ARVO 2015 Annual Meeting Abstracts
372 Stem cell biology
Tuesday, May 05, 2015 3:45 PM–5:30 PM
Exhibit Hall Poster Session
Program #/Board # Range: 3572–3588/A0001–A0017
Organizing Section: Retinal Cell Biology
Program Number: 3572 Poster Board Number: A0001
Presentation Time: 3:45 PM–5:30 PM
Generation of neuroretina from induced pluripotent stem cells
and effects of Sonic Hedgehog
Camille Yvon, Conor Ramsden, Amanda-Jayne F. Carr, Matthew
Smart, Michael Powner, Amelia Lane, Britta Nommitse, Lyndon da
Cruz, Pete Coffey. Institute of Ophthalmology, UCL, London, United
Kingdom.
Purpose: Induced pluripotent stem cells (iPSCs) have the potential
to provide robust models for the study of human retinogenesis and
treatment therapies for retinal diseases. iPSC differentiation into
neural retina (NR) can be driven in vitro by lineage-determining
transcription factors. However, there exists a great source of
variability between published protocols. We sought to determine if
iPSCs could generate optic vesicle like structures or NR, by varying
culture conditions of Matrigel™ and Sonic Hedgehog (Shh).
Methods: We compared culture conditions for NR differentiation
using two batches of Matrigel™ (M1 and M2), and variable
concentrations of Shh trapped in the Matrigel coating the
plate. Early changes in retinal development were monitored by
immunocytochemistry and quantitative polymerase chain reaction.
Results: We report the generation of optic vesicle and cup
structures from iPSCs, using our modified protocol. It was found
that embryoid bodies cultured in M2 showed higher expression
of retinal development association transcription factors such as
Pax6, Rax, Otx2 and Chx10 compared to M1. Embryoid bodies
cultured in elevated Shh concentrations induced a greater percentage
of organized structures and increased expression of eye field
transcription factors.
Conclusions: Our modified protocol using Shh delivered via plate
coating provides practical and efficient ways of generating NR from
iPSCs, thus helping to optimise the differentiation protocol.
Commercial Relationships: Camille Yvon, None; Conor Ramsden,
None; Amanda-Jayne F. Carr, None; Matthew Smart, None;
Michael Powner, None; Amelia Lane, None; Britta Nommitse,
None; Lyndon da Cruz, None; Pete Coffey, None
Program Number: 3573 Poster Board Number: A0002
Presentation Time: 3:45 PM–5:30 PM
WNT signaling during patterning of human iPSC-derived optic
vesicle-like structures
Anna Petelinsek1, Lynda S. Wright1, Elizabeth E. Capowski1, Jessica
Lien1, Isabel Pinilla Lozano2, 5, Jishnu Saha1, Joe Phillips1, David
M. Gamm3, 4. 1University of Wisconsin-Madison, Madison, WI;
2
Ophthalmology, University Hospital Lozano Blesa, Zaragoza, Spain;
3
Department of Ophthalmology and Visual Sciences, University
of Wisconsin-Madison, Madison, WI; 4McPherson Eye Research
Institute, Madison, WI; 5Aragon Health Sciences Institute, IIS
Aragon, Zaragoza, Spain.
Purpose: VSX2, a homeodomain transcription factor expressed in
neural retina progenitor cells (NRPCs), has a prominent role in optic
vesicle (OV) patterning. Recently, we used hiPSCs to demonstrate
that an R200Q mutation in the DNA binding region of VSX2 caused
a partial cell fate switch from NR to RPE. RNAseq data comparing
NR from wild type (WT) and (R200Q)VSX2 hiPSCs suggested that
the WNT pathway was altered in VSX2 mutant OVs. To further
investigate this finding, we examined the spatiotemporal expression
of MITF, VSX2, and canonical WNT signaling proteins during
hiPSC-OV patterning.
Methods: hiPSC-OVs were generated from an individual bearing
homozygous R200Q mutations in VSX2 and an unaffected sibling
using our established methods. We performed ICC to localize
expression of VSX2, MITF, and the WNT markers b-catenin, WLS,
and LEF1 at days 14, 18, 35 and 50 of differentiation.
Results: Retinal differentiation of WT hiPSCs resulted in induction
of a MITF+ population of OV cells at d14, a subset of which initially
coexpressed VSX2. Also at d14, expression of the WNT markers
WLS and LEF1, along with nuclear localization of the canonical
WNT pathway effector b-catenin, were detected in MITF+ cells
but seldom in VSX2+ cells. By d18 of differentiation, WT OV
cells exclusively expressed either MITF or VSX2, with the latter
developing as NR and the former adopting an RPE fate. However,
when (R200Q)VSX2 hiPSCs were differentiated toward a retinal
fate, VSX2 and MITF remained coexpressed as late as d18. Nuclear
localization of b-catenin was robustly observed in VSX2+/MITF+
cells at d14 in (R200Q)VSX2 OV cells, and WLS and LEF1
displayed aberrant coexpression with VSX2 as well. However,
once NR and RPE were established, differences in canonical WNT
signaling were not detected between WT and mutant. WLS and LEF1
were observed in maturing RPE but not NR in both WT and mutant
OVs, and nuclear localization of b-catenin was absent in both by d35.
Conclusions: We demonstrated that canonical WNT signaling
proteins colocalized with MITF+ OV and RPE cells during early
hiPSC-OV patterning, but were downregulated in NRPCs following
expression of WT VSX2. Early NRPCs expressing mutant (R200Q)
VSX2 displayed prolonged expression of MITF and WNT markers,
along with nuclear localization of b-catenin. This data suggests that
VSX2 DNA binding activity is associated with downregulation of
canonical WNT signaling during NRPC specification.
Commercial Relationships: Anna Petelinsek, None; Lynda S.
Wright, None; Elizabeth E. Capowski, None; Jessica Lien, None;
Isabel Pinilla Lozano, None; Jishnu Saha, None; Joe Phillips,
None; David M. Gamm, Cellular Dynamics International (S)
Support: This work was supported by National Institutes of Health
grant numbers R01 EY21218, P30 HD03352; the Foundation
Fighting Blindness Wynn-Gund Translation Research Award; the
Retina Research Foundation (Kathryn and Latimer Murfee and
Emmett A Humble Chairs); the Choroideremia Research Foundation;
and the McPherson Eye Research Institute (Sandra Lemke Trout
Chair in Eye Research).
Program Number: 3574 Poster Board Number: A0003
Presentation Time: 3:45 PM–5:30 PM
Neural Potential of Human Limbal Cells
Xiaoli Chen1, Pawez Hossain1, 2, Helen Griffiths1, Jennifer
Scott1, Andrew J. Lotery1, 2. 1Faculty of Medicine, University of
Southampton, Southampton, United Kingdom; 2Eye Unit, University
Hospital Southampton NHS Foundation Trust, Southampton, United
Kingdom.
Purpose: Previous studies have shown that neural colonies
(neurospheres) derived from adult mouse limbus are neural crest
originated stem/progenitor cells, and can differentiate into functional
neurons and/or cells expressing retinal specific markers in vitro. The
aim of this study is to investigate whether limbal neurosphere cells
(LNS) can be derived from aged human limbus, and differentiate into
retinal like cells in vitro and in vivo.
Methods: Human limbal tissues used in this study were from two
resources: (1) aged donor eyes from Bristol Eye Bank (age ranged
from 72-97), and (2) healthy limbal tissue from live patients who
underwent conjunctival surgery (size 1 mm2, age ranged from 34-
©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
85). Human limbus cells were isolated and cultured in the presence
of mitogens. Following co-culture with developing retinal cells or
in the presence of extrinsic factors, LNS and their progeny were
characterized using immunocytochemistry and/or RT-PCR. Enhanced
green fluorescent protein-tagged LNS were transplanted into the
subretinal space of neonatal mice. The potential for limbal cells to
differentiate into retinal like cells and integrate into the host retina
was assessed by immunohistochemistry after 2-5 weeks.
Results: Human LNS were successfully generated from aged donor
limbal tissues through a serum free sphere forming assay. Human
LNS expressed neural stem cell markers, including Sox2 (31.2 ±
10.2%) and Nestin (34.8 ± 2.2%). For the superficial limbal tissues (1
mm2) obtained from live patients, no apparent LNS were generated,
but cells expressing Nestin (3-5%) and early differentiated neuronal
marker beta-III tubulin (15-20%) can be grown through explant
culture in the presence of serum and mitogens. Following co-culture
with developing retinal cells or in presence of extrinsic factors,
low levels of retinal progenitor markers, such as Lhx2, Pax6 and
Rx were detected in human LNS at the transcription level. Mature
photoreceptor specific markers were not observed in human LNS
either in vitro or in vivo.
Conclusions: Here we demonstrate that cells with neural potential
can be derived from aged human limbal tissue or 1 mm2 of superficial
limbal tissues from adult patients. Other approaches are needed to
promote human limbal cells transdifferentiation into retinal lineage.
However, their surgical accessibility and presence in aged individuals
make them an attractive cell resource for autologous cell rescue of
degenerative retinal diseases.
Commercial Relationships: Xiaoli Chen, None; Pawez Hossain,
None; Helen Griffiths, None; Jennifer Scott, None; Andrew J.
Lotery, None
Support: National Eye Research Centre (NERC), Rosetrees Trust,
T.F.C. Frost Charity and the Gift of Sight Appeal.
Program Number: 3575 Poster Board Number: A0004
Presentation Time: 3:45 PM–5:30 PM
In vitro modeling of human retinogenesis with pluripotent stem
cells
Akshayalakshmi Sridhar1, Sarah Ohlemacher1, Jason S. Meyer1,
2 1
. Biology, Indiana Univ Purdue Univ Indianapolis, Indianapolis,
IN; 2Stark Neurosciences Research Institute, Indiana University,
Indianapolis, IN.
Purpose: Human pluripotent stem cells (hPSCs) provide a unique
ability to study some of the earliest events of human development,
particularly some of the earliest events in human retinogenesis
such as the establishment of a definitive retinal fate from a more
primitive neural progenitor source. In this role, hPSCs may provide
an in vitro model for understanding the complex interplay of
transcription factors involved in the acquisition of a retinal fate from
an unspecified pluripotent cell population.
Methods: hPSCs were differentiated as previously described
and samples were collected every two days, starting from the
undifferentiated state through when cells acquired either retinal or
non-retinal forebrain identities. Immunocytochemistry and qRTPCR approaches were undertaken to identify candidate transcription
involved in retinal fate establishment. Lentiviral-mediated
overexpression and shRNA knockdown approaches assessed the role
of candidate transcription factors in the specification of a retinal fate
apart from other non-retinal neural fates. Furthermore, epigenetic
approaches assessed the role of DNA methylation in retinal and
forebrain fate determination.
Results: Candidate transcription factors were identified underlying
the establishment of a retinal fate apart from other neural lineages.
Neural transcription factors including PAX6 and OTX2 were
expressed early while retinal-associated transcription factors
such as SIX6 were expressed at slightly later timepoints. Upon
establishment of an anterior neural identity, expression patterns of
certain transcription factors such as RAX became more restricted
to subpopulations of cells, indicating the emergence of retinal
and forebrain populations from the same primitive anterior neural
population. Gene overexpression and knockdown experiments
investigated the mechanism of action of these candidate transcription
factors. Furthermore, epigenetic analysis demonstrated that DNA
methylation could potentially account for differential gene expression
in the establishment of retinal phenotypes apart from alternate neural
lineages.
Conclusions: Preliminary results begin to elucidate the complex
interplay of transcription factors involved in the specification of a
retinal fate from differentiating hPSCs. Overall, these results will
help to better establish hPSCs as a valuable in vitro system with
which to study some of the earliest events of human retinogenesis.
Commercial Relationships: Akshayalakshmi Sridhar, None;
Sarah Ohlemacher, None; Jason S. Meyer, None
Support: NH Grant R01 EY024984-01, BrightFocus G2012027
Program Number: 3576 Poster Board Number: A0005
Presentation Time: 3:45 PM–5:30 PM
Characterizing Sox2+cell in the adult mouse optic nerve lamina
Yan Guo, Zara Mehrabyan, Steven L. Bernstein. Ophthalmology,
Univ of Maryland Sch of Medicine, Baltimore, MD.
Purpose: The optic nerve lamina (ONL) is a unique optic nerve
structure bordering the retina and optic nerve. The ONL has a rich
vasculature supply compared with rest of the optic nerve, and plays
an important role in many optic nerve diseases. We recently found
that there are abundant Sox2+ cells in the adult lamina. Sox2 is a
nuclear transcription factor, essential for the pluripotency of adult
neural stem cells (NSC) in central nervous system (CNS). We
wanted to characterize these Sox2+ cells in order to have a better
understanding of this unique region.
Methods: 6 wild type mice (C57BL/6J) at age postnatal day
30-36 were utilized in the study. Mice were perfused with 4%
paraformaldehyde. The optic laminae with approximately 0.5mm
optic nerve were dissected and post fixed in PFA over night, and
transferred to PBS, then 30% sucrose, and embedded in OCT
and quickly frozen in dry ice. Ten micron thick frozen sections
were prepared for immunohistochemical analysis. We performed
immunohistochemistry using antibodies to Sox2, Nestin, GFAP,
Ki67, Ng2, NeuN, Laminin. Slides were examined on an Olympus
900 laser confocal microscope.
Results: Sox2+ cells are abundant in the ONL and fewer in the
distal ON. In the retina, Sox2+ cells are located in the inner nuclear
and ganglion cell layers. Sox2+ cells in ONL and ON are strongly
associated with GFAP (glial astrocyte and neural stem cell marker),
as well as nestin (neural stem cell marker). Some Sox2+ cells in the
ONL are Ki67 positive. The Sox2 expressing cells in the ONL and
their association with the other cell markers are very similar to those
seen in the CNS subgranular zone (SGZ) in mice of same age. No
association of Sox2+ and iba1 (macrophage marker) was seen, nor
were Ng2 (oligodendrocyte precursor) or laminin (blood vessel). In
the retina, Sox2+ cells are not clearly associated with GFAP, or nestin.
They do not co-localize with NeuN (neuron marker) or Ki67.
Conclusions: Sox2+ cells are abundantly expressed in the adult
mouse ONL. Their expression pattern and their associations with
other stem cell markers such as nestin, and mitotic markers such as
KI67, are similar to what have seen in SGZ. Sox2+ cells in the retina
do not associate with GFAP, implying that these cells may be of a
©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
different type. Collectively, these findings suggest that similar to the
pluripotent neural stem cells seen in the SGZ, ONL Sox2+ cells may
possess the ability to develop into other cell types.
Commercial Relationships: Yan Guo, None; Zara Mehrabyan,
None; Steven L. Bernstein, None
Support: EY-015304
Program Number: 3577 Poster Board Number: A0006
Presentation Time: 3:45 PM–5:30 PM
Scalable and reliable generation of retinal cells from human
transgene-free induced pluripotent stem cells under defined xenofree and feeder-free conditions
Olivier Goureau1, Amelie Slembrouck1, Angelique Terray1, Giuliana
Gagliardi1, Celine Nanteau1, Jose A. Sahel1, 2, Sacha Reichman1.
1
Institut de la Vision, INSERM U968; Sorbonne Universités UPMCParis 06; CNRS UMR7210, Paris, France; 2Centre Hospitalier
National d’Ophtalmologie des Quinze-Vingts, Paris, France.
Purpose: For retinal cell therapy based on human induced pluripotent
stem (iPS) cells, one of the major challenges is to develop essential
culture conditions for the use of these cells for future clinical
purposes. Until recently, iPS cell culture (maintenance and/or
differentiation) has been carried out using feeder cells and/or culture
media that contain animal products. Here, we adapted our new retinal
differentiation method using confluent human iPS cells, bypassing
cell clumps or embryoid body formation and in absence of Matrigel
or serum (Reichman et al. PNAS 2014; 111:8518), in a well-defined
xeno-free / feeder-free (XF/FF) system
Methods: Integration-free iPS cells cultured on mouse embryonic
fibroblasts were transferred onto vitronectin-coating plates and
cultured with xeno-free medium. Confluent iPS cells obtained in
these XF/FF conditions were directed toward a retinal lineage in a
serum free proneural medium containing N2 supplement. Emergent
neural retina (NR)-like structures were isolated and cultured in
floating conditions for their maturation with a serum free proneural
medium. Capacity for retinal differentiation was determined by
immunohistochemistry and qRT-PCR analysis triggering specific
developmental and mature retinal markers
Results: In less than one month, confluent iPS cells are able to
generate self-forming NR-like structures containing multipotent
retinal progenitor cells (RPCs). Floating cultures of isolated
neuroretinal tissue enabled the differentiation of RPCs into all types
of retinal cells. Early-born retinal cells (i.e. ganglion, amacrine and
horizontal cells) were identified after one month in culture, and lateborn retinal cells (i.e. photoreceptors, Muller glial and bipolar cells)
started to appear after two months
Conclusions: These data demonstrate that human iPS cell lines can
be maintained and directed to differentiate into retinal cell types
under XF/FF conditions that are required for translation to clinical
applications. In this context the reliable generation of retinal ganglion
cells and photoreceptor precursors could find important applications
in regenerative medicine
Commercial Relationships: Olivier Goureau, None; Amelie
Slembrouck, None; Angelique Terray, None; Giuliana Gagliardi,
None; Celine Nanteau, None; Jose A. Sahel, None; Sacha
Reichman, None
Support: ANR [GPiPS: ANR-2010-RFCS005]; [ANR-11IDEX-0004-02] in the frame of the LABEX LIFESENSES [ANR-10LABX-65]; Regional Council of Ile-de-France [DIM-Biothérapies];
SATT LUTECH
Program Number: 3578 Poster Board Number: A0007
Presentation Time: 3:45 PM–5:30 PM
Biasing early primitive ectoderm-like cells toward retinal cone
photoreceptors
Andrea S. Viczian1, 2, Kimberly A. Wong1, 2, Michael Trembley3.
1
Ophthalmology, Center for Vision Res, SUNY Upstate Medical
Univ, Syracuse, NY; 2SUNY Eye Institute, Syracuse, NY;
3
Department of Pharmacology & Physiology, University of Rochester
School of Medicine and Dentistry, Rochester, NY.
Purpose: In the developing embryo, primitive ectoderm formation
is lineage-restricted in response to extrinsic factors. The extrinsic
factor, Noggin, plays a key role in inducing retinal cell markers in
cultured human embryonic stem (ES) cells. In contrast, mouse ES
cells do not express retinal markers when exposed to Noggin. Human
ES cells have been shown to share more characteristics with mouse
primitive ectoderm than with mouse ES cells. We conducted our
study to determine if driving mouse ES cells to a primitive ectoderm
lineage would allow them to respond to Noggin, and induce retinal
cell markers.
Methods: Mouse ES cells were treated with conditioned media and
transformed into primitive ectoderm-like (EPL) cells. EPL-induced
cells were then treated with Noggin and subsequently grown in
differentiation media. Immunocytochemical and qPCR were used to
characterize the cells.
Results: If first converted to primitive ectoderm, Noggin treatment
resulted in a dose-dependent reduction of pluripotent markers and
an increase in neural and retinal progenitor markers. Interestingly,
we also found a substantial number of cells expressing markers for
cone photoreceptors in the EPL-driven cultures. These results suggest
that first restricting mouse ES cells to a primitive ectoderm lineage
creates an environment where Noggin can induce retinal cell marker
expression.
Conclusions: We are currently determining the underlying molecular
mechanism that drives ES cells to retinal progenitors and further,
cone photoreceptors
Commercial Relationships: Andrea S. Viczian, None; Kimberly
A. Wong, None; Michael Trembley, None
Support: EY019517
Program Number: 3579 Poster Board Number: A0008
Presentation Time: 3:45 PM–5:30 PM
Experimental Analysis of Signaling Pathways Underlying Retinal
Cell Specification Using Human Pluripotent Stem Cells
Jason S. Meyer1, 2, Akshayalakshmi Sridhar1, Dana Oakes1, Elyse
Feder1, Sarah Ohlemacher1. 1Biology, Indiana Univ- Purdue Univ
Indianapolis, Indianapolis, IN; 2Stark Neurosciences Research
Institute, Indiana University, Indianapolis, IN.
Purpose: Human pluripotent stem cells can be differentiated to yield
all of the major cell types of the retina, with important implications
for studies of retinogenesis as well as degenerative disorders
affecting the retina. However, the precise mechanisms underlying
the specification of certain retinal cell types over others has been
largely ignored. Thus, efforts were undertaken to elucidate signaling
pathways responsible for the directed differentiation of major retinal
cell types.
Methods: Human pluripotent stem cells were differentiated to
a highly enriched retinal progenitor cell population following
previously established protocols. The ability to influence the
differentiation of these retinal progenitor cells was tested through
the activation and inhibition of classical signaling pathways, and the
effect of these signaling factors on retinal cell fate determination was
assessed by immunocytochemistry and qRT-PCR. Differences in cell
proliferation and differentiation toward specific retinal cell types was
©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
then assessed and quantified, and significant differences between
treatment conditions determined.
Results: Highly enriched populations of retinal progenitor cells
could be derived from human pluripotent stem cells within a total of
30 days of differentiation, expressing progenitor markers including
CHX10. By default, these progenitor cells are capable of giving
rise to all of the major cell types of the retina, with retinal ganglion
cells and photoreceptors the most abundant cell types generated by
a total of 70 days of differentiation. Activation and/or inhibition of
classical signaling pathways, including Wnt and Hedgehog pathways,
demonstrated the ability to alter the differentiation of retinal
progenitor cells, biasing their differentiation toward specific retinal
neurons.
Conclusions: The ability to influence the differentiation of human
pluripotent stem cell-derived retinal progenitor cells allows for
the unique ability to study critical events of human retinogenesis,
yielding enriched populations of retinal neurons. Such an ability
also has profound implications for the study of retinal degenerative
disorders such as glaucoma or age-related macular degeneration,
as enriched populations of specific retinal cell types will likely be
required for the development of therapeutic strategies to combat the
degenerative processes associated with these diseases.
Commercial Relationships: Jason S. Meyer, None;
Akshayalakshmi Sridhar, None; Dana Oakes, None; Elyse Feder,
None; Sarah Ohlemacher, None
Support: NIH R01 EY024984-01, BrightFocus G2012027
Program Number: 3580 Poster Board Number: A0009
Presentation Time: 3:45 PM–5:30 PM
Mitochondrial metabolism and structural analysis of Induced
Pluripotent Stem Cell (iPSC) derived RPE from Age-Related
Macular Degeneration (AMD) Patients
Jie Gong1, Mark Fields1, Ernesto F. Moreira1, Hannah Bowrey1,
Zsolt Ablonczy1, Baerbel Rohrer1, Craig C. Beeson2, Lucian V. Del
Priore1. 1Ophthalmology, MUSC Storm Eye Institute, Charleston,
SC; 2College of Pharmacy, MUSC, Charleston, SC.
Purpose: Retinal pigment epithelium (RPE) derived from human
iPSC may provide a promising source of cells for transplantation.
Physiological tests are required to confirm that these cells can
function as RPE cells prior to cell transplantation. Herein we
analyzed critical RPE functions such as the development of
Transepithelial Resistance (TER), polarized secretion of vascular
endothelium growth factor (VEGF) and pigment epithelium-derived
factor (PEDF) as well as energy metabolism.
Methods: Fibroblasts cultured from axillary skin biopsies
obtained from a dry AMD patient and an age-matched control
were reprogramed into iPSC by using Yamanaka factors (Oct3/4,
Sox2, Klf4, c-Myc) delivered using non-integrating Sendai viruses.
iPSC cells were subsequently differentiated into RPE cells. iPS
(IMR90 Wi-cell Wisconsin USA) was also used as a young control.
Pigmented iPSC derived RPE were grown into monolayers on
permeable transwells and fed with medium containing 1% serum.
Media collected from each chamber were examined for VEGF
and PEDF levels using commercially available ELISA kits. TER
was measured using an epithelial voltohmmeter. Mitochondrial
respiratory function and glycolysis were measured by determining
the oxygen consumption rate (OCAR) and extracellular acidification
rate (ECAR) of iPS derived RPE using a Seahorse Bioscience XF96
instrument.
Results: iPS derived RPE cells from dry AMD and aged control
patients formed a hexagonal monolayer exhibiting an RPE phenotype
based on pigmentation, the expression of RPE (MITF) and tight
junction markers (ZO-1). They form a monolayer with TER of an
average of 335+36 Ω*cm2. Polarized VEGF and PEDF secretion
was observed [VEGF: basal>apical, (apical 1059+93 pg/ml; basal:
1299+29 pg/ml, p=0.03), PEDF secretion: apical>basal (apical
2185+196 ng/ml basal: 1220+460 ng/ml, p=0.009)]. Mitochondrial
respiration and glycolysis revealed an apparent age-dependent decline
in metabolic function. However no significance differences between
cells from the AMD and the age-matched control sample could be
identified.
Conclusions: iPS derived RPE cells exhibited physiological
properties typically obtained from adult RPE cells. Additional
samples are required to confirm this observation and expand it to
determine whether there are differences between dry AMD and agematched control samples.
Commercial Relationships: Jie Gong, None; Mark Fields,
None; Ernesto F. Moreira, None; Hannah Bowrey, None; Zsolt
Ablonczy, None; Baerbel Rohrer, None; Craig C. Beeson, None;
Lucian V. Del Priore, None
Support: Foundation Fighting Blindness, Research to Prevent
Blindness and IRB
Program Number: 3581 Poster Board Number: A0010
Presentation Time: 3:45 PM–5:30 PM
Investigating the Effect of PAX6 Gene Expression on
Differentiation of CD133+Human Cord Blood Stem Cells
Sahar Balagholi1, 4, Sedigheh Amini Kafiabadi1, Zahra-Soheila
Soheili2, Mozhgan Rezaeikanavi4, 3, Shahram Samiei1. 1Iranian
Blood Transfusion Organization, Tehran, Iran (the Islamic Republic
of); 2National Institute of Genetic Engineering and Biotechnology,
Tehran, Iran (the Islamic Republic of); 3Ophthalmic Research Center,
Shahid Beheshti University of Medical Sciences, Tehran, Iran (the
Islamic Republic of); 4Ocular Tissue Engineering Research Center,
Shahid Beheshti University of Medical Sciences, Tehran, Iran (the
Islamic Republic of).
Purpose: To assess the influence of PAX6 gene expression on
differentiation of CD133+ human cord blood stem cells.
Methods: Human cord blood mononuclear cells were isolated by
ficoll. CD133+ cells were obtained using the CD133MicroBead Kit
in combination with the autoMACS Separator and then cultured
in the Stem Span media. HEK293T packaging cells were cotransfected with PLEX-MCS, PsPAX2, and pMD2G via calcium
phosphate method. The resultant lentiviral vectors were collected
and concentrated by PEG 6000. The appropriate amount of viruses
was used for infecting CD133+ cells. The successful transduced
cells were selected by puromycin resistance. After two weeks, the
expression of rhodopsin, CHX10, Thy1, nestin, and PAX6 proteins
were assayed by immunocytochemistry.
Results: Two weeks after transfection, CD133+ cells expressed
rhodopsin, CHX10, Thy1, nestin, and PAX6 proteins, as compared to
the HEK293T cells that expressed rhodopsin and nestin.
Conclusions: Transfected CD133+ human cord blood stem cells with
PAX6 gene are able to differentiate into progenitor retinal neural- and
ganglion-like cells.
Commercial Relationships: Sahar Balagholi, None; Sedigheh
Amini Kafiabadi, None; Zahra-Soheila Soheili, None; Mozhgan
Rezaeikanavi, None; Shahram Samiei, None
©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
Program Number: 3582 Poster Board Number: A0011
Presentation Time: 3:45 PM–5:30 PM
Modeling retinal degeneration using induced pluripotent stem
cells from patients with the common P347L mutation in the
RHODOPSIN gene
Angelique Terray1, Celine Nanteau1, Amelie Slembrouck1, Jose A.
Sahel1, 2, Sacha Reichman1, Isabelle S. Audo1, 2, Olivier Goureau1.
1
Institut de la Vision, INSERM U968 ; Sorbonne Universités, UPMCParis 06 ; CNRS UMR7210, Paris, France; 2Centre Hospitalier
National d’Ophtalmologie des Quinze-Vingts, Paris, France.
Purpose: Inherited retinal degenerations, associated with
photoreceptor loss leading to blindness or visual impairment, affect
more than one million people throughout the world. The recent
discovery of direct reprogramming of somatic cells into induced
pluripotent stem (iPS) cells offers the opportunity to model in vitro
the effects of mutations on human retinal development. We have
focused on the most prevalent form of autosomal dominant retinitis
pigmentosa (adRP) in European cohorts, resulting from a mutation
(substitution P347L) on the gene coding for the visual pigment
RHODOPSIN (RHO).
Methods: Reprogramming of fibroblasts is performed by a non
integrative approach using Sendai Virus expressing the four
reprogramming genes, OCT4, KLF4, SOX2 and C-MYC. The
stemness status of the induced pluripotent stem (iPS) cells is assessed
phenotypically and by the expression of specific markers both by
qPCR and immunohistochemistry. Differentiation of iPS cell lines
towards retinal and photoreceptor lineages is performed using a wellestablished protocol (Reichman et al. PNAS 2014; 111:8518).
Results: Fibroblasts of three patients from the same family (including
one healthy patient) have been successfully reprogrammed into iPS
cells. All generated iPS cell lines expressed the specific markers
of pluripotency SSEA-4, TRA-1-81, OCT4 and NANOG. Q-PCR
analysis confirmed the absence of the Sendai viral reprogramming
genes and each iPS cell lines exhibited a normal karyotype. Normal
and RHODOPSIN-mutated iPS cell lines were able to generate neural
retina (NR)-like structures, containing retinal progenitors within 2
weeks. Culturing these NR-like structures in floating conditions, we
have obtained photoreceptor (precursors and matures) from both
normal and mutated cells. However after long-term cultures rod
and cone cell numbers were significant lower in NR-like structures
differentiated from RHODOPSIN-mutated iPS cell lines than in
structures differentiated from normal iPS cells.
Conclusions: From these results we conclude that mature
photoreceptors differentiated from RHODOPSIN-mutated iPS cells
degenerate in a RP-specific manner, demonstrating the ability of
patient-specific photoreceptors to recapitulate the disease phenotype
in vitro.
Commercial Relationships: Angelique Terray, None; Celine
Nanteau, None; Amelie Slembrouck, None; Jose A. Sahel, None;
Sacha Reichman, None; Isabelle S. Audo, None; Olivier Goureau,
None
Support: Agence Nationale de la Recherche [GPiPS: ANR-2010RFCS005], Région Ile de France (DIM –Biothérapies) and Fondation
de France (B. Fouassier).
Program Number: 3583 Poster Board Number: A0012
Presentation Time: 3:45 PM–5:30 PM
The mechanism of mTOR signaling pathway in the regulation of
differentiation of iPS into retinal pigment epithelial cells
sijia ding, Chao Jiang, Chen Zhao. The first affiliated hospital with
Nanjing Medical University, Nanjing, China.
Purpose: To explore the role of mTOR signaling pathway in the
regulation of differentiation of iPS into retinal pigment epithelial
(RPE) cells.
Methods: After embryoid bodies were formed by cultured iPS in
suspension condition, they were induced to differentiate into RPE
cells. The expressions of RPE specific proteins (RPE65, LRAT,
ZO-1) in iPS-RPE cells during differentiation were detected by
immunocytochemistry. Q-PCR and Western Blotting were carried
out to analyze RPE specific genes, proteins and mTOR activity in
iPS-RPE at different time points of differentiation (after one month,
two months, three months). Finally, iPS-RPE were treated with
mTOR inhibitor rapamycin and RPE specific protein expression was
evaluated.
Results: The RPE specific proteins (RPE65, LRAT, zo-1) of iPS-RPE
cells were observed after one month of differentiation by fluorescence
microscope. Compared with the control iPS, Q-PCR results showed
that iPS-RPE exhibited significant higher level of RPE specific genes
(RPE65, Best1, MerTK, CK18) after three months of differentiation
(P <0.01). Western Blotting also showed that the expressions of RPE
specific proteins BEST1, catenin and MerTK significantly increased
in iPS-RPE cells as differentiation process went on. The activity
of the mTOR was inhibited in this process. In addition, rapamycin
treated cells exhibited higher expression of catenin, but expression of
BEST1, MerTK and CK18 was not changed.
Conclusions: We established an efficient method to obtain iPS-RPE
cells. And mTOR signaling pathway is gradually suppressed during
the process of iPS differentiation into RPE cells in vitro.
Commercial Relationships: sijia ding, None; Chao Jiang, None;
Chen Zhao, Jiangsu Province’s Key Provincial Talents Program
(No. RC201149) (F)
Support: Jiangsu Province’s Key Provincial Talents Program
(RC201149 to C.Z.)
Program Number: 3584 Poster Board Number: A0013
Presentation Time: 3:45 PM–5:30 PM
Retinal Pigment Epithelial Cells Derived from Induced
Pluripotent Stem Cells exhibit Cytokine Profiles Similar to Other
Human RPE Cell Lines
Aya Yanagida, Kaitlen Knight, Jennifer R. Chao. University of
Washington, Seattle, WA.
Purpose: Age-related macular degeneration is the leading cause of
blindness in elderly populations in the developing countries, and
currently there are no effective long-term treatments available. In
order to better understand AMD disease pathology, there has been
significant interest in studying patient-specific iPSC-derived retinal
pigmented epithelium (RPE) cells, which are known to play a central
role in AMD. To confirm that iPSC-derived RPE cells recapitulate the
cell morphology and function of native RPE, we compared them to
cultures often used to study AMD, including human fetal RPE, ARPE
19 cells and human embryonic stem cell (hESC) derived RPE.
Methods: iPSC- and hESC-derived RPE cells, human fetal RPE
(gestational age 18-20 weeks), and ARPE-19 cells were cultured
on transwell filter membranes in order to establish cell polarity and
monolayers that replicate native RPE. After 4 and 8 weeks, cell
cultures were assessed by light, confocal, and transmission electron
microscopy (TEM). They were also assessed by transepithelial
resistance (TER) measurements in order to detect the formation of
tight junctional complexes. Secreted proteins in media (both apical
and basal) were analyzed by multiplex protein analysis after 4 and 8
weeks in culture.
Results: All cell types expressed RPE markers (CRALBP, ZO-1, and
Otx2) by 4 weeks in culture. iPSC-RPE, hESC-RPE, and human fetal
RPE, but not ARPE-19 cells, developed tight junctional complexes
©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
and apical microvilli, as determined by TEM. In addition, all cell
lines except ARPE-19 cells exhibited TER measurements similar to
that of native RPE by 8 weeks, indicating the establishment of tight
junctions. In a profile of 44 secreted proteins in apical and basal
media, iPSC-RPE cells were most similar to hESC-RPE and least
similar to ARPE-19 cells. iPSC-RPE were similar to fetal RPE in a
polarized (basal > apical) secretion of VEGF-A and apolipoprotein E,
two important factors in AMD disease development.
Conclusions: Human iPSC-derived RPE cells have similar structure
and cytokine profiles compared to native RPE and are the most
similar to ES cell derived RPE cells in our study. Our findings
provide support for the use of patient–specific iPSC-derived RPE in
studying the pathology of retinal degenerative diseases, including
AMD.
Commercial Relationships: Aya Yanagida, None; Kaitlen Knight,
None; Jennifer R. Chao, None
Support: Research to Prevent Blindness, NIH K08 EY019714
Program Number: 3585 Poster Board Number: A0014
Presentation Time: 3:45 PM–5:30 PM
Use of induced pluripotent stem cell technology to understand
photoreceptor cytoskeletal dynamics in retinitis pigmentosa
Roly Megaw1, Carla Mellough2, Baljean Dhillon1, Alan Wright3,
Majlinda Lako2, Charles ffrench-Constant1. 1Scottish Centre for
Regenerative Medicine, University of Edinburgh, Edinburgh, United
Kingdom; 2Newcastle University, Newcastle, United Kingdom;
3
University of Edinburgh, Edinburgh, United Kingdom.
Purpose: Mutations in RPGR account for 20% of all Retinitis
Pigmentosa (RP). RPGR’s function is unknown. It has no cure.
Animal rpgr models provide conflicting evidence as to the nature
of disease. We set out to establish a novel, human, cell-based model
for RPGR disease to test the hypothesis that RPGR mutations lead to
retinal degeneration due to a dysregulation of the actin cytoskeleton.
Methods: iPSCs were generated from skin biopsies of patients with
RPGR mutations (g.ORF15+689_692del4) and unaffected relatives.
Our published protocol was modified to produce self-organising
3 dimensional eye cups. RGPR-mutated and control cultures were
compared.
Results: Mutant and wild-type iPSC lines were generated and
characterised. Differentiation of wild type and mutant lines resulted
in the generation of optic cups in a self-organising manner. After
100 days in culture, these cups contained organised, mature
photoreceptors (PRs), as evidenced by morphology and both RNA
and protein expression (recoverin and rhodopsin). RPGR is localised
to the PR connecting cilium. Western blot analysis shows expression
of a truncated RPGRORF15 protein splice variant in mutated PR
cultures compared to controls. PR cultures from RGPR-mutated iPS
cells had increased actin polymerisation compared with controls
(confocal pixel intensity counts : 59.02 (SD 16.24) vs 23.70 (SD
8.128) p<00081).This finding was confirmed by assessment of
F-actin with western blot.
Pathways regulating actin turnover were explored. Western blot
analysis showed reduction in both Src and ERK phosphorylation in
RGPR-mutated PR cultures. An unbiased protein array confirmed
this reduction. Several other pathways were also shown to be
dysregulated in the RGPR-mutated PR cultures; confirmed by
western blots of repeat cultures from varying patient and control cell
lines.
Conclusions: This study supports the hypothesis that RPGR
mutations lead to actin dysregulation. We have identified several
pathways which are interupted in RPGR-mutant photoreceptor
cultures and could be contributing to disease. This study is the first
use (to our knowledge) of human iPSCs with retinitis pigmentosa-
causing RPGR mutations to look at pathophysiology of disease. It
suggests a role for RPGR in facilitating Rhodopsin transport to PR
outer segments by regulating actin turnover.
Commercial Relationships: Roly Megaw, None; Carla Mellough,
None; Baljean Dhillon, None; Alan Wright, None; Majlinda Lako,
None; Charles ffrench-Constant, None
Support: Wellcome Trust Grant (100470/Z/12/Z)
Program Number: 3586 Poster Board Number: A0015
Presentation Time: 3:45 PM–5:30 PM
Immunological properties of human embryonic stem cell-derived
retinal pigment epithelial cellsImmunological properties of
human embryonic stem cell-derived retinal pigment epithelial
cells
Qiuhui Liu, Lai Wei, Jing Wang, Shaofen Lin, Xiao Wang, Bing
Huang, Lin Lu, Yan Luo. Zhongshan Ophthalmic Center, Guangzhou,
China.
Purpose: The subretinal transplantation of human embryonic stem
cell-derived retinal pigment epithelial cells (hES-RPE) is thought
to be one of the most promising therapies for dry AMD and retinitis
pigmentosa. Although previous studies indicated that the subretinal
space possess immune privilege, the potential immune rejection
limits the clinical application of hES-RPE due to the immunological
properties of the ES-RPE cells are still unclear. It is necessary to
investigate the immunological properties of the ES-RPE cells and
develop new strategies to induce immune tolerance of allogeneic
transplants. This study was aimed to define the immunological
properties of the hES-RPE.
Methods: Human embryonic stem cells were induced to RPE cells
by adding DKK-1, Noggin and IGF to the medium. Then hES-RPE
cells were seed onto matrigel and co-cultured with CD 4+ T cells
for 72 hours. The expression of MHC class I and II molecules in the
hES-RPE cells before and after co-cultured with CD 4+ T cells were
detected by flow cytometry. The production of IL-1 beta, IL-6, TGFbeta 1 and TGF-beta 2 was analyzed by ELISA.
Results: The hES-RPE cells expressed specific RPE marker Mitf
and RPE65 after 28 days culture, the matured hES-RPE cells had
phagocytic ability and could secret PEDF. hES-RPE cells expressed
moderate levels of MHC class I molecules and negative for MHC
class II molecules. After co-cultured with CD 4+ T cell, the levels of
the MHC class I and II molecules slightly increased. Compared with
hRPE cells, the level of IL-1 beta and IL-6 produced by ES-RPE
cells was less. Cells pretreated with TGF-beta expressed less MHC-II
molecules after co-cultured with T cell.
Conclusions: ES-RPE cells showed less immunogenicity than hRPE
cells; however it still can express MHC class I and II molecules
when co-cultured with CD 4+ T cells, indicating it may be rejected
after transplantation. TGF-beta might partly suppress the immune
rejection.
Commercial Relationships: Qiuhui Liu, None; Lai Wei, None;
Jing Wang, None; Shaofen Lin, None; Xiao Wang, None; Bing
Huang, None; Lin Lu, None; Yan Luo, None
Support: National Basic Research Development Program of China
(973 program: 2013CB967000) ; the National Natural Science
Foundation of China to YAN LUO (81371020)
©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
Program Number: 3587 Poster Board Number: A0016
Presentation Time: 3:45 PM–5:30 PM
Altered RB and p27 expression patterns in hESC-derived versus
fetal retinal cone-precursors
Dominic Shayler1, Hardeep P. Singh2, Narine Harutyunyan2, David
Cobrinik2, 3. 1Developmental, Stem Cell and Regenerative Medicine,
Keck School of Medicine, University of Southern California, Los
Angeles, CA; 2The Vision Center, Division of Ophthalmology and the
Saban Research Research Institute, Children’s Hospital Los Angeles,
Los Angeles, CA; 3USC Eye Institute, Department of Ophthalmology,
Keck School of Medicine, University of Southern California, Los
Angeles, CA.
Purpose: Methods have recently been developed to generate retinal
tissue from human embryonic stem cells (hESCs). These hESCderived “retinal spheres” potentially could model aspects of human
retinal development and disease more accurately than live animal
models. For example, in human but not mouse retinas, maturing
cone arrestin (CA)-positive fetal cone precursors have been shown
to upregulate RB protein, MDM2 and MYCN, and to downregulate
p27.These changes have been implicated in the human cone precursor
response to RB loss in retinoblastoma development (Xu et al., 2014,
Nature). To investigate whether hESC-derived cone precursors
display similar features to fetal cone precursors, as might be needed
in a retinoblastoma model, RB, MDM2, MYCN and p27 were
examined in spheres of multiple ages.
Methods: H9 embryonic stem cells were differentiated into
retinal tissue as described (Nakano et al. 2012). Retinal spheres
were collected from multiple preparations, frozen sectioned and
immunostained. p27, RB and CA expression was observed at days
27, 41, 60/69, 97 and 132, while MDM2 and CA expression was
observed at the same ages as well as at day 123. Fetal retina of weeks
15 and 21 served as positive controls. All sections were imaged under
confocal microscopy.
Results: The earliest CA-positive cells appeared at day 41. Older
spheres displayed greater numbers of CA-positive cells with
increasing numbers on the apical surface. p27 was downregulated
in these cells in fetal retina but not in spheres. RB was expressed at
higher levels in cone precursors compared to other retinal neuron cell
types in fetal retina, but was expressed at lower levels at all ages in
hESC-derived tissue. MDM2 was expressed at higher levels in CApositive cells than other retinal cell types both in retinal spheres and
in fetal retina.
Conclusions: These data demonstrate that developing CA-positive
cells do not share all protein expression patterns with fetal retina.
MDM2 displayed similar expression in both systems, but RB was
not prominent and p27 remained highly expressed in cone precursors
as compared to other retinal cell types in the sphere system. This
indicates that there is a difference in expression of proteins that are
relevant to retinoblastoma development in the hESC-derived retina
model.
Commercial Relationships: Dominic Shayler, None; Hardeep P.
Singh, None; Narine Harutyunyan, None; David Cobrinik, None
Support: The Larry and Celia Moh Foundation, Research to Prevent
Blindness, A.B. Reins Foundation
Program Number: 3588 Poster Board Number: A0017
Presentation Time: 3:45 PM–5:30 PM
Exogenous factors induce rod photoreceptor-specific progenitors
from adult mouse retinal stem cells
Brian G. Ballios1, Saeed Khalili2, Kenneth Grisé2, Laura
Donaldson3, Gilbert Bernier4, Derek van der Kooy2. 1MD/PhD
Program, University of Toronto, Toronto, ON, Canada; 2Molecular
Genetics, University of Toronto, Toronto, ON, Canada; 3Division
of Ophthalmology, McMaster University, Hamilton, ON, Canada;
4
Centre de recherché, Pavillon Marcel-Lamoureux, 4MaisonneuveRosemont Hospital, Montréal, QC, Canada.
Purpose: Adult retinal stem cell (RSCs) derived from the ciliary
epithelium (CE) of mice can give rise to all retinal cell types. Taurine,
retinoic acid and FGF2/heparin (T+RA+FH) added to differentiating
clonal RSC colonies increases the number of rods to 90% of all
progeny; RSC progeny produce 10% rods when differentiated in
1%FBS+FH (pan-retinal conditions). We hypothesized that T/RA acts
on RSC progeny in an instructive, rather than permissive, manner
to bias photoreceptor differentiation through the enrichment of rodspecific progenitors.
Methods: RSCs were clonally isolated from the CE of 4-6 week old
mice. We used limiting dilutions (<1 clone / well) of a fluorescent
retroviral construct to label individual progenitor clones in vitro. In
addition, single cell sorting isolated non-pigmented and pigmented
cells in wells, which were then treated with T/RA for 28 d. Survival,
clone size, and phenotype were assessed by immunocytochemistry.
Results: Clonal retroviral labeling revealed enrichment in the
percentage of rod-only clones between 1%FBS (13%) to T/RA
(over 70%), without affecting clone size or overall cell survival.
This strongly argues against selective survival of rod progenitors
or differential survival of post-mitotic rods within a clone. In
1%FBS, clones derived from single non-pigmented progenitors
were distributed between non-rod and mixed clones, with a minority
of rod-only clones (100% Rhodopsin-positive; n=4 of 28 clones).
Clones derived from pigmented cells in 1%FBS never gave rise to
rod-only clones. In T+RA conditions, all clones derived from nonpigmented progenitors (n=34) were rod-only clones, while those
derived from pigmented progenitors (n=47 of 48) were almost all norod clones. Of note, one rod-only clone (the largest) was derived from
a single pigmented cell in T+RA conditions, suggesting potential
neural lineage plasticity in a very early, pigmented progenitor.
Survival rates of non-pigmented cell derived clones were similar in
T+RA and 1%FBS. Similar experiments using Wnt, BMP4 and TGFβ
inhibition increases the number of RSC-derived cones to >60% of all
progeny.
Conclusions: This study marks an important step in the
characterization of a rod-specific progenitor – no markers exist and
literature is divided on their existence in vivo. Our study suggests a
critical role for exogenous signals instructing early lineage decisions
between fate-restricted retinal progenitors.
Commercial Relationships: Brian G. Ballios, None; Saeed Khalili,
None; Kenneth Grisé, None; Laura Donaldson, None; Gilbert
Bernier, None; Derek van der Kooy, None
Support: Canadian Institutes of Health Research (CIHR),
Foundation Fighting Blindness (FFB) / Krembil 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.