gentle dissociation.
Conclusions: This study demonstrates robust production of hiPSCderived neural retina across multiple cGMP-compliant culturing
platforms. In our hands, E8/vitronectin is the most ideal hiPSC
maintenance medium/substrate. Current studies are focused on full
characterization of super donor hiPSC lines and their neural retinal
derivatives and optimizing cryopreservation methods.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
Commercial Relationships: David M. Gamm, Cellular Dynamics
International (C); Neehar Bhatia, None; Anna Petelinsek, None;
Jee Min, None; Elizabeth E. Capowski, None; Travis Cordie,
None; Diana Drier, None; Connor Lyons, None; Derek Hei, None;
Joe Phillips, None
Support: Foundation Fighting Blindness Wynn-Gund TRAP
Award, NIH Product Assistance for Cellular Therapies, Retinal
Research Foundation Emmett A. Humble Distinguished Directorship,
McPherson Eye Research Institute (Sandra Lemke Trout Chair), Carl
and Mildred Reeves Foundation, NIH P30HD03352, Muskingum
County Community Foundation, Choroideremia Research Foundation
Program Number: 3171
Presentation Time: 11:30 AM–11:45 AM
The RPE as stem-like cells for retinal regeneration in adult
mouse
Shu-Zhen Wang, Li He, Run-Tao Yan. Department of Ophthalmology,
University of Alabama at Birmingham, Birmingham, AL.
Purpose: Regenerating neurons and neural connection has been
selected as NEI Audacious Goal. Our laboratory investigates the
possibility of using a regulatory gene, ngn1 or ngn3, to reprogram the
RPE for retinal regeneration in the mouse eye. In previous studies we
found ectopic retinal tissue/cells in some PVMD2-ngn1 and PRPE65-ngn3
transgenic mice. This study examines whether (1) the RPE in adult
mouse could undergo the gene-directed reprogramming, (2) the RPE,
which plays a crucial role in the function and health of the retina,
was preserved in those transgenic animals, and (3) Müller glia was
involved.
Methods: Cryosections of eyes from adult transgenic mice with
photoreceptor-like cells in the subretinal space were immunostained
with antibodies that recognize RPE proteins, photoreceptor protein
Recoverin (Rcv), or Müller glia. The presence of a hybrid or
mixed cell type, i.e., a cell co-expressing markers that otherwise
are exclusive for either RPE or photoreceptor cells, was used as an
indication of the cell amid RPE-to-photoreceptor reprogramming
process.
Results: At places with Rcv+ cells in the subretinal space, the
highly melanized RPE was present and was immuno-positive
for RPE proteins: CRALBP, Cytokeratin-18, Ezrin, and RPE65.
Ezrin and RPE65 were also detected in some of the ectopic Rcv+
cells, especially in those containing some pigment granules, which
otherwise are abundantly present in normal RPE cells. Otx2, a
transcriptional factor known to be important for RPE differentiation
and maintenance of RPE properties and to be down-regulated during
the RPE-to-retina transdifferentiation observed in amphibians and
chicken, was detected in the RPE at most places and not in regions
where RPE was seemingly undergoing RPE-to-photoreceptor
reprogramming. Some cells within the territory occupied by the
subretinal Rcv+ cells were immuno-positive for Müller glia makers
GS and CRALBP, and a few Rcv+/GS+ double-labeled cells were
observed.
Conclusions: The results suggest that the RPE in adult mice can be
guided by ngn1 or ngn3 to produce retinal cells, including Müller
glia, and can regenerate itself afterward. This raises a possibility
using the RPE as stem-like cells for retinal regeneration in adult
mammals.
Commercial Relationships: Shu-Zhen Wang, None; Li He, None;
Run-Tao Yan, None
Support: NIH/NEI grant EY011640, Research to Prevent Blindness,
and NIH/NEI core grant P30 EY003039
Program Number: 3172
Presentation Time: 11:45 AM–12:00 PM
Fabrication and characterization of tissue-engineered multilayer
outer retinal grafts
Kristan S. Worthington, Emily Kaalberg, Robert F. Mullins, Edwin M.
Stone, Budd Tucker. Ophthalmology and Visual Sciences, University
of Iowa, Iowa City, IA.
Purpose: As AMD associated photoreceptor cell death is typically
preceded by loss of cells of the underlying retinal pigment epithelium
(RPE) and choroidal vasculature, an effective restorative treatment
strategy will likely require transplantation of multi layered cellular
grafts. Recent data suggests that autologous induced pluripotent stem
cells may be the optimal cell source for such application. However,
current bolus cell delivery methods typically result in significant
cell loss (as much as 99%) and limited cellular integration following
transplantation. The purpose of this study is to overcome these
limitations by combining state of the art stem cell and biodegradable
tissue engineering technologies to fabricate multi-layered outer
retinal grafts.
Methods: Cross-linked methacrylate-functionalized tropoelastin and
chitosan served as biomaterial substrates for the delivery of RPE and
photoreceptor precursor cells, respectively. Chemical reactions were
confirmed by NMR. The mechanical properties of the constructs were
measured using dynamic mechanical analysis while cellular survival,
identity, morphology and directionality were characterized using rtPCR, Western blot, and immunocytochemical approaches.
Results: Tropoelastin and glycol chitosan were both successfully
modified with methacrylate groups by reaction with methacrylic
anhydride and AOHPMA, respectively. Tropoelastin based Bruch’s
Membrane mimics were fabricated using photo-crosslinking with
365nm light, while chitosan hydrogel based outer nuclear layer
mimics were fabricated with 650nm light. The mechanical properties
of materials were tuned to match the compressive modulus of native
retinal tissue by adjusting the polymer cross-linking density. RPE and
photoreceptor precursor cells survived on their respective materials
and exhibited lineage-specific markers.
Conclusions: These results lay the foundation for testing of
autologous cell/polymer-based therapeutic modalities in vivo in
animal models of retinal degenerative diseases. The constructs
developed can be used as transplantation grafts or as closer in
vitro approximations to the in vivo retinal environment than those
offered by traditional cell culture methods. Further, the inclusion of
additional layers in future studies, such as a 3D-printed choroid-like
structure, could result in a powerful regenerative graft for a variety of
retinal degenerative diseases of the outer retina.
Commercial Relationships: Kristan S. Worthington, None; Emily
Kaalberg, None; Robert F. Mullins, None; Edwin M. Stone, None;
Budd Tucker, None
Program Number: 3173
Presentation Time: 12:00 PM–12:15 PM
Characterization of Human Stem Cells Generated from AMD
Patients
Lucian V. Del Priore, Jie Gong, Ernesto F. Moreira, Hannah E.
Bowrey, Zsolt Ablonczy, Mark Fields. Ophthalmology, Storm Eye
Institute, Charleston, SC.
Purpose: Retinal pigmented epithelium (RPE) derived from human
induced pluripotent stem (iPS) cells is a promising source of cells
for the replacement of degenerated RPE in patients with age-related
macular degeneration (AMD), as use of patient-specific iPS will
avoid graft rejection. Here we demonstrate that iPS can be derived
from fibroblasts obtained from older patients with AMD, and that
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
these cells can be used to generate autologous RPE despite advanced
patient age and the presence of severe AMD in the donors.
Methods: Patients with advanced AMD exhibiting either geographic
atrophy or exudative AMD were recruited. Fibroblasts obtained from
skin biopsies were grown to confluence (dry AMD n=8, wet AMD
n=7, age-matched control n=5) in DMEM containing 10% Fetal
Bovine Serum (FBS). Supernatant was collected 24 hours later and
the VEGF levels were measured by ELISA. iPS were generated from
these fibroblasts using Sendai virus reprogramming and functional
RPE tests were performed.
Results: Fibroblasts were obtained successfully from over 20 patients
with advanced AMD and 5 age-matched controls. VEGF secretion
from these fibroblasts was as follows: dry AMD 185 + 60 pg/ml,
wet AMD 118 + 38 pg/ml, aged control 230 + 191 pg/ml. VEGF
secretion by both dry and wet AMD patients were significantly lower
than aged-matched controls (dry AMD vs aged control P < 0.0001,
wet AMD vs aged control p=0.02). VEGF secretion in wet AMD
patients was significantly lower than dry AMD patient (P=0.0002).
Patient-specific iPS have been generated from 6 of these samples, and
5 RPE cell lines have been generated. iPS cells were all positive for
stem cell markers including SSEA4, TRA-1-60, OCT4, and TRA1-8. Cells differentiated towards an RPE fate formed a hexagonal
monolayer within 30 days with a transepithelial resistance of 154.8
+ 0.23 Ω*cm2. Monolayers were uniformly positive for Bestrophin,
MITF, RPE65 and ZO-1. iPS-derived RPE were able to ingest
outer segments, and this phagocytic ability was blocked by specific
addition of avB5 integrin antibodies.
Conclusions: Patient-specific fibroblasts have been reprogrammed
into iPS from AMD patients up to age 80 with no decline is this
ability with advancing patient age. Interestingly, supernatant VEGF
levels were lower in fibroblasts from AMD patients compared to agematched controls. iPS can be differentiated towards RPE with cells
expressing RPE markers and performing phagocytic functions.
Commercial Relationships: Lucian V. Del Priore, None; Jie Gong,
None; Ernesto F. Moreira, None; Hannah E. Bowrey, None; Zsolt
Ablonczy, None; Mark Fields, None
Support: Foundation Fighting Blindness
Program Number: 3174
Presentation Time: 12:15 PM–12:30 PM
Towards the identification of deep-intronic ABCA4 mutations in
Stargardt patients by using induced pluripotent stem cell-derived
photoreceptor progenitor cells
Silvia Albert1, Riccardo Sangermano1, Nathalie Bax3, Susanne
Roosing4, L. I. van den Born2, Anke den Engelsman-van Dijk1,
Angelique Ramlal1, Edwin M. Stone5, 6, Carel C. Hoyng3, Frans
Cremers1, 3. 1Human Genetics, Radboud Universit Medical Centre,
Nijmegen, Netherlands; 2The Rotterdam Eye Hospital, Rotterdam,
Netherlands; 3Ophthalmology, Radboud University Medical Center,
Nijmegen, Netherlands; 4Pediatric Brain Diseases, The Rockefeller
University, New York, NY; 5Ophthalmology and Visual Sciences,
University of Iowa Carver College of Medicine, Iowa, IA; 6Howard
Hughes Medical Institute, Iowa, IA.
Purpose: We previously found that ~40% of persons with autosomal
recessive Stargardt disease (STGD1) carry only one, and ~10% no
ABCA4 mutations. We hypothesized that the lacking mutations are
either heterozygous deletions not detected by exon PCR and Sanger
sequencing, or located deep in the introns, most likely affecting
ABCA4 mRNA. We have shown previously that ABCA4 mRNA is
expressed at very low levels in lymphoblasts and fibroblasts but
robustly in photoreceptor cells that have differentiated from human
stem cells. We propose to perform sequence analysis of ABCA4
mRNA isolated from photoreceptor progenitor cells (PPCs) that
were differentiated from reprogrammed patient-specific induced
pluripotent stem cells (iPSCs).
Methods: We performed Sanger sequencing of the ABCA4 promoter
region in a cohort of 45 patients with STGD1 or STGD1 with conerod dystrophy phenotypes carrying one ABCA4 mutation, multiplex
ligation-dependent probe amplification (MLPA) of the 50 ABCA4
exons in 27 cases of the cohort, and Sanger sequencing of 20 weak
deep-intronic splice sites in 45 cases. Skin biopsy fibroblasts were
cultured from eight STGD1 patients and reprogrammed into iPSCs.
iPSCs were differentiated into PPCs, and the presence of the ABCA4
transcript was evaluated by quantitative RT-PCR (qRT-PCR) and
immunocytochemistry (ICC). The ABCA4 transcript was also
analysed by RT-PCR and Sanger sequencing to identify insertions or
deletions.
Results: MLPA and Sanger sequencing revealed heterozygous
deletions in two maculopathy families, deep-intronic variants in
six families, and a combination of a heterozygous deletion and a
deep-intronic variant in one family. Patient-derived fibroblasts were
successfully reprogrammed into iPSCs, and differentiated into PPCs,
in seven and five probands, respectively. qRT-PCR and ICC of the
cultured cells confirmed their differentiation into PPCs. The ABCA4
transcripts produced revealed the presence of several abnormal cDNA
fragments.
Conclusions: Heterozygous deletions and deep-intronic variants
were identified in nine of 45 (20%) maculopathy families illustrating
that deep-intronic variants are a significant cause of macular disease.
To identify additional deep-intronic variants, we successfully
obtained cultures of PPCs that strongly expressed ABCA4 after four
weeks of differentiation.
Commercial Relationships: Silvia Albert, None; Riccardo
Sangermano, None; Nathalie Bax, None; Susanne Roosing, None;
L. I. van den Born, None; Anke den Engelsman-van Dijk, None;
Angelique Ramlal, None; Edwin M. Stone, None; Carel C. Hoyng,
None; Frans Cremers, None
Program Number: 3175
Presentation Time: 12:30 PM–12:45 PM
Human LCA16 iPSC-RPE recapitulates the phenotype associated
with the nonsense mutation c.158 G>A (p.W53X)
Bikash R. Pattnaik1, 2, Pawan Shahi3, 2, Katarzyna Borys4, 2, Dalton
Hermans3, Nathaniel York3, 2, Elizabeth E. Capowski4, 2, Simran
Brar3, De-Ann M. Pillers3, 2, David M. Gamm4, 2. 1Pediatrics Ophthal
& Visual Sci, Univ of Wisconsin, Madison, WI; 2McPherson
Eye Research Institute UW-Madison, Madison, WI; 3Pediatrics,
University of Wisconsin, Madison, WI; 4Waisman Center, University
of Wisconsin, Madison, WI.
Purpose: Mutations in the KCNJ13 gene lead to a dysfunctional
inwardly-rectifying potassium channel, Kir7.1, and result in one
variant of Lebers Congenital Amaurosis (LCA16). Kir7.1 acts in the
RPE apical processes to control the subretinal space environment
which affects vision. We sought to determine the ability of using
human iPSC-RPE to recapitulate aspects of the LCA16 phenotype
associated with the nonsense mutation c.158 G>A (p.W53X) in vitro.
Methods: Skin biopsy (5mm punch) was obtained from a male
LCA16 patient with a confirmed W53X mutation (proband) and from
a healthy male after informed consent. The study was approved by
UW-Madison Institutional Review Board. Fibroblast cells were used
to generate iPSCs and were differentiated to RPE cells in culture
according to described methods. Pigmented and hexagonal colonies
were purified and used for propagating iPSC-RPE cells maintained
in 6 mm inserts for 6-8 weeks with media replacement twice weekly.
We used sequencing to confirm the presence of the LCA16 mutation.
Immunocytochemistry was performed to determine the expression
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2015 Annual Meeting Abstracts
of RPE-specific proteins. We performed whole-cell patch clamp
to register Kir7.1 current from iPSC-RPE cell lines. Results were
compared with current recordings using heterologous expression of
wild-type and mutant proteins in CHO-K1 cells.
Results: iPSC-RPE cells appeared pigmented with characteristic
cobblestone morphology. Both the proband and the sibling control
cell lines expressed the RPE-specific markers Mitf and ZO-1 protein
and developed a resistance of more than 800 MOhm. A truncated
Kir7.1 protein was detected in LCA16 cells by Western blot analysis.
Control cells measured Kir7.1 current that reversed at -53 mV
compared to a depolarized membrane potential of -30 mV for LCA16
cells. Rb+ generated about 9-fold increase in inward current measured
at -150 mV potential in control cells, but had no effect on the LCA16
cells.
Conclusions: The Kir7.1 channel controls both potassium recycling
around the resting membrane potential as well as a hyperpolarized
membrane potential, and both were evident in control cells.
TheLCA16 mutation W53X resulted in a dysfunctional truncated
protein that depolarized the membrane potential. Using patientderived iPSC-RPE cells, we have successfully reproduced a
measurable aspect of the Kir7.1 phenotype which gives us the
potential to safely test novel therapies for LCA16 in vitro.
Commercial Relationships: Bikash R. Pattnaik, None; Pawan
Shahi, None; Katarzyna Borys, None; Dalton Hermans, None;
Nathaniel York, None; Elizabeth E. Capowski, None; Simran
Brar, None; De-Ann M. Pillers, None; David M. Gamm, None
Support: UW-Foundation
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.