Session 160 Stem Cell Engineering and Fundamental

ARVO 2016 Annual Meeting Abstracts
160 Stem Cell Engineering and Fundamental Mechanisms
Sunday, May 01, 2016 3:15 PM–5:00 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 1130–1150/B0349–B0369
Organizing Section: Retinal Cell Biology
Program Number: 1130 Poster Board Number: B0349
Presentation Time: 3:15 PM–5:00 PM
Xeno-free defined conditions for differentiation of proteininduced pluripotent stem cells towards retinal precursor cells
Ramesh Kaini, Heuy-Ching H. Wang. Ophthalmology, United States
Army Inst of Surgical Rsrch, San Antonio, TX.
Purpose: Induced pluripotent stem cells (iPSCs) have been
successfully differentiated into different retinal cell types, thus
instilling hopes for stem cell-based therapy in various retinal
degenerative diseases and trauma. However, iPSCs used in these
studies were reprogrammed using retroviral or lentiviral approaches
and differentiated using animal derived products that may have
potential risks of oncogenesis, zoonotic disease transmission, and
immune rejection. Therefore, we sought to differentiate genetic
integration free, protein-induced pluripotent stem cells (piPSCs)
towards retinal lineage and generate clinical-grade retinal precursor
cells in defined xeno-free conditions.
Methods: Commercially available piPSCs were maintained in
xeno-free TeSR-E8 medium and vitronectinXF coated culture
plates for at least 5 passages. piPSCs were differentiated towards
retinal lineage by using a modified protocol by Sridhar A et al.
Cells were dissociated using ReLeSRTM and then generated as
uniform sized EBS of 9000 cells/EB using Aggrewell. EBs were
gradually transitioned to neural induction medium (NIM) on day
3 of differentiation and maintained in suspension culture in rotary
cell culture system (Synthecon) for 7 days. EBs were plated on
vitronectinXF coated plates on day 8. On Day 17, aggregates were
dislodged and transferred to rotary cell culture system in retinal
differentiation medium (RDM).
Results: piPSCs maintained in vitronectinXF coated plates with
TeSR-E8 medium retained the expression of pluripotent markers,
including OCT4, Nanog, SSEA-3, and TRA-1-60 after five passages.
During retinal differentiation, piPSCs acquired expression of various
neural and retinal specification markers including Otx2, Sox2, LHX2,
RAX, PAX6, and CHX10 at different time points of differentiation.
Conclusions: In this study, we have confirmed the capacity of
piPSCs to differentiate into retinal precursor cells using a modified
xeno-free retinal differentiation protocol. Our results suggest that
pluripotent stem cells developed using protein-based reprogramming
can be a viable option for generating a safe source of patient-specific
cells for retinal regenerative therapies.
Commercial Relationships: Ramesh Kaini; Heuy-Ching H. Wang,
Program Number: 1131 Poster Board Number: B0350
Presentation Time: 3:15 PM–5:00 PM
Generating cone photoreceptor-like cells by inhibiting BMP and
Activin/TGFβ signaling in early primitive ectoderm-like cells
Kimberly A. Wong1, 2, Michael Trembley6, Ichiro Hiratani5,
David M. Gilbert4, Andrea S. Viczian3, 2. 1Neuroscience & Physiology,
SUNY Upstate Medical University, Syracuse, NY; 2Center for
Vision Research, SUNY Upstate Medical University, Syracuse,
NY; 3Ophthalmology, SUNY Upstate Medical University, Syracuse,
NY; 4Biological Science, Florida State University, Tallahassee,
FL; 5RIKEN Center for Developmental Biology, Kobe, Japan;
Pharmacology & Physiology, Aab Cardiovascular Research
Institute, University of Rochester School of Medicine and Dentistry,
Rochester, NY.
Purpose: Photoreceptor cells derived from mouse embryonic
stem (mES) cells are an attractive source for studying
photoreceptor-replacement therapies; however, current studies
have been unsuccessful in generating a sufficient number of cone
photoreceptors, due to a lack of understanding about how cones
are specified. In Xenopus, we have found that inhibition of bone
morphogenetic protein (BMP) and Activin/TGFβ signaling pathways
in primitive ectoderm is sufficient to bias cells to retinal progenitor
cells. We hypothesize that a similar treatment with BMP and Activin/
TGFβ inhibitors will direct mES-derived primitive ectoderm to
become retinal progenitors, including cone photoreceptors.
Methods: Mouse D3 ES cells were treated with conditioned media
in suspension and transformed into primitive ectoderm-like (EPL)
spheroids. Cultures were then supplemented with Noggin, or the
chemical inhibitors dorsomorphin and SB431542, to inhibit BMP
and Activin/TGFβ signaling, respectively. After differentiation, we
assayed for the presence of various neural and retinal cell types
by immunofluorescence and qRT-PCR. We also conducted this
assay with transgenic Crx-GFP ES cells, which allowed for visual
identification and sorting of GFP+ photoreceptor precursors.
Results: If first converted to primitive ectoderm, treatment with
high concentrations of BMP and activin/TGFβ inhibitors resulted
in a dose-dependent reduction of pluripotency markers and increase
in neural and retinal progenitor markers. Interestingly, we found
that 99% of the inhibitor-treated aggregates expressed markers for
photoreceptors (crx), and 93% of aggregates expressed cone-specific
opsins (M-opsin, S-opsin). Within these spheres, 62 ±12% of cells
were crx+ photoreceptor progenitors, and that 23.8 ± 0.7% of cells
expressed S-opsin after 9 days of culture.
Conclusions: These findings suggested that first restricting mES
cells to a primitive ectoderm lineage creates an environment where
BMP and activin/TGFβ inhibition can induce significant cone
photoreceptor generation in vitro. These results allow a better
understanding of the signaling responsible for directing cells towards
photoreceptor lineages. We are currently developing a method to
differentiate cone and rod progenitors in our cultures, in order to
identify cone-specific signaling factors.
Commercial Relationships: Kimberly A. Wong, None;
Michael Trembley, None; Ichiro Hiratani, None;
David M. Gilbert, None; Andrea S. Viczian, None
Support: NEI Grant EY019517, Research to Prevent Blindness
Unrestricted Award, and the Lions of District 20-Y1
Program Number: 1132 Poster Board Number: B0351
Presentation Time: 3:15 PM–5:00 PM
BMP/SMAD-signaling is necessary for the formation of
proliferating Müller glia-derived progenitors
Levi Todd, Isabella Palazzo, Natalie Squires, Andrew J. Fischer.
Neuroscience, The Ohio State University, Columbus, OH.
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ARVO 2016 Annual Meeting Abstracts
Purpose: Müller glia are the predominant glial cell type in the retina
and normally perform homeostatic functions. However, in response
to acute retinal injury or treatment with growth factors, Müller glia
can become proliferating neurogenic progenitors with the ability to
replace lost neurons. Therefore, understanding the mechanisms that
orchestrate the ability of Müller glia to transition into Müller gliaderived progenitor cells (MGPCs) is imperative to harnessing the
regenerative capacity of the retina. This study investigates whether
BMP/SMAD-signaling influences the formation of neurogenic
MGPCs in the in vivo chick retina.
Methods: All experiments utilized hatched chicks (7-21 days old).
Compounds were delivered via intraocular injections where one
eye served as the “experimental” eye and the contralateral eye
served as the “control”. Compounds included recombinant BMP4 to
activate BMP/SMAD-signaling, the small molecule inhibitor DMH1
to selectively block the BMP receptor, and recombinant FGF2 to
activate MAPK-signaling and stimulate proliferating Müller glia.
Retinas were damaged by a single injection of N-Methyl-D-aspartate
(NMDA). Intraocular injections of BrdU/EdU were applied during
experimental paradigms to label proliferating cells. Following
injection paradigms, retinas were harvested and processed for indirect
immunofluorescence and digital photomicroscopy. Significance of
difference (*p<0.05; n≥5) was determined by using a paired, twotailed t-test.
Results: We found that pSMAD1/5/8, a readout of active BMP/
SMAD-signaling, accumulates selectively in Müller glia in response
to injections with BMP4, NMDA or FGF2. We found that DMH1
specifically inhibits the up-regulation of pSMAD induced by BMP4.
We also found that inhibition of BMPR1 suppressed the formation of
proliferating MGPCs in NMDA and FGF2-treated retinas. Evidence
is provided using immunohistochemistry that inhibition of BMP/
SMAD-signaling may impact the formation of MGPCs by disrupting
Müller glia’s ability to up-regulate cFos, pS6 (a readout of mTorsignaling), and the retinal stem cell factor Pax6.
Conclusions: Our data suggests that active BMP/SMAD-signaling
is necessary for the formation of MGPCs. This work provides novel
data that the BMP/SMAD-signaling is an important component of
the network of cell-signaling pathways that regulate the formation or
MGPCs and retinal regeneration.
Commercial Relationships: Levi Todd, None; Isabella Palazzo,
None; Natalie Squires, None; Andrew J. Fischer, None
Support: NIH-EY022030-3
Program Number: 1133 Poster Board Number: B0352
Presentation Time: 3:15 PM–5:00 PM
Differential effects of early BMP4 exposure on optic vesicle
production across multiple hiPSC lines
Jishnu Saha1, Elizabeth E. Capowski1, Divya Sinha1, Jee Min1,
Joe Phillips1, 3, David M. Gamm2, 3. 1Waisman Center, University of
Wisconsin-Madison, Madison, WI; 2Department of Ophthalmology
and Visual Sciences, University of Wisconsin-Madison, Madison,
WI; 3McPherson Eye Research Institute, University of WisconsinMadison, Madison, WI.
Purpose: To test the effect of brief, early BMP4 treatment (Kuwahara
et al. (2015) Nat Commun. 6:6286) on the production of 3-D optic
vesicle-like structures (OVs) from multiple hiPSC lines.
Methods: hiPSC lines derived from 8 individuals were maintained
on Matrigel in mTesR1 and differentiated to OVs according to
our established stepwise protocol, which minimizes the use of
exogenous factors (Meyer et al. (2011) Stem Cells 29: 1206; Phillips
et al. (2012) IOVS 53:2007). On day 6 of differentiation, each
line was treated with or without 55 ng/ml BMP4, followed by 1:1
media exchanges every 3 days until day 16, when neuroectodermal
colonies were lifted from the culture plates. Efficiency of hiPSC-OV
production was assessed by counting OVs and non-OV neurospheres
at day 25.
Results: In the absence of BMP4, one of the hiPSC lines routinely
generated >50% OVs (high producer), 6 generated <10% OVs
(low producer), and one never generated OVs or any other
neuroectodermal derivative (non-producer). The low producing
lines had the greatest responses to BMP4 treatment, significantly
increasing OV production an average of 8.4 ± 2.1-fold (p =
0.0038) per 6 well starting plate of undifferentiated hiPSCs. The
non-producing hiPSC line also responded to BMP4 treatment by
generating neuroectoderm, but did not yield OVs. However, the
high producing line showed no difference in OV production with or
without BMP4 treatment.
Conclusions: BMP4 treatment is capable of significantly increasing
the yield of OVs in low producing hiPSC lines. These results
extend the previously published findings of Kuwahara et al. (2015)
and demonstrate both the utility and the limitations of this culture
Commercial Relationships: Jishnu Saha; Elizabeth E. Capowski,
None; Divya Sinha, None; Jee Min, None; Joe Phillips, None;
David M. Gamm, None
Support: Foundation Fighting Blinding Wynn-Gunn TRAP
(Translational Research Acceleration Program) Research Award,
Research to Prevent Blindness, Retina Research Foundation (Kathryn
and Latimer Murfee and Emmett A. Humble Chairs) McPherson Eye
Research Institute (Sandra Lemke Trout Chair), NIH P30HD03352,
and NIH R01EY21218
Program Number: 1134 Poster Board Number: B0353
Presentation Time: 3:15 PM–5:00 PM
The role of SIX6 in the development of optic vesicle-like
structures derived from human pluripotent stem cells
Jessica A. Cooke1, Akshayalakshmi Sridhar1, Sarah Ohlemacher1,
Jason S. Meyer1, 2. 1Biology, IUPUI, Indi, IN; 2Stark Neurosciences
Research Institute, Indiana University, Indianapolis, IN.
Purpose: Three-dimensional optic vesicle-like structures are capable
of being derived from human pluripotent stem cells (hPSCs). Highly
enriched for retinal progenitor cells, these populations express a
number of transcription factors associated with retinal development.
Among these is the homeobox transcription factor SIX6, which is
highly expressed in retinal progenitor cells yet its precise role in early
retinogenesis remains largely unclear. In the current study, efforts
were undertaken to elucidate the role(s) of SIX6 in the formation of
hPSC-derived three-dimensional optic vesicle-like structures.
Methods: hPSCs were differentiated to yield three-dimensional
optic vesicle-like structures following existing protocols. Following
differentiation, optic vesicle-like structures were readily identified
and isolated apart from non-retinal forebrain populations of cells.
The retinal progenitor nature of these cells was confirmed by both
immunocytochemistry and qRT-PCR. To identify a role for SIX6,
the gene was either overexpressed or knocked down in hPSCs using
a lentiviral vector and its effects on proliferation and differentiation
were characterized.
Results: Within the first thirty days of differentiation, hPSCderived optic vesicle-like structures were isolated and the retinal
progenitor nature of these cells was confirmed by their expression
of developmental transcription factors including CHX10, RAX,
and PAX6. Analysis of the expression of SIX6 within these cells
demonstrated a specific localization within hPSC-derived optic
vesicle-like structures, in which nearly all cells were SIX6positive. Further analysis of the role of SIX6 by overexpression and
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ARVO 2016 Annual Meeting Abstracts
knockdown demonstrated important roles in the proliferation and
differentiation of hPSC-derived optic vesicle-like structures.
Conclusions: Overall, the results of this study build a foundation
for understanding the role of SIX6 in retinal development. SIX6 was
highly expressed specifically within optic vesicle-like structures,
indicating an important role in the establishment of a retinal fate,
as well as the differentiation of retinal cell types. Such results
help to establish hPSCs as a valuable in vitro model system for
studies of human retinogenesis, with important implications for the
differentiation of specific cell types for translational applications.
Commercial Relationships: Jessica A. Cooke, None;
Akshayalakshmi Sridhar, None; Sarah Ohlemacher, None;
Jason S. Meyer, None
Support: NEI RO1 EY024940
Program Number: 1135 Poster Board Number: B0354
Presentation Time: 3:15 PM–5:00 PM
Elucidating the role of RAX in human retinogenesis with
pluripotent stem cells
Akshayalakshmi Sridhar1, Casey A. Miller1, Kimberly Ho-A-Lim1,
Jessica A. Cooke1, Sarah Ohlemacher1, Jason S. Meyer1, 2. 1Biology,
Indiana Univ Purdue Univ Indianapolis, Indianapolis, IN; 2Stark
Neurosciences, Indiana University, Indianapolis, IN.
Purpose: Human pluripotent stem cells (hPSCs) allow for the
unprecedented ability to recapitulate the earliest stages of human
retinogenesis, including the establishment of a definitive retinal fate
from a more primitive neural progenitor source. Recent studies have
clearly illustrated this ability, with hPSCs possessing the capacity to
give rise to three-dimensional optic vesicle-like structures in vitro. In
this role, hPSCs provide an attractive in vitro model for elucidating
complex mechanisms of human retinogenesis in an easily accessible
in vitro system.
Methods: hPSCs were directed to a retinal lineage as via previously
described, resulting in three-dimensional optic vesicle-like structures.
Initially, the expression patterns of critical eye field transcription
factors were analyzed over a timecourse of differentiation via
immunocytochemistry and quantitative RT-PCR. Lentiviralmediated overexpression and shRNA knockdown approaches were
also utilized to evaluate the role of critical transcription factors in
retinal differentiation. Additionally, DNA methylation analyses were
performed to identify epigenetic modifications associated with early
retinal fate determination.
Results: At early stages of differentiation, RAX was observed to be
expressed broadly throughout most cells, followed by its restriction to
a subpopulation of cells shortly thereafter. This restriction was closely
associated with the onset of more definitive retinal transcription
factors such as SIX6 and CHX10. Interestingly, the expression of
RAX was also absent from any cells adopting a non-retinal forebrain
phenotype, suggesting a critical role for RAX in retinal specification.
Methylation analysis of the RAX promoter suggested that epigenetic
mechanisms could potentially account for the restricted expression of
RAX in early retinal differentiation. Overexpression and knockdown
of RAX further demonstrated its requirement in the establishment
of a retinal fate, as well as the specification of particular retinal cell
Conclusions: Overall, the results of this study help to demonstrate
the suitability of hPSCs as an effective in vitro model of human
retinogenesis, with RAX demonstrated to be critical for the initial
establishment of a retinal fate as well as the specification of particular
retinal cell types. Such results help to establish hPSCs as a valuable
in vitro model system for studies of human retinogenesis.
Commercial Relationships: Akshayalakshmi Sridhar, None;
Casey A. Miller; Kimberly Ho-A-Lim, None; Jessica A. Cooke,
None; Sarah Ohlemacher, None; Jason S. Meyer, None
Support: NEI R01 EY024940, BrightFocus Foundation Grant
G2012027, Indiana CTSI (NIH/NCRR Grant UL1TR001108)
Program Number: 1136 Poster Board Number: B0355
Presentation Time: 3:15 PM–5:00 PM
Development of a step-wise protocol for the generation of human
induced pluripotent stem cell-derived choroidal endothelial cells
Allison E. Songstad, Luke A. Wiley, Erin R. Burnight,
S Scott Whitmore, Emily E. Kaalberg, Megan J. Riker,
Edwin M. Stone, Robert F. Mullins, Budd A. Tucker. Stephen A.
Wynn Institute for Vision Research, Carver College of Medicine,
University of Iowa, Iowa City, IA.
Purpose: Age-related macular degeneration (AMD) involves
dysfunction of choroidal endothelial cells (CEC), retinal pigment
epithelial cells (RPE), and photoreceptor cells (PRs). Human induced
pluripotent stem cell (iPSC)-based strategies to replace PRs and RPE
are a major scientific focus. However, success of these approaches
may require the replacement of damaged and lost CECs, which
degenerate early in the pathogenesis of AMD. The purpose of this
study is to design a step-wise differentiation protocol to generate
iPSC-derived CECs.
Methods: Fibroblasts from a patient with normal ocular history
were isolated from a skin biopsy and reprogrammed into iPSCs via
transduction with a non-integrating Sendai virus driving expression
of OCT4, SOX2, KLF4, and c-MYC. Pluripotency was assessed via
TaqMan Scorecard Assay and immunocytochemistry (ICC). RNA
sequencing analysis of cultured monkey RF/6A CECs and 2-D gel/
mass spec analysis of RF/6A-conditioned medium identified seven
secreted proteins known to be involved in vascular development.
Using the Taguchi statistical strategy, media were developed using
combinations of the seven proteins to drive CEC differentiation.
In a separate strategy, each protein was eliminated one at a time
from the differentiation protocol to identify those that were crucial
for CEC differentiation. The iPSC-CECs from both methods were
characterized via rt-PCR and ICC for EC- and CEC-specific markers.
Results: Using each approach, patient-specific iPSCs cultured in
media containing CTGF, TWEAKR, and VEGF-B differentiated into
CECs that expressed the EC-specific markers CD31, CD34, eNOS,
FOXA2, ICAM1, TIE2, and VE-Cadherin, and the CEC-restricted
markers CA4 and TTR. CTGF is required for CEC differentiation,
whereas TWEAKR and VEGF-B enhanced CEC differentiation.
CTNNB1, PDPN, RTN4, and SHC1 did not play a major role in CEC
differentiation. The iPSC-CECs fed CTGF, TWEAKR, and VEGF-B
formed vascular tube networks morphologically identical to those
formed in primary human CEC cultures.
Conclusions: We describe two strategies used to develop a stepwise
differentiation protocol for the derivation of CECs from patientspecific iPSCs. In addition to being useful for cell replacement,
patient-specific CECs, in combination with RPE and PRs, will be
invaluable to accurately interrogate the pathophysiology of AMD.
Commercial Relationships: Allison E. Songstad, None;
Luke A. Wiley, None; Erin R. Burnight, None; S Scott Whitmore,
None; Emily E. Kaalberg, None; Megan J. Riker, None;
Edwin M. Stone, None; Robert F. Mullins, None; Budd A. Tucker,
Support: Wynn Institute Endowment for Vision Research, Elmer and
Sylvia Sramek Charitable Foundation, NIH RO1 - EY 024605
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ARVO 2016 Annual Meeting Abstracts
Program Number: 1137 Poster Board Number: B0356
Presentation Time: 3:15 PM–5:00 PM
A flow cytometry sorting method for the production of high
fidelity retinal pigmented epithelial cells
Lisa R. Conti, Monte J. Radeke, Carolyn Radeke, Tyler Jean,
Stephanie LaBouve, Pete J. Coffey. Neuroscience Research Institute,
University of California at Santa Barbara, Santa Barbara, CA.
Purpose: Establishing renewable sources of retinal pigmented
epithelial cells (RPE) and creating patient-specific cell lines that
produce high fidelity RPE is valuable to the field of degenerative
eye research. Prevailing techniques for iPSC-derived RPE (iPSCRPE) production rely on manual dissection or passive enrichment
by passage. These methods are often labor intensive, variable and
restricted to cell lines with a propensity to produce high quantities of
pigmented cells. This study sought to determine whether granularity,
conferred by pigment, is sufficient to purify iPSC-RPE cells from
mixed differentiated cultures via flow cytometry.
Methods: Five iPSC lines were established from fRPE donor lines.
Following differentiation using three methods, cells were dissociated
and sorted based on side and forward scatter-properties. Pigmented
cells were collected and, following an expansion step, transcriptome
profiles were compared to their fRPE counterparts. Cultures were
further subject to immunocytochemistry (ICC) characterization.
Results: A single round of sorting yielded cell populations that
formed uniform pigmented epithelial monolayers. Intra-donor
specific transcriptome comparison between iPSC-RPE and fRPE
cell lines revealed gene markers consistent with strong RPE
cell signatures. In the few cases where sorted cultures displayed
heterogeneous pigment patterns, contaminant cell marker signatures,
confirmed with ICC, coincided. In these cases, a second round of
sorting established culture purity. High fidelity, high purity RPE
cultures were expandable up to 1000 fold. To date, we have applied
this enrichment method to generate seven iPSC-RPE lines, producing
banks that range from 3 to 55 million cells per line
Conclusions: Sorting based on pigment is an effective technique for
enriching iPSC-RPE cell lines. Notably, sorting is applicable to iPSC
lines with limited pigmented cell production, providing a resource
where samples are scarce. Further, this technique can eliminate the
need for cell line termination following failed enrichment attempts. In
all, we have established a flow cytometry pigment based method for
the reliable, user-independent production of high fidelity RPE.
Commercial Relationships: Lisa R. Conti, None;
Monte J. Radeke, None; Carolyn Radeke, None; Tyler Jean, None;
Stephanie LaBouve, None; Pete J. Coffey
Support: CIRM LA1-02086, Garland Initiative for Vision Research,
Arnold and Mabel Beckman Initiative for Macular Research
Program Number: 1138 Poster Board Number: B0357
Presentation Time: 3:15 PM–5:00 PM
Adipose tissue derived mesenchymal stem cells migrate towards
RPE, rescue apoptotic RPE under oxidative stress, and have the
potential to differentiate into RPE
Aya Barzelay, Sebastian Katz, Shira Wheisthal, Moshe Ben - Hemo,
Anat Loewenstein, Adiel Barak. Division of Ophthalmology, Tel Aviv
Medical Center, Tel Aviv, Israel.
Purpose: Oxidative stress plays a cardinal role in the
pathophysiology of AMD, and leads to apoptosis of RPE. Adipose
tissue derived mesenchymal stem cells (ASCs) may serve as a
therapeutic tool to regenerate RPE. Here we evaluated the activity
of ASCs when exposed to RPE under oxidative stress in vitro;
we assessed their migratory capacity towards injured RPE, and
their ability to prevent apoptosis of RPE. We also studied the
differentiation potential of ASCs into RPE.
Methods: Human ASCs were harvested from subcutaneous fat of
patients undergoing abdominoplasty. Primary cultures of human
RPE cells were subjected to oxidative stress by exposure to 1.5mM
hydrogen peroxide (H2O2). Conditioned medium of “stressed” RPE
was collected. The migratory capacity of ASCs towards conditioned
medium of “stressed” RPE or towards conditioned medium of
normoxic RPE was determined by a scratch assay. In order to study
the preventive effect of ASCs on apoptosis of RPE, “stressed” RPE
were treated with ASCs’ conditioned medium or with standard, nonconditioned medium. H2O2 induced RPE apoptosis was measured by
Annexin V/ propidium iodide staining and flow cytomtery analysis.
Finally, the differentiation potential of ASCs into RPE was evaluated
by culturing ASCs with 10nM nicotine amide.
Results: ASCs exhibited enhanced migration towards RPE that
were subjected to oxidative stress (193%±0.4 increase cells per
area, p<0.05). Treatment of “stressed” RPE with ASCs’ conditioned
medium prevented H2O2 induced apoptosis (50±0.7% decrease cells
number, p <0.05). After two weeks in differentiation medium, ASCs
underwent marked morphological changes forming spheroid bodies
in culture and upregulating early eye field markers (PAX6 2.2±0.1,
RX 2.1±0.8, BF1 4.7±0.7, NestinA 2.3 ±0.38 folds).
Conclusions: ASCs migrate towards RPE under oxidative stress
and reduce H2O2 induced apoptosis of RPE. Moreover, ASCs
demonstrate a differentiation potential into RPE evident by a
morphological change and upregulation of eye field markers. These
data may imply to a therapeutic potential of ASCs in regenerating
ASCs exhibited enhanced migration when exposed to conditioned
medium of RPE under oxidative stress (A- 0 hrs, C-24 hrs) compared
to control(B-0 hrs, D-24 hrs).
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ARVO 2016 Annual Meeting Abstracts
was observed by IF staining until 2 weeks after implantation. Longterm expression of Phagocytosis-related receptors was observed
1 month and 4 months after implantation
Conclusions: Subretinal implanted hESC-RPE cell might not
conduct its biology function in short-term after implantation.
Microenvironment of degenerated retina or surgical trauma might
attribute to the delayed expression of functional receptors of hESCRPE cell.
Commercial Relationships: LI ZHANG; Danhong Zhu,
None; Hossein Nazari Khanamiri, None; Biju Thomas, None;
Mark S. Humayun, None
After two weeks in culture with differentiation medium, ASCs
exhibited morphological change forming spheroid bodies.
Commercial Relationships: Aya Barzelay, None; Sebastian Katz,
None; Shira Wheisthal, None; Moshe Ben - Hemo, None;
Anat Loewenstein, None; Adiel Barak, None
Support: Moxie foundation grant
Program Number: 1139 Poster Board Number: B0358
Presentation Time: 3:15 PM–5:00 PM
Expression of phagocytosis-related receptors of subretinal
implantated hESC-RPE cells in RCS rats
LI ZHANG1, 2, Danhong Zhu2, Hossein Nazari Khanamiri3, 2,
Biju Thomas2, Mark S. Humayun2. 1Eye Center, Second Affiliated
Hospital, Medical School of Zhejiang University, Hangzhou, China;
Ophthalmology, Keck School of Medicine, University of Southern
California, Los Angeles, CA; 3University of Texas Medical Branch,
Galveston, TX.
Purpose: Retinal degeneration, including retinitis pigmentosa and
age-related macular degeneration, are leading cause of blinding
diseases worldwide. Efficient phagocytosis of photoreceptor outer
segment (POS) fragments by retinal pigment epithelial (RPE) cells,
which depends on the apical distribution of the integrin family
adhesion receptor αvβ5 and Mer tyrosine kinase (MerTK) receptors,
plays a key role in biological renewal and maintenance of retina
health. hESC-RPE cell based treatment for retinal degeneration is
heading towards clinical trial. How does the implanted hESC-RPE
cell conduct its biology function and express functional-related
receptors, remains currently unknown. This study aimed to observe
the expression of functional-related receptors of hESC-RPE after
implanted into the subretinal space of retinal degenerated animal
Methods: Polarized monolayer hESC-RPE cell cultured on an
ultrathin scaffold were transplanted into the subretinal space of
RCS rats, which has been wildly used as an animal model of
retinal degeneration. Eyeball samples were fixed and dissected
at different time point (1 week, 2 weeks, 1 month and 4 months)
after implantation. Optical coherence tomography (OCT) and H&E
staining were used to assess the position of implanted scaffold in
retina. Expression of human cell marker (TRA-1-85) and RPE
specific marker (RPE65) were observed by Immunofluorescence (IF)
staining. Phagocytosis-related receptors, including Integrin αvβ5,
MerTK and Rhodopsin were stained with corresponding antibodies.
Results: OCT and H&E staining confirmed the presence of
monolayer of hESC-RPE attached to parylene up to 4 months postimplantation. No expression of Integrin αvβ5, MerTK or Rhodopsin
Program Number: 1140 Poster Board Number: B0359
Presentation Time: 3:15 PM–5:00 PM
Utilization of an extracellular flux assay to measure
mitochondrial respiration in hPSC-RPE
Divya Sinha1, 2, Gyda C. Beeson3, Molly M. Wilson1, Janis T. Eells4,
Craig C. Beeson3, David M. Gamm5, 2. 1Waisman Center,
University of Wisconsin-Madison, Madison, WI; 2McPherson Eye
Research Institute, University of Wisconsin-Madison, Madison,
WI; 3Department of Drug Discovery and Biomedical Sciences,
Medical University of South Carolina, Charleston, SC; 4Department
of Biomedical Sciences, University of Wisconsin-Milwaukee,
Milwaukee, WI; 5Department of Ophthalmology and Visual Sciences,
University of Wisconsin-Madison, Madison, WI.
Purpose: Human pluripotent stem cell-derived RPE (hPSC-RPE)
cells are being utilized to model retinal disease in vitro, as well as
to potentially treat patients with certain blinding disorders via cell
transplantation. In vivo, RPE cells form a highly metabolically
active monolayer that performs several important functions requiring
efficient mitochondrial oxidative metabolism. Thus, efforts to monitor
and optimize hPSC-RPE health should at least in part take into
account mitochondrial function. Toward this end, we examined the
behavior of hPSC-RPE in a well-established, rigorous extracellular
flux (XF) assay after optimizing critical assay parameters.
Methods: RPE cells were differentiated from human embryonic
stem cells (hESC) or human induced pluripotent stem cells (hiPSCs)
using protocols established by our lab. Pure monolayer cultures
of hPSC-RPE were dissociated and then re-seeded at densities of
5000, 10000 or 30000 cells per well on XFe96-well microplates,
and cultured for time periods varying from 1 day to two weeks. An
XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to
measure basal respiration, ATP-coupled and maximal mitochondrial
respiration via oxygen consumption rates (OCR). Data was analyzed
using XF Wave software (Seahorse Bioscience).
Results: A seeding density of 30,000 cells/well on Seahorse 96-well
microplates works optimally to reproducibly measure OCR for
hPSC-RPE. At lower seeding densities, confluent monolayers are not
formed in 1-2 days resulting in variable OCRs. Basal OCR levels can
reach greater than 100 pmol/min when seeded hPSC-RPE cells are
maintained longer than 3 days in culture and maximal OCR can be
1.5 – 3-fold of basal.
Conclusions: Efforts to optimize the XF assay for hPSC-RPE
succeeded in producing highly robust and reproducible results
when seeding density and length of time in culture were taken
into consideration. hPSC-RPE demonstrated active mitochondrial
respiration in these assays, indicative of overall excellent cell health.
Commercial Relationships: Divya Sinha, None; Gyda C. Beeson,
None; Molly M. Wilson, None; Janis T. Eells, None;
Craig C. Beeson, None; David M. Gamm, None
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ARVO 2016 Annual Meeting Abstracts
Support: Research to Prevent Blindness Catalyst Award,
International Retina Research Foundation, Foundation Fighting
Blindness, NIH R01EY024588, 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, SIG
1S10OD018039-01 for Seahorse XFe96 analyzer, Support Grant P30
CA014520 to SMSSF (core service of the University of Wisconsin
Carbone Cancer Center)
Program Number: 1141 Poster Board Number: B0360
Presentation Time: 3:15 PM–5:00 PM
Rescue of MerTK retinitis pigmentosa using a human induced
pluripotent stem cell disease model
Conor M. Ramsden1, 2, Britta Nommiste1, Amelia Lane1,
Michael Powner1, Amanda-Jayne F. Carr1, Andrew Webster1,
Anthony T. Moore1, Lyndon da Cruz2, Pete Coffey1. 1Ophthalmology,
Institute of Ophthalmology, London, United Kingdom; 2Vitreoretinal,
Moorfields Eye Hospital, London, United Kingdom.
Purpose: Inherited retinopathies are fast becoming the leading
cause of blindness in developed countries. In order to counter this
heterogeneous group of diseases, adequate disease models need to
be developed in order to better understand pathology and to test
potential therapies. Induced pluripotent stem cells offer us a new and
radicle way to recapitulate the disease in the dish and give us almost
limitless material to drive discovery.
Methods: Fibroblast derived stem cells were used to generate
RPE from a patient suffering from retinitis pigmentosa due to a
deficiency in MerTK. The MerTK deficiency arises from a nonsense
mutation causing a premature stop codon. These RPE cells were
fully characterised and then exposed to a drug screen of read-through
agents to force expression of the whole length protein.
Results: Two candidate read-through agents were able to force
translation through the premature stop codon. The G418 and PTC124
compounds had the best effect at restoring a full length MerTK
protein phenotype.
Conclusions: These findings represent a promising avenue of drug
discovery in order to treat this rare and devastating disease.
Commercial Relationships: Conor M. Ramsden, None;
Britta Nommiste, None; Amelia Lane, None; Michael Powner,
None; Amanda-Jayne F. Carr, None; Andrew Webster, None;
Anthony T. Moore, None; Lyndon da Cruz, None; Pete Coffey,
Support: MRC/CIRM - G1000730
Program Number: 1142 Poster Board Number: B0361
Presentation Time: 3:15 PM–5:00 PM
Maintenance of phenotype following Expansion of iPS cell
derived RPE cultures through multiple passages
Alan D. Marmorstein, Benjamin Gillies, Lori Bachman,
Joshua Upton, Samuel Cross, Adiv A. Johnson, Lihua Y. Marmorstein.
Ophthalmology, Mayo Clinic, Rochester, MN.
Purpose: The use of induced pluripotent stem (iPS) cells in research
on the retinal pigment epithelium (RPE) is limited by the ability to
grow sufficient numbers of iPSC-derived RPE cells. Methods for
the differentiation of iPSC-derived RPE require expensive media
supplements, frequent feeding, extended periods of growth, and
yet often yield small numbers of cells. This study sought to define
conditions for the growth of large numbers of iPS-derived RPE cells
using inexpensive media.
Methods: The iPS cell line 006-BIOTR-0001 was obtained from the
Mayo Clinic BioTrust and cultured in mTeSR1™ media on Geltrex™
coated plates. Differentiation of iPSCs to RPE was performed on
Matrigel™ coated plates following a protocol using defined media.
Differentiation was assessed using morphologic, biochemical, and
functional criteria. Cells were then subject to additional passages
during which they were switched to DMEM containing 1% FBS at
0, 7, 14, or 28 days after plating. Cells were passaged using one of 4
different enzymatic preparations.
Results: iPSC-derived RPE formed a pigmented monolayer of
hexagonal cells 60-80 days after initiation of differentiation.
Differentiated cells formed monolayers of cuboidal epithelia with
apical microvilli, melanin granules, and junctional complexes.
The RPE marker protein Best1 was expressed and localized to the
basolateral plasma membrane. Western blot demonstrated that cells
expressed RPE markers Best1, CRALBP, PEDF, and RPE65. 80 days
into the differentiation protocol we passaged cells at a 1:3 ratio using
Accumax, Tryp LE, trypsin/EDTA, or dispase. Phenotype was best
preserved using Accumax or Tryp LE. Cells were then transferred
from DMEM containing B27 supplement to DMEM containing
1% FBS at 0, 7, 14, or 28 days, followed by passaging at 30 or 60
days using Accumax (1x). Following passage feeding was reduced
from daily to 3x/week. Phenotype was preserved through at least 3
additional passages in cells following transfer to DMEM/1% FBS
and was most effective after cells were preconditioned to DMEM/
FBS for at least 16 days prior to passage.
Conclusions: Using these data we have developed a protocol that
permits a single 6 cm plate of iPSCs to yield up to 81 x 6 cm plates of
iPSC-derived RPE cells in as little as 170 days with the majority of
those plates maintained in DMEM/FBS and fed three times per week.
Commercial Relationships: Alan D. Marmorstein;
Benjamin Gillies, None; Lori Bachman, None; Joshua Upton,
None; Samuel Cross, None; Adiv A. Johnson, None;
Lihua Y. Marmorstein, LAgen Laboratories LLC (I), LAgen
Laboratories LLC
Program Number: 1143 Poster Board Number: B0362
Presentation Time: 3:15 PM–5:00 PM
Xeno- and feeder-free differentiation of human pluripotent stem
cells to ocular epithelial cells
Heidi Hongisto, Tanja Ilmarinen, Alexandra Mikhailova,
Heli Skottman. BioMediTech, University of Tampere, Tampere,
Purpose: As human pluripotent stem cells (hPSCs) provide
a promising cell source for ocular cell replacement therapy
applications, more standardized and xenogeneic-free culture and
differentiation protocols for hPSCs are desired. We aimed to develop
robust, xeno- and feeder cell-free culture system for undifferentiated
hPSCs and efficient differentiation methods thereafter to produce
ocular epithelial cells suitable for clinical transplantation.
Methods: Several human embryonic stem cell (hESC) and human
induced pluripotent stem cell (hiPSC) lines were transferred to
the hPSC culture system based on the Essential 8TM (E8, Thermo
Fisher Scientific) culture medium and human recombinant laminin
matrix. Differentiation to retinal pigment epithelial cells (RPE)
was performed with two xeno-free media: X-VIVOTM 10 (Lonza)
and xeno-free differentiation medium (XF-RPEbasic) using
both embryoid body and adherent differentiation methods on a
combination of extracellular matrix (ECM) proteins. Cells were
analyzed for differentiation efficiency, RPE gene (RT-PCR) and
protein (immunofluorescence staining and confocal microscopy)
expression, barrier properties (TER) and functional maturity
(phagocytosis assay and ELISA).
Results: Human PSCs were successfully maintained in the defined,
xeno- and feeder-free culture system using single cell passaging.
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ARVO 2016 Annual Meeting Abstracts
The hPSCs demonstrated correct undifferentiated morphology,
hPSC marker expression, full pluripotency, and normal karyotype.
Prolonged culture led to recurrent karyotype changes conveying
culture and differentiation advantage. Both RPE differentiation
media enabled efficient differentiation to RPE that was enhanced by
small molecule induction in a media and cell line specific manner. A
combination of ECM proteins mimicking the natural RPE basement
membrane allowed for proliferation and maturation of tight epithelial
monolayers with excellent TER values, correct expression and
localization of RPE proteins and RPE functionality. Xeno-free
cryopreservation protocols for both pluripotent hPSCs and hPSCRPE were optimized for cell banking.
Conclusions: A robust and reproducible, xeno- and feeder-free
culture method for hPSCs as well as RPE differentiation and
cryobanking was established. The method allows for easy transition
to Good Manufacturing Practice-quality for generation of clinical
grade cells, and can also be used to generate corneal epithelial cells.
Commercial Relationships: Heidi Hongisto, None;
Tanja Ilmarinen, None; Alexandra Mikhailova, None;
Heli Skottman, None
Program Number: 1144 Poster Board Number: B0363
Presentation Time: 3:15 PM–5:00 PM
Differentiation of Human Embryonic Stem cells into
Photoreceptor Precursor – In-Vitro and In-Vivo Study
Yossi Mandel1, 2, Yoav Chemla2, Astar Shamul2, Amos Markus2,
Nairouz Farah1, 2, Ron S. Goldstein2. 1Optometry and Visual Science,
Bar Ilan University, Israel, Ramat Gan, Israel; 2The Mina and Everard
Goodman Faculty of Life Sciences, Bar Ilan University, Israel, Ramat
Gan, Israel.
Purpose: There are several methods for inducing the differentiation
of human embryonic stem cells (hESCs) into photoreceptors
precursors. Herein, we studied photoreceptors generation in twodimensional (2D) or a three-dimensional (3D) cell culture model. In
addition, we studied the retention of labeled photoreceptor cells in the
subretinal space of rats by fluorescence imaging of the retina.
Methods: hESCs (H9, US National Stem Cell Bank) differentiation
into photoreceptor precursors was investigated in two cell culture
models: a 2D model where photoreceptor differentiation was induced
in monolayer cultures over 17 days using media including Dkk1,
Noggin and IGF1, or a 3D model, where differentiation was induced
in suspension cultures over 21 days with media containing endo-IWR
1, SAG, CHIR 99021. Differentiation was studied by immunostaining
(CRX, PAX6) and mRNA expression (CRX,VSX2,PAX6). Cells
that were subjected to the differentiation protocol were labeled
by infection with GFP-expressing adenovirus and then injected
into the subretinal space of Long-Evans rats (100,000 cells/8 µl).
The retention of the cells was studied over a month by repeated
fluorescence imaging of the retina by a Micron-IV Phoenix rodent
imaging system.
Results: hESC were differentiated into photoreceptor precursors
in the 2D and 3D both models, as was revealed by staining or
expression of CRX. Higher efficiency was found in the 3D model as
compared to the 2D with up to 80 percent of cells stained for CRX.
Transplanted cells diffused evenly in the subretinal space without
producing cells clumps. Retention of cells was low with less than
10% of the transplanted cells being be detected by fluorescence
imaging at 1 month post transplantation.
Conclusions: Both 2D and 3D protocols were efficient in
photoreceptor generation with some advantage of the 3D over the 2D
method. Retention of injected cells differentiated from hESC in the
subretinal space of rats is poor. Further studies should be conducted
in order to improve the transplantation protocol.
Commercial Relationships: Yossi Mandel; Yoav Chemla, None;
Astar Shamul, None; Amos Markus, None; Nairouz Farah, None;
Ron S. Goldstein, None
Program Number: 1145 Poster Board Number: B0364
Presentation Time: 3:15 PM–5:00 PM
Modulation of photoreceptor outer segment phagocytosis and
degradation by environmental stressors in an hiPSC-derived
model system
Sonal Dalvi1, Ali Hashim1, Chad Galloway1, Leslie MacDonald1,
Ruchira Singh1, 2. 1Department of Ophthalmology and Biomedical
Genetics, University of Rochester, Rochester, NY; 2Center for Visual
Science, University of Rochester, Rochester, NY.
Purpose: Macular degenerative diseases are multifactorial, the sum
of both genetic as well as environmental factors which often affect
the retinal pigment epithelium (RPE) cells. Amongst environmental
factors, iron toxicity has been implicated in age related macular
degeneration (AMD) while cigarette smoke is the number one
preventable factor contributing to AMD. The ability to derive RPE
from human inducible pluripotent stem cells (hiPSCs) now allows
investigation of the additive effect of environmental factors within the
genetic background of macular disease patients. This study utilized
hiPSC-RPE cells in culture to evaluate the effect of environmental
stressors (iron overload, cigarette smoke) on maculopathy-associated
physical and functional attributes of RPE cells.
Methods: Established monolayers of hiPSC-RPE were treated either
chronically (1 week –1 month) or acutely (24 h) with exogenous iron
(ferric ammonium citrate, FAC) or cigarette smoke extract (CSE).
Subsequently, morphologic, gene/protein expression and functional
characteristics of hiPSC-RPE-treated with environmental stressors
(FAC, CSE) were compared to untreated control hiPSC-RPE.
Results: The expression of genes involved in the pigmentation
(TYRP1, SILV) and iron homeostasis (FTH1, SLC40A1) pathway(s)
were altered in long-term FAC-treated hiPSC-RPE compared to
untreated control hiPSC-RPE. Furthermore, both FAC and CSE
treatments affected the rate of photoreceptor outer segment (POS)
phagocytosis/degradation in hiPSC-RPE cultures. Interestingly, the
effect of FAC and/or CSE was different in RPE cultures that had been
chronically stressed with POS compared to RPE cultures without any
prior POS exposure.
Conclusions: Two significant contributing factors in the progression
of macular degenerative disease, iron toxicity and cigarette smoke,
alter an obligate function of RPE, phagocytosis/degradation of POS,
in hiPSC derived RPE. In addition, chronic iron supplementation
also alters metal ion homeostasis, plausibly contributing to RPE
dysfunction in hiPSC-derived cultures. Altogether, our data show
that hiPSC-RPE cultures are a suitable platform to study the effect
of environmental stressors on RPE physiology and thus may be a
suitable tool to dissect the contribution of gene vs. environment in
specific eye diseases, like macular degeneration.
Commercial Relationships: Sonal Dalvi, None; Ali Hashim, None;
Chad Galloway, None; Leslie MacDonald, None; Ruchira Singh,
Support: Brightfocus Foundation Macular Degeneration Research
Grant, David Bryant Trust, Knight Templar Eye Foundation Research
Starter Grant, Research to prevent Blindness (Career Development
Award, Singh- PI), Research to Prevent Blindness (unrestricted
grant, Feldon- PI) Retina Research Foundation Pilot Study Grant,
University Research Award (University of Rochester).
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ARVO 2016 Annual Meeting Abstracts
Program Number: 1146 Poster Board Number: B0365
Presentation Time: 3:15 PM–5:00 PM
Micro-culture arrays to control the generation of retinal
Sarah Decembrini1, Nathalie Brandenberg2, Sylke Hoehnel2,
Yvan Arsenijevic1, M.P. Lutolf2. 1Unit of Gene Therapy & Stem
Cell Biology, Jules-Gonin Eye Hospital, FAA, Lausanne,
Switzerland; 2Laboratory of stem cell and bioengineering, institute of
bioengineering, school of life sciences, Lausanne, Switzerland.
Purpose: The feasibility to recapitulate mammalian retinogenesis in
vitro by growing self-organizing retinal organoids from pluripotent
stem cells was recently demonstrated by our and other laboratories.
We observed that retinal organoids are not completely synchronized
in culture, resulting in retinas with different sizes, shapes, and apicalbasal orientation suggesting the need to improve the adopted cell
culture conditions.
Methods: We attempt here to standardize an approach, based
on microwell arrays, that can be used to screen compounds to
reproducibly generate retinal organoids meanwhile saving time, space
and materials. Different biomaterials were tested as culture substrates
of microwell arrays. The stiffness, quantity of hydrogels, well shape
and size, volume of the reaction and medium composition as well as
the effect of cell number were evaluated in respect to the efficiency to
generate retinal organoids.
Results: Different qualities and stiffness of hydrogels were tested
in order to establish the best culture substrate supporting retinal
organoid generation. Agarose, noble agarose, polyethylene-glycol
(PEG) and polydimethylsiloxane (PDMS) were assessed. The
results highlight the higher propensity of 5% w/vol PEG, to generate
retinal organoids in comparison to all the other tested materials.
AggreWellTM (Stemcell Technologies) served in primis as template
to construct the desired well topography. Flat- or V- bottom wells,
round or square with a diameter of 400 or 800 µm were tested. The
shape and size of the microwells tested were not able to support the
generation of the retinal organoids. A standard photolithography
was developed to generate an unlimited well size and geometry. The
optimal microwell size and shape was identified in 1.5mm height
(1.7 max), 1.7mm width, U-bottom wells. Such microwells allow
the aggregate formation in less than 6 hours. Different medium
conditions, supplements, matrigel concentrations and surviving
factors were assessed to fine-tune the retinal organoid production.
The results suggest the requirement of factors to induce retinal
organoids different than those utilized with plastic wells revealing
some distinctive properties of the microwells that are currently under
Conclusions: We conclude that the microwell plates support the
survival and differentiation of retinal organoids and can be used to
screen morphogens aimed to improve the development of retinal
Commercial Relationships: Sarah Decembrini;
Nathalie Brandenberg, None; Sylke Hoehnel, None;
Yvan Arsenijevic, None; M.P. Lutolf, None
Program Number: 1147 Poster Board Number: B0366
Presentation Time: 3:15 PM–5:00 PM
Self organization of human induced pluripotent stem cell (iPSC)
derived retinal progenitor cells on a 3D scaffold
Laurel T. Tainsh2, 1, Deepti Singh1, 2, SHAO-BIN WANG2, 1,
Maryam Ghiassi-Nejad2, 1, Bo Chen2, Ron A. Adelman2,
Lawrence J. Rizzolo1, 2. 1Surgery, Yale University, New Haven, CT;
Ophthalmology, Yale University, New Haven, CT.
Purpose: Stem cell-derived retinal progenitor cells (RPC) have
potential for treating retinal degenerations. Although RPC can form
retina-like neurospheres, planar structures would be more appropriate
for implantation. We investigated whether RPC could be reconfigured
on a planar scaffold.
Methods: Human iPSCs were differentiated in DMEM/F12
supplemented with N2 and B27 according to published protocols.
After 3 or 8 weeks of differentiation, retinal cups (RCs) were
seeded as intact spheres or after dissociation with trypsin onto a
scaffold composed of gelatin, hyaluronic acid, and chondroitin
sulfate. Differentiation of intact and dissociated RCs on scaffolds
was monitored with qPCR at monthly time points for up to
14 weeks as compared to control RCs grown in suspension.
Immunohistochemistry, confocal microscopy, and scanning electron
microscopy were used to assess cell penetration, proliferation, and
differentiation within scaffolds. In some experiments, RPC scaffolds
were co-cultured with RPE. Effects on the RPE were assessed by the
transepithelial electrical resistance (TER).
Results: Intact RCs attached as clusters and proliferated to invade
the scaffold forming distinct subdomains expressing ganglion cell,
interneuron, and photoreceptor markers. Intact cell clusters on
scaffolds continued to grow in size for up to 6 weeks. Trypsinized
RCs populated the scaffold more uniformly reforming as clusters on
the surface and laminating within scaffold pores. Scaffolds seeded
with dissociated RCs remained uniformly populated for up to 14
weeks. RT-qPCR results showed that both intact and dissociated RCs
on scaffolds differentiate along a timeline as well or better than RCs
grown in suspension. Intact and dissociated RCs expressed eyefield,
ganglion cell, interneuron, and early photoreceptor markers such as
recoverin, but did not express the more mature photoreceptor markers
opsin and rhodopsin. With co-culture TER increased 3x for the RPE,
while PAX6 decreased and OTX2 increased in the RPC.
Conclusions: Both intact and dissociated iPSC derived RCs
penetrate, proliferate, and differentiate within HA scaffolds.
Dissociating RCs with trypsin before seeding results in a more
uniformly populated, laminated, and planar scaffold. Co-culture
furthered the maturation of RPE and RPC. Ongoing experiments aim
to optimize this approach.
Commercial Relationships: Laurel T. Tainsh, None; Deepti Singh;
SHAO-BIN WANG, None; Maryam Ghiassi-Nejad, None;
Bo Chen, None; Ron A. Adelman, None; Lawrence J. Rizzolo,
Support: NIH CTSA TL1TR000141, Department of Defense
MR130036, CT Regenerative Medicine Research Fund 14-SCBYale-18, Leir Family Fund, Alonzo Family Fund
Program Number: 1148 Poster Board Number: B0367
Presentation Time: 3:15 PM–5:00 PM
Integrin and extracellular matrix (ECM) protein expression
in differentiating neural retina generated from human ES cell
(hESC) cultures
Debjani Phillips1, 2, Douglas Streeten3, Isabel Pinilla4, 3,
William J. Brunken5, 6, David M. Gamm7, 2. 1Waisman Center,
University of Wisconsin Madison, Madison, WI; 2McPherson Eye
Research Institute, Madison, WI; 3University of Wisconsin Madison,
Madison, WI; 4Ophthalmology, University Hospital Lozano Blesa,
Zaragoza, Spain; 5Ophthalmology, Upstate Medical University,
Syracuse, NY; 6SUNY Eye Institute, Syracuse, NY; 7Department
of Ophthalmology and Visual Sciences, University of Wisconsin
Madison, Madison, WI.
Purpose: During retinal development, integrin heterodimers
associate with ECM proteins to regulate axon guidance, spatial
organization and ECM assembly, thus directing cell fate. The β1
integrin subunit has been implicated in the expansion of neural
progenitor cells and is integral for maintaining the collective integrity
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ARVO 2016 Annual Meeting Abstracts
of the developing retina. Using our established hESC model of retinal
development, we sought to profile the expression of β1 integrin and
its partners, α1-6 integrins and αv integrin, and the ECM proteins
with which they associate at varying stages of human neural retinal
Methods: Optic vesicles (OVs) generated from H9CRX17tdTomato
hESCs were differentiated to day (D) 45, D67, D92 and D100.
Expression of β1 integrin subunit, its associated α integrin subunits,
and the ECM proteins fibronectin, collagen IV and laminin were
observed using immunocytochemistry (ICC). Levels of integrin
expression on developing CRX+ cells were analyzed by flow
cytometry. The CRX+ population was gated according to tdTomato
reporter expression levels.
Results: The β1 integrin subunit levels remained elevated at D45 and
D100 of differentiation. In contrast, expression levels of α integrin
subunits were low or undetectable at D45 but increased at D100.
Fibronectin, collagen IV, and laminin expression were prevalent
during differentiation of hESCs into neural retina and integrins
appeared clustered alongside tdTomato-expressing CRX+ cells.
Conclusions: Our hESC-OV neural retina model is a useful tool
for identifying integrins and ECM proteins that have the capacity to
contribute to development and perhaps fate determination of neural
retinal cells. Positive identification of the β1 integrin subunit along
with all of its associated α integrin subunits in these 3-D cultures
provides further evidence of their utility as models of human retinal
development. Knowledge of the expression profiles of integrins and
their ECM binding partners during different stages of development
improves our understanding of the developing human retina and
provides guidance for the manipulation of culture techniques in order
to direct cells to a desired fate.
Commercial Relationships: Debjani Phillips, None;
Douglas Streeten; Isabel Pinilla, None; William J. Brunken, None;
David M. Gamm, None
Support: Retinal Research Foundation Emmet 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: 1149 Poster Board Number: B0368
Presentation Time: 3:15 PM–5:00 PM
Scaffold Composition Affects the Proliferation and
Differentiation of Retinal Progenitor Cells
Maryam Ghiassi-Nejad1, Deepti Singh1, SHAO-BIN WANG1,
Laurel T. Tainsh1, Bo Chen2, Ron A. Adelman2, Lawrence J. Rizzolo1.
Surgery/Ophthalmology, Yale University, New Haven, CT;
Ophthalomology, Yale University, New Haven, CT.
Purpose: Retinal progenitor cells (RPC) can form neurospheres
that resemble retinal structure, but their small spherical geometry
limits their utility for experimentation and implantation into diseased
retinas. We examined different scaffolds for biocompatibility, and
ability to foster stem cell growth and differentiation.
Methods: Scaffolds were fabricated from electrospun
polycaprolactone (PCL) or from alginate or gelatin that was decorated
with hyaluronic acid and chondroitin sulfate. The later scaffolds were
freeze-dried to create large pores, rehydrated, and sectioned into
discs varying from 60-120 microns thick. Pore size was evaluated
by scanning electron microscopy, and potential toxicity was tested
by implanting the scaffolds into mouse retinas. Human embryonic
stem cells (WA09) were differentiated, and in some experiments,
co-cultured with human fetal (hfRPE). The cell differentiation was
monitored using qRT-PCR and immunofluorescence every two weeks
for two months. The cultures were followed up to three months and
monitored by quantitative RT-PCR (qPCR) or immunofluorescence.
Results: Scaffolds had interconnected pores 50-100 micron in
diameter, but for PCL pores were flattened in the XZ plane. Stem
cells penetrated PCL only 20-30 micron, and the cultures were not
uniform. RPC were able to differentiate on PCL and gelatin scaffolds,
but growth and differentiation was impaired on alginate scaffolds.
We proceeded with gelatin-based scaffolds. Scaffolds degraded
slowly in culture, but were degraded within three weeks following
implantation into the subretinal space. There was no evidence of
overt inflammation, retinal edema, or degeneration. When plated
at low density and co-cultured with hfRPE, RPC transformed from
spherical cells to broad, flattened cells that extended neurite-like
projections. When plated at high density, cells uniformly populated
the full thickness of scaffolds as thick as 120 microns and expressed
retinal markers.
Conclusions: Scaffolds based on freeze-dried gelatin were
most successful at satisfying 3 criteria: full penetration of the
scaffolds, uniform cell density in the xy-plane, and no cytotoxicity
upon implantation. Retinal lamina began to form, but further
experimentation is required to achieve the structures found in
Commercial Relationships: Maryam Ghiassi-Nejad,
None; Deepti Singh, None; SHAO-BIN WANG, None;
Laurel T. Tainsh, None; Bo Chen, None; Ron A. Adelman, None;
Lawrence J. Rizzolo, None
Support: Dept. of Defense MR 130036, CT Regan Med Rsch Fund
14-SCB-Yale-18: Leir Family Fund: Alonzo Family Fund; Richard K
Greshon Fellowship.
Program Number: 1150 Poster Board Number: B0369
Presentation Time: 3:15 PM–5:00 PM
Evaluating the effects of ECM and elastic modulus on human
pluripotent stem cell-derived photoreceptor enrichment
Jee Min1, Joe Phillips1, 2, William Daly3, 4, William Murphy3, 4,
David M. Gamm5, 2. 1Waisman Center, University of Wisconsin
- Madison, Madison, WI; 2McPherson Eye Research Institute,
Madison, WI; 3Department of Materials Science and Engineering,
University of Wisconsin Madison, Madison, WI; 4Department
of Biomedical Engineering, University of Wisconsin-Madison,
Madison, WI; 5Department of Ophthalmology and Visual Sciences,
University of Wisconsin - Madison, Madison, WI.
Purpose: Human pluripotent stem cell (hPSC)-based photoreceptor
replacement strategies require efficient generation and isolation
of photoreceptors, as well as the removal of proliferative retinal
progenitor cells (RPCs). Extrinsic factors such as the extracellular
matrix (ECM), as well as the stiffness (elastic modulus) of the
extracellular environment are known to modulate cellular adherence,
differentiation, and maturation. The purpose of this study was
to determine the potential of these extrinsic factors to promote
photoreceptor enrichment while reducing the number of RPCs.
Methods: Using our established protocols, hPSC-derived
optic vesicle-like (OV) structures were generated, isolated, and
differentiated to day 80. OVs were then dissociated and plated onto
various substrates and maintained for one week. Two experiments
were performed to evaluate the following substrates: Experiment 1)
96 well plates were pre-coated with ECMs and adhesion promoting
molecules, including fibronectin, laminin, vitronectin, collagen,
matrigel, poly-L-lysine (PLL), poly-D-lysine (PDL), and poly-Lornithine (PLO). Experiment 2) Polyethylene glycol based (PEG)
hydrogels were spotted onto 384 well plates. A slow degrading
and a fast degrading MMP-labile crosslinker, and a non MMPlabile cross-linker were added to PEG at varying concentrations to
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ARVO 2016 Annual Meeting Abstracts
vary stiffness and degradability of the hydrogel. Furthermore, the
optimal concentration of integrin binding peptides were also probed.
Quantitative immunocytochemistry analysis was performed on the
Operetta High Content Screening System to determine the number
of photoreceptors (RECOVERIN+) and RPCs (Ki67+) in both
Results: Initial results suggested that PLO and PLL promoted the
greatest degree of photoreceptor enrichment and RPC reduction
in experiment 1. In experiment 2, medium stiffness Tryptophan
crosslinked hydrogels with a high concentration of IKVAV provided
the most photoreceptor enrichment and RPC reduction.
Conclusions: In this study, high content screening was used to
identify target substrates for photoreceptor enrichment. Knowledge
gained from this study can be used to improve photoreceptor
production and isolation methods. Additionally, these results are also
being used to inform hydrogel design, as transplanting photoreceptors
in an encapsulating hydrogel may improve transplant efficiency and
cell survival.
Commercial Relationships: Jee Min, None; Joe Phillips, None;
William Daly, None; William Murphy, None; David M. Gamm,
Support: Foundation Fighting Blindness Wynn-Gund TRAP
Award, NIH RO1 EY21218, Retinal Research Foundation Emmett
A. Humble Distinguished Directorship, McPherson Eye Research
Institute (Sandra Lemke Trout Chair), Research to Prevent
Blindness, Carl and Mildred Reeves Foundation, NIH P30HD03352,
Muskingum County Community Foundation, Choroideremia
Research Foundation
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