ARVO 2014 Annual Meeting Abstracts 218 Stem Cell I Monday, May

ARVO 2014 Annual Meeting Abstracts

218 Stem Cell I

Monday, May 05, 2014 8:30 AM–10:15 AM

Exhibit/Poster Hall SA Poster Session

Program #/Board # Range: 1358–1388/A0114–A0144

Organizing Section: Retinal Cell Biology

Contributing Section(s): Retina

Program Number: 1358 Poster Board Number: A0114

Presentation Time: 8:30 AM–10:15 AM

The formation of primitive ocular structures and stratified neural retina from human pluripotent stem cells

Carla B. Mellough 1 , Joseph F. Collin 1 , Mahmoud Khazim 1, 2

3 , David Steel 1

, Evelyne

Sernagor 2 , Nicholas Wride 3 , Majlinda Lako 1 . Institute of Genetic Medicine and North East Stem Cell Institute (NESCI),

2

Newcastle University, Newcastle upon Tyne, United Kingdom;

Institute of Neuroscience, Newcastle University, Newcastle upon

Tyne, United Kingdom; 3 Sunderland Eye Infirmary, Sunderland,

United Kingdom .

Purpose: Diseases of the outer retina are a leading cause of irreversible blindness worldwide, yet no treatments currently exist for many forms. The ability to generate retinal tissue for disease modelling, drug screening or transplantation would be extremely useful in order to resolve this important challenge. Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) can be differentiated in vitro to develop towards retinal cells including photoreceptors and retinal pigmented epithelium (RPE). Their ability to self-organise into complex retinal tissue, similar to embryonic human retina, has recently been demonstrated and represents a leap towards in vitro modelling of disease phenotypes and the study of human retinal ontogenesis. The reproducibility and efficacy of this capability is however not well established across multiple hESC/ hiPSC lines. We have built upon our previous work to devise a simple and reliable method that produces laminated retinal tissue in vitro using one factor only.

Methods: hESC/hiPSC lines were expanded on mouse embryonic fibroblasts then differentiated under 3-dimensional conditions for 90 days in a ventral neural induction medium either alone or supplemented with human insulin-like growth factor (IGF).

Results: The addition of IGF during differentiation orchestrates the formation of ocular-like structures from hESC/hiPSC with high frequency compared to controls. IGF-generated structures contain not only RPE and neural retina, but other eye elements including primitive lens and corneal epithelium. Retinal organisation is reminiscent of developing human retina and comprises multiple phenotypes including photoreceptors, amacrine and ganglion cells, which establish synaptic connections and form visible plexiform layers. Photoreceptors exhibit primitive rod- and cone-like inner and outer segments and functional cyclic nucleotide gated channel properties. Inhibition of IGF pathway signalling abolished the ability of hESC/hiPSC to give rise to viable laminated retina, establishing an important role for this pathway in retinal histogenesis.

Conclusions: The efficient derivation of ocular derivatives including laminated retinal tissue can be achieved from hESC and hiPSC using

IGF, offering exciting new opportunities to study retinal development and disease and to provide an expandable source of transplantable derivatives for cell replacement studies.

Commercial Relationships: Carla B. Mellough , None; Joseph F.

Collin , None; Mahmoud Khazim , None; Evelyne Sernagor , None;

Nicholas Wride , None; David Steel , None; Majlinda Lako , None

Support: Fight for Sight (FFS) and Retinitis Pigmentosa Fighting

Blindness (RPFB).



Identification and characterization of neural crest stem cells in rat olfactory mucosa as a use for cell-mediated neuroprotection as treatment of glaucoma

Maayke Kuijten

Steve Brocchini

1, 2 , Claire Ginn

1, 2 . 1

2 , Peng Khaw 1 , G Astrid Limb 1 ,

National Institute for Health Research (NIHR)

Biomedical Research Centre at Moorfields Eye Hospital NHS

Foundation Trust and UCL Institute of Ophthalmology, London,

United Kingdom; 2 UCL School of Pharmacy, London, United

Kingdom .

Purpose: The olfactory mucosa (OM) is an area of constant neural regeneration populated by olfactory ensheathing cells (OEC), which may constitute a source of neurotrophic support for spinal cord damage and glaucoma treatment. However, the heterogeneous nature of these cells makes their isolation difficult for therapeutic use. We examined the OM to identify neural crest stem cell (NCSC) populations, from which OECs originate and which may constitute an alternative source for neuroprotection in glaucoma.

Methods: We performed immunohistochemistry and confocal microscopy to characterise the rat OM. Different cell sorting strategies such as flow cytometry and differential adhesion were used to separate different cell populations. Analyses were performed using

Western blotting and RT-PCR techniques.

Results: Confocal analysis of postnatal day 16 OM tissue showed the presence of three different cell populations in the lamina propria

(LP). Positive staining for mesenchymal stem cell (MSC) markers in this tissue indicates the presence of mesenchymal-like stem cells and their location seems to indicate a MSC niche in the LP of OM tissue. Expression of neural stem cell markers including Sox2, Pax6 and nestin was found in the LP, which could indicate the presence of different stem cell populations. The NCSC markers Sox10, snail and HNK-1 were also found, indicating the presence of a NCSC population in the LP. Differential adhesion has been used as a means of selecting different cell populations. In these cell populations, expression of the NCSC genes p75, Sox10, HNK-1 and snail was also seen. Expression of the above markers was further confirmed by examination of mRNA and protein lysates of whole OM. In addition, mRNA samples of the obtained cell population and OM tissue have been investigated for neurotrophin expression, such as BDNF, CNTF,

GDNF, NGF, NT3 and NT4/5, which are all expressed.

Conclusions: The OM is a complex tissue that contains at least one or possibly more stem cell populations residing in the LP. Based on the gene expression profile, Western blotting of tissue and cell lysates and staining of tissue and cell populations, neural crest stem cells were shown to be present in the OM. In addition, the obtained cell populations and whole OM express several neurotrophin genes, suggesting their potential neuroprotective ability.

Commercial Relationships: Maayke Kuijten , None; Claire Ginn ,

None; Peng Khaw , University College Moorfields (P); G Astrid

Limb , None; Steve Brocchini , None

Support: UCL School of Pharmacy, NIHR Moorfields Biomedical

Research Centre, Helen Hamlyn Trust, John Nolan, Michael and Ilse

Katz Foundation, Fight for sight

©2014, 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

.




Biological and synthetic membranes for human induced pluripotent stem cell-based transplantation therapy

Joe Phillips 1, 2 , Eric Clark 1 , Enio T. Perez 1

Patrick M. Barney 1 , Luke Beardslee 3

, Samantha T. Reshel 1

,

,

Norman D. Radtke 4

, Dyson Hickingbotham 4

, Magnus Bergkvist 3 , David M. Gamm 2,

5 . 1 Waisman Center, University of Wisconsin, Madison, WI;

2 McPherson Eye Research Institute, University of Wisconsin-

Madison, Madison, WI; 3 College of Nanoscale Science and

Engineering, University at Albany-SUNY, Albany, NY; of Louisville, Louisville, KY; 5

4 University

Department of Ophthalmology and

Visual Sciences, University of Wisconsin-Madison, Madison, WI .

Purpose: Scaffolds may improve transplantation therapy by providing an organized platform for cell delivery. In this study, we evaluated two membranes for human induced pluripotent stem cell (hiPSC)-based transplantation therapy, an acellular biological membrane derived from porcine small intestine submucosa (SIS), as well as a synthetic micro-patterned membrane (SU-8).

Methods: SIS membrane (Cook Biotech), a porous extracellular matrix sheet roughly 15 m m thick, was cultured using Snapwell transwell inserts (Corning). SU-8 membranes were generated with standard photolithography techniques, resulting in free-standing porous microstructures that were 8 m m thick with 5 m m pores. Both membranes were pretreated with laminin, and then coated with hiPSC-derived RPE or hiPSC-derived neural retina (NR) cells.

Cellular gene and protein expression was monitored with RT-PCR and immunocytochemistry. Cell growth, viability, and polarity were analyzed by standard light and confocal microscopy. Subretinal insertion of cell-impregnated membranes (1mm in diameter) into the

Long Evans rat retina was performed using a patented transplantation device (Eyevation). Human cells within the rat retina were identified with a human cytoplasm specific antibody (Stem Cell Inc.).

Results: Both membranes promoted cell adhesion and growth in vitro. hiPSC-RPE formed polarized, pigmented monolayers and expressed mature markers of RPE. hiPSC-NR cells, including numerous RCVRN+ photoreceptors, grew in layers on both membranes and expressed characteristic NR markers. Following transplantation, the SIS membrane was generally well tolerated by the host rat retina, although Muller glia became activated. Both hiPSC-NR cells and hiPSC-RPE transplanted on the SIS membrane survived for up to one month in vivo, the latest time point examined.

The SU-8 membrane has also been successfully transplanted and analysis is currently underway.

Conclusions: We demonstrate that both synthetic and biological scaffolds permit cell adherence, growth, and differentiation of hiPSC-RPE and NR in vitro. Furthermore, both can be transplanted into the rat retina. These scaffolds provide an organized structure for hPSC-based cellular transplantation and may also improve in vitro modeling.

Commercial Relationships:

Enio T. Perez

Barney , None;

Eyevation (P);

Bergkvist

Support:

, None; Samantha T. Reshel

Luke Beardslee

Norman D. Radtke

, None;

Joe Phillips

, None;

David M. Gamm

Dyson Hickingbotham

, Eyevation (P);

, None

, None;

Foundation, Choroideremia Research Foundation

Eric Clark

, None; Patrick M.

Magnus

, None;

Institute (Sandra Lemke Trout Chair), Carl and Mildred Reeves

,

Foundation Fighting Blindness Wynn-Gund Translational

Research Award, Retina Research Foundation (Kathryn and Latimer

Murfee and Emmett A. Humble Chairs), McPherson Eye Research

Foundation, NIH P30HD03352, Muskingum County Community



Generation and characterization of retinal ganglion cells from glaucoma patient iPSCs

Yvonne Ou 1 , Karen L. Chu 1 , Erik M. Ullian 1, 2 . 1 Ophthalmology,

University of California, San Francisco, San Francisco, CA;

2 Physiology, University of California, San Francisco, San Francisco,

CA .

Purpose: Glaucoma, the leading cause of irreversible blindness worldwide, is a degenerative optic neuropathy characterized by gradual loss of peripheral vision and eventual blindness. The underlying cause of visual dysfunction is the progressive loss of retinal ganglion cells (RGCs) and their axons. Recently, retinal ganglion cell-like cells have been derived from human embryonic stem cells and human induced pluripotent stem cells (hiPSCs).

However, the proportion of cells in these cultures that are iPSCderived RGCs (iPSC-RGCs) is typically low. Here we describe a modified protocol based on existing methods to increase the proportion of iPSC-RGCs derived from glaucoma patients in order to establish a human glaucoma cell culture model.

Methods: Skin biopsies were obtained from glaucoma patients seen at the UCSF Department of Ophthalmology’s Glaucoma Service.

Multiple hiPSC lines were established using nonintegrating episomal reprogramming vectors consisting of OCT 3/4, c-Myc, KLF4, and

SOX2 . The glaucoma patient-derived hiPSCs were differentiated into

RGCs in the presence of various BMP and Wnt inhibitors, as well as IGF-1 and Sonic hedgehog (Shh). The retinal progenitor cells and differentiated neurons produced were then assayed using RT-PCR and immunofluorescence microscopy.

Results: Glaucoma patient-derived hiPSC lines were established and then differentiated into retinal progenitors as assayed by expression of eye-field transcription factors. The addition of DAPT, a Notch signaling pathway inhibitor, increased the proportion of cells expressing neuronal and RGC-specific markers. The inclusion of Shh increased the proportion of Brn3a-expressing cells in a concentration dependent manner (25.4% at 100 ng/ml vs. 11.4% at 10 ng/ml; p<0.05). These glaucoma patient-derived iPSC-RGCs were also capable of forming anatomic synapses by synaptic marker expression.

Conclusions: We have modified existing protocols to enhance the differentiation of RGCs from glaucoma patient-derived hiPSCs, providing an abundant source of cells for the establishment of an in vitro cell culture model of human glaucoma.

Commercial Relationships: Yvonne Ou , None; Karen L. Chu ,

None; Erik M. Ullian , None

Support: Alcon Research Institute Young Investigator grant,

Research to Prevent Blindness Career Development Award, NIH-NEI

Core Grant EY002162



Spontaneously Differentiated Human Embryonic Stem Cell derived Retinal Pigment Epithelium Cells express RPE-65 and

ZO-1 proteins

K V Chalam, Sankarathi Balaiya, William Scott, Lee R. Ferguson.

Ophthalmology, Univ of Florida-Jacksonville, Jacksonville, FL .

Purpose: To determine the protein expression profile of the retinal pigment epithelium-65 (RPE65) and zonular occludins-1 (ZO-1) markers during spontaneous maturation of precursor cells into human embryonic stem cell derived RPE (hESC-RPE)

Methods: WA09-DL-11 feeder dependent human embryonic stem cells (hESC) were seeded onto a confluent layer of Mitomycin

C inactivated mouse embryonic fibroblasts. hESC growth and differentiation was monitored for the presence of pigmented



.

ARVO 2014 Annual Meeting Abstracts embryoid body (EB) development. Pigmented EB clumps were isolated following dissection from hESC colonies. EBs were seeded onto 6-well gelatin coated plates and allowed to form an expansive monolayer of hESC-RPE cells. Cells were then passaged 2 – 3 weeks after plating EB. Following passage cells were re-plated onto four gelatin coated wells. Each well represented different time points of extraction: 5, 13, 21, and 28 days post passage. Cells were lysed and then centrifuged to acquire supernatant proteinaceous material.

Cellular lysate supernatant was then quantified and subjected to western blot analysis. Antibodies to RPE65 and ZO-1 were used for the analysis. Following western blot procedure densitometry was performed to quantify protein expression at various time points.

Results: hESC-RPE post passage day 5 densitometry value was

1782.841 optical density units (odu) for RPE65 and 295.021 odu for

Zo-1. On day 13 there was a 1.4 fold increase for RPE65 (2556.024 odu) and 16.5 fold change in expression for ZO-1 (4856.589 odu).

Protein expression continued to rise for RPE65 and ZO-1, as day 21 levels showed a 1.6-fold (2832.953 odu) and 20.5-fold (6052.439 odu) increase, respectively, when contrasted to day 5 values. Finally on day 28, expression profiles plateaued as RPE65 levels only showed a 1.2-fold increase from baseline (2303.79 odu), while ZO-1 demonstrated a 20.4-fold increase from baseline (6019.196 odu).

Conclusions: demonstrates RPE-65 and ZO-1 expression as early as 5 days with a plateau apparent at day 28. Further investigation into days prior to day 5 and after day 28 are needed for precise delineation of onset of expression for these two markers. This study demonstrates the pattern of RPE65 and ZO-1 expression, which provides further insight into the proper characterization of hESC-RPE cells as true RPE cells.


Balaiya

Precursor marker characterization of hESC-RPE

, None;

K V Chalam

William Scott , None;

, None; Sankarathi

Lee R. Ferguson , None



Small Molecules and Adult Ciliary Epithelium Cells

Reprogramming

Carolina B. Del Debbio, Dânia E. Hamassaki. Cell and

Developmental Biology, University of Sao Paulo, Sao Paulo, Brazil .

Purpose: The Ciliary Epithelial cells (CE) of adult mammalian eyes are a quiescent population of cells able to proliferate and generate neurospheres with retinal progenitor profile. Despite the potential of CE to generate retinal progenitors, the efficiency is still low, probably due to the inefficient reprogramming and the presence of reminiscent epithelial parental properties. It is known that small molecules have been effective in stem and iPS cells reprogramming and, for that reason, we evaluate the effect of two small molecules on CE reprogramming efficiency,the Compound C (CC), inhibitor of

AMP-activated protein kinase and bone morphogenetic protein, and

NSC23766, a Rac1 GTPase inhibitor

Methods: After 4 consecutive intraocular injections of the small molecules (CC =10ng/eye, NSC = 200ng/eye) in adult Wistar rats, we dissected the CE, sectioned in cryostat or extracted the RNA and synthesized the cDNA. Later, the expression of the pluripotent genes c-Myc, Oct4 and Sox2 was analyzed. For in vitro experiments,

CE cells were dissected and cultured in the presence of growth factors (FGF and EGF), to form neurospheres, and small molecules.

Proliferation rates and progenitor profile were analyzed

Results: Our data indicated that CC injections increased the expression of c-Myc and Oct4 (6 and 14 folds change (fc), respectively), in comparison to controls (PBS), but no effect was observed in Sox2 expression. Rac1 inhibition increased the expression of c-Myc and Oct4 (5 and 9 fc), however, Sox2 expression was decreased (4 fc). Furthermore, Rac1 inhibition increased CE cells proliferation rates observed through Ki67 immunostaining

(32.8

± 0.20, control = 5.10

E. Hamassaki

Support:

, None

FAPESP, CNPq, and Pró-Reitoria de Pesquisa/ USP

(NAPmir and NAPNA)

± 0.87), and quantitative PCR (5 fc).

Methyltransferase transcripts analysis indicated that both molecules decreased the expression of DNMT1 and DNMT3b (CC = 1.5 and 1.2 folds; NSC = 1.6 and 2 folds), indicating the regulation of epigenetic factors. Neurospheres treated in the presence of small molecules showed an apparently increased expression of proliferation transcripts (ki67 and cyclins), and decreased epithelial properties genes (palmdelphin and Rab27), in comparison to controls

Conclusions: Our results suggest that the use of small molecules can be efficient for CE reprogramming, and the information gleaned from this study may provide valuable insight into the cellular and molecular events that underlie the reprogramming response of CE cells and the mechanism of retinal recovery

Commercial Relationships: Carolina B. Del Debbio , None; Dânia



Evaluation of Degree of Pigmentation as an Indicator of

Maturation status in Human iPSC-RPE

Hiroyuki Kamao 1, 2 , Michiko Mandai

Kiryu 1 , Masayo Takahashi 2

2 , Katsutoshi Goto 1 , Junichi

. 1 Ophthalmology, Kawasaki Medical

School, Kurashiki, Japan; 2 Laboratory for Retinal Regeneration,

RIKEN Center for Developmental Biology, Kobe, Japan .

Purpose: In transplantation of human induced pluripotent stem cell derived retinal pigment epithelium (hiPSC-RPE), the determination of maturation status of these cells is an essential factor and the degree of pigmentation (dPG) can serve as a good indicator. The aim of this study was to establish a method of evaluating dPG in a group of hiPSC-RPE objectively and quantitatively.

Methods: Bright field images of hiPSC-RPE were recorded sequentially (0w, 2w, 4w, 6w after hiPSC-RPE reached confluence) under the same condition. Two observers, expert and inexpert, determined dPG subjectively by observation the recoded images as follows: a single cell dPG was classified into three different stages

(low, mediate, high) and the overall dPG was compared between two cell groups to choose the one with higher dPG. The κ coefficient was applied to assess inter-observer reproducibility. Subsequently, dPG was objectively determined in single cells and cell groups by converting the recorded images into those with 256 gray-levels and the correlation with the subjective evaluation and time dependent change of dPG was investigated.

Results: The κ coefficient was 0.96 and 0.87 in a single cell and cell group observation respectively, meaning that inter-observer reproducibility of dPG was excellent. The objective dPG as gray-level of single cells highly correlated with the subjectively determined dPG (low: 16.03, mediate: 71.11, high: 131.55, P <

0.001). However, in comparison between 2 cell groups, the subjective determination of high dPG cell groups was not a complete match with objective determination and sometimes the observers could not distinguish dPG between 2 cell groups (dPG subjective/objective: high/high 86%, high/low 3% undetermined 11%). The objective dPG in cell groups increased in a time-dependent manner (0w: 27.21, 2w:

51.85, 4w: 80.25, 6w: 90.03, P < 0.001).

Conclusions: culture.

The determination of dPG in cell groups of hiPSC-

RPE using gray-scale image was reliable and excelled subjective difficulties in monitoring hiPSC-RPE maturation indicated by dPG in



.



Mandai , None; Katsutoshi Goto

Masayo Takahashi , None

Hiroyuki Kamao

, None;

, Fizer (F);

Junichi Kiryu

Michiko

, Fizer (F);



Derivation, Characterization and Retinal Neural Induction of

Human Tenon’s -Derived iPS Cells

Jian Ge, Deng Fei, Liu Ying, Xiong Yunfan. Glaucoma, Zhongshan

Ophthalmic Center, Guangzhou, China .

Purpose: To determine if induced pluripotent stem cells (iPSCs) derived from human Tenon’s capsule fibroblasts (HTFs) could express retinal progenitor cell (RPC)-related genes with the capacity to directly differentiate into retinal neurons in vitro.

Methods: HTFs harvested from fresh samples were reprogrammed by retroviral transduction to iPSCs. The HTF-derived iPSCs (TiPSCs) were characterized for pluripotency by morphology, gene expression, surface antigens, alkaline phosphatase activity analysis and a teratoma formation assay. Human ESC colonies were used as the positive control. The resulting TiPSCs were induced to differentiate into retinal cells by stepwise treatment with the defined factor combination of Dkk1,Noggin,Lefty-A, DAPTand overexpression of

ATOH7, which follows the human retinal development timeline. The induced retinal cells were analyzed with phase contrast microscopy, real-time PCR, immunofluorescence, FACS analysis, and calcium imaging analysis.

Results: The resulting TiPS colonies were indistinguishable from human ESC colonies according to standard criteria. Upon retinal differentiation, embryoid bodies (EBs) were formed from TiPSCs by suspension culture in serum-free medium with the increase of

RPC-related gene expressions such as Pax6, Sox2 and Nestin. In matrigel-coated culture, acquisition of neuroepithelial colonies at day

10 with an early eye field fate was enhanced through the addition of DKK1, NOGGIN and Lefty-A, as determined by qPCR and immunofluorescence. Further overexpression of ATOH7 combined with DAPT-treated cultures, TiPSCs can generate retinal neural cells that express Chx10,Brn3b, Atoh7, Syn,Crx,Mitf,GFAP,etc.

Conclusions: generated from HTFs and through a stepwise neural differentiation strategy, the TiPSCs can generate retina-specific cells in vitro, which should aid in the investigation of ophthalmological regenerative medicine.


Ying , None;

Support:

These findings demonstrate that iPSCs can be

Xiong Yunfan

Jian Ge

, None

, None; Deng Fei , None;

NSFC Grant 81170846; NSFC Grant 30973266

Liu



Epigenetic and Transcriptional Regulation of RAX in Retinal

Fate Determination using Human Induced Pluripotent Stem Cells

Akshayalakshmi Sridhar 1 , Sarah Ohlemacher 1

1

, Jason S. Meyer 1, 2 .

Biology, Indiana Univ Purdue Univ Indianapolis, Indianapolis, IN;

2 Stark Neurosciences, Indianapolis, IN .

Purpose: Human pluripotent stem cells (hPSCs) allow for the unprecedented ability to study the earliest events in human retinal fate determination in vitro . Previous studies have been successful in the derivation of retinal cell types from hPSCs, however the mechanism of retinal specification from an unspecified pluripotent population remains to be clearly defined. Thus, efforts were undertaken to better elucidate the transcriptional and epigenetic mechanisms underlying retinal fate specification from hPSCs.

Methods: Following the differentiation of hPSCs to neural and retinal lineages, expression patterns of candidate transcription factors were characterized using immunocytochemistry and qRT-PCR. Based on these studies, the transcription factor RAX (retinal and anterior neural fold homeobox) was identified as influential in retinal fate due to its early widespread expression followed by restricted expression in retinal progenitor cells. To further investigate the role of RAX, gene overexpression and shRNA mediated knockdown approaches were undertaken. Furthermore, methylation analyses of CpG islands in the RAX promoter region were investigated at various stages of retinal development to better elucidate epigenetic modifications associated with early retinal fate determination.

Results:

Sight

Analyses by immunocytochemistry and qRT-PCR demonstrated that the expression of RAX peaked early and then became restricted to a subpopulation of cells, specifically retinal progenitor populations identified by subsequent transcriptional analysis. Additionally, the expression of RAX was subsequently lost from non-retinal neural populations. Methylation analysis of the

RAX promoter region suggested that epigenetic mechanisms could be regulating the expression of RAX in the establishment of a retinal fate. The ability of RAX to regulate the adoption of a retinal fate was subsequently tested through lentiviral-mediated overexpression and shRNA experiments.

Conclusions:

Support:

Overall, the results of this study help to elucidate the role of RAX in the establishment of a retinal fate. These studies will assist in the establishment of more efficient methods to generate retinal cells from hPSCs for translational purposes, and serve to further establish hPSCs as an important in vitro model system for studies of the earliest stages of human retinal development.


Sarah Ohlemacher , None;

Akshayalakshmi Sridhar

Jason S. Meyer , None

, None;

BrightFocus Foundation Grant No. G2012027, Fight for


Presentation Time: 8:30 AM–10:15 AM c-Kit positive cells isolated from human fetus eyes are a new population of stem cells

1

Guang-hua Peng 1, 2 , Pengyi Zhou 2 , Haiwei Xu 1 , Zhengqin Yin 1 .

Ophthalmolgy, Ophthalmology, Xinan Hospital, Chongqing, China; 2

Zhengzhou Unervisity, Zhengzhou, China .

Purpose: Despite intensive research on the potential use of retinal progenitor cells in the treatment of degenerative eye diseases, the definitive markers for these prognitors are still lacking. Our current study focused on CD117 (c-kit), a cell surface marker for haematopoietic stem cells and progenitor cells. Retinal progenitor cells were isolated from human fetus and the c-kit + and SSEA4- retinal cells were sorted out for further biological characterization

Methods: and GFAP.

hRPCS were isolated from human retina of 10 to 16 weeks gestational age (GA). Immunohistochemical staining was performed using antibody against c-kit + to determine the distribution of c-kit+/

SSEA-4- cell in the eyes of fetus. The c-kit + / SSEA4- retinal progenitor cells sorted out by flow cytometry were subjected to cell-mediated immunity fluorescent and cell cycle analysis for their capablity to proliferation and differentiation.

Results: A few c-kit + / SSEA4- cells were detected in the inner part of fetal retina. Sorted c-kit + / SSEA4- cells expressed some retinal stem cell markers including Pax6, Sox2 and Nestin. Over 80% of the cells expressed ki67, and cell cycle analysis demonstrated that more than 40% of the cells were in their proliferation phase. These results clearly demonstrate proliferation property of these cells. When cultured in differentiation medium, these cells expressed markers found in photoreceptor cells and glial cells such as CRX, recoverin



.


Conclusions: c-kit can be used as a surface marker for retinal progenitor cells. The c-kit + / SSEA4- retinal progenitor cell isolated fetal eyes exhibit the ability to self-renew and differentiate into retinal cells.

Commercial Relationships: Guang-hua Peng , None; Pengyi Zhou ,

None; Haiwei Xu , None; Zhengqin Yin , None

Support: National key basic research program of china(Project

No.2013CB967001 to Peng)



Allogenic iPSC-derived RPE cell transplants induce immune response in pigs

2

Elliott H. Sohn 1, 2 , Chunhua Jiao 2 , Robert Mullins 2 , Edwin M. Stone 1,

, Budd A. Tucker 2

Iowa City, IA; 2

. 1 Retina Service, Univ of Iowa Hosp & Clinics,

Stephen A. Wynn Institute for Vision Research,

University of Iowa Dept of Ophthalmology, Iowa City, IA .

Purpose: Stem cell strategies focused on replacement of RPE cells for the treatment of geographic atrophy are under intense investigation. Although the eye has long been considered immune privileged, a limited number of large animal studies focused on the post-transplant immune response have been performed. The purpose of this study was to determine if allogenic iPSC-derived RPE cells delivered to the subretinal space of the pig would survive and fail to induce an immune response in non-diseased eyes.

Methods: 250,000 iPSC-derived RPE cells, generated from GFPpositive outbred domestic swine, were injected subretinally into

12-week-old vitrectomized Yucatan mini swine (a subset of eyes received BSS vehicle control only). Eyes were enucleated at 3 weeks post-op, fixed in 4% paraformaldehyde, cryosectioned and immunostained with antibodies targeted against GFP, ZO1, macrophages (BA4D5), CD45, GFAP, nestin, Ki67, and PCNA.

Vitreous samples extracted at the time of vitrectomy and again at post-op week 3 were assayed for cytokine levels using a swine cytokine Quantibody array kit (RayBiotech, Inc). Data were analyzed using Student’s t-test and one-way ANOVA followed by Fisher’s

LSD test.

Results: GFP-positive cells expressing the RPE marker ZO-1 were identified in the subretinal space at 3 weeks post-injection.

Accompanying GFP-negative cells positive for CD45 and macrophage markers were also identified. All cells were negative for

GFAP as well as the cell cycle markers nestin, Ki67, and PCNA. At post-op week 3, vitreous TGF-beta1 levels were elevated in the iPSC-

RPE group compared to BSS controls and native vitreous. IL-12 levels were greater in post-op week 3 compared to native vitreous but not post-op week 3 BSS controls.

Conclusions: Subretinal injection of allogenic iPSC-RPE cells into wild-type mini-pigs can induce a positive immune response. These findings suggest that immunologically matched or autogenic donor cells may be required for clinical RPE cell replacement.

Commercial Relationships: Elliott H. Sohn , None; Chunhua Jiao ,

None; Robert Mullins , None; Edwin M. Stone , None; Budd A.

Tucker , None



Xeno-free 3D retinal differentiation of human inducedpluripotent stem cells

Ramesh Kaini, Anthony J. Johnson, Teresa A. Burke, Dallas Golden,

Heuy-Ching H. Wang. Ocular Trauma, USAISR, Fort Sam Houston,

TX .

Purpose: Advances made in recent years to generate different retinal precursor cells from pluripotent stem cells have instilled hopes for cell replacement therapy in retinal degeneration diseases and retinal trauma. However, stem cells must be derived, maintained and differentiated in xeno-free condition for potential clinical use in human. In this study, we sought to differentiate human inducedpluripotent stem (iPS) cells towards neural retinal lineage and produce clinical-grade retinal progenitor cells.

Methods: Commercially available iPS cells, IMR90-4, were maintained in VitronectinXF coated culture plates with xenofree TeSR-E8 medium. iPS cells were dissociated and quickly reaggregated using Sumilon PrimeSurface96V culture plates in

GMEM medium containing 20% KnockOut Serum Replacement

XenoFree . To start the xeno-free 3D differentiation, VitronectinXF

(10 ug/ml) was added in the retinal differentiation medium from day 2 onwards to day 18. 3D cell aggregates were treated with Wnt inhibitor, IWR-1-endo, for the first 12 days. KnockOut SR Growth

Factor cocktail was added on day 12 and Smoothened Agonist (SAG) on day 15. Aggregates were then switched to DMEM/F12-Glutamax



.

ARVO 2014 Annual Meeting Abstracts medium with N2 supplement on day 18. Expression of different markers of eye field and retinal progenitor cells were studied at different time points.

Results: iPS cells 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.

Aggregates of xeno-free 3D differentiation started expressing different neural and eye field markers, including Otx2, Sox2, Rx,

LHX2, Six6, PAX6, MITF, and CHX10 at different time points of differentiation. Retinal progenitor cell marker, CHX10 was observed on day 16 onwards.

Conclusions: In this study, we observed that neural retinal lineage cells can be derived from human iPS cells in a 3D culture system using defined, xeno-free components. We are in the process of developing stratified neural retina from iPS cells under defined, xenofree condition and generating different retinal precursor cells for cell replacement therapy.

Commercial Relationships: Ramesh Kaini , None; Anthony J.

Johnson , None; Teresa A. Burke , None; Dallas Golden , None;

Heuy-Ching H. Wang , None

Support: National Research Council Research Associateship

Program, US Army Clinical and Rehabilitative Medical Research

Program (CRMRP)



Ex vivo evaluation of intravitreal mesenchymal stem cell viability using bioluminescence imaging

Marcelo J. Silva, Priscila C. Ferreira, Rubens C. Siqueira, Rodrigo

Jorge, Andre Messias, Maria L. Rodrigues, Rodrigo J. Calado,

Dimas T. Covas, Jayter S. Paula. Opthalmology, FMRP University of

São Paulo, Ribeirão Preto, Brazil .

Purpose: eye.

Methods: m

Bone marrow-derived mesenchymal stem cells (MSC) therapy is a promising treatment for several degenerative diseases, including retinopathies and glaucoma, however no reproducible method of monitoring these cells into the eye has been established.

The aim of this study was to describe a successful bioluminescence imaging (BLI) to detect viable luciferase-expressing MSC into the

Human donors’ MSC in culture were infected with 50ul of luciferase lentiviral vector (3,000 viral particles/cell) prior to intraocular injections. Three eyes of two rabbits were evaluated through BLI after receiving 1x106 luciferase-expressing MSC intravitreally with (E1) or without (E2) D-luciferin (40mg/ml - 300

L of PBS), via pars plana. D-luciferin (40mg/ml – 300 method to address these issues.

m L of PBS) without cells was injected in a third eye at beginning (E3) and after one hour in E2.

Results: E1 showed high BLI radiance report and decay in eight hours. After D-luciferin infusion, E2 also displayed high average radiance, with similar decay rate of E1. No signal was observed in E3. Figure 1 show the bioluminescence imaging acquired from the three experimental eyes and the figure 2 show the distribution of total amount of captured photons from the three eyes, using bioluminescence, during the eight hours period.

Conclusions: Identification of cell location and viability is still an important problem regarding the use of MSC for eye diseases. This is maybe the first ex vivo study demonstrating BLI is useful and reliable

Commercial Relationships: Marcelo J. Silva , None; Priscila C.

Ferreira , None; Rubens C. Siqueira , None; Rodrigo Jorge , None;

Andre Messias , None; Maria L. Rodrigues , None; Rodrigo J.

Calado , None; Dimas T. Covas , None; Jayter S. Paula , None



Upregulation of the Notch and Wnt signalling pathways by HB-EGF in adult human Müller stem cells in vitro

Angshumonik Angbohang, Karen Eastlake, Silke Becker, Na Wu,

G Astrid Limb. National Institute for Health Research (NIHR)

Biomedical Research Centre at Moorfields Eye Hospital NHS

Foundation Trust and UCL Institute of Ophthalmology, UCL,

London, United Kingdom .

Purpose: Müller glial stem cells (MCSs) spontaneously regenerate the retina of adult zebrafish after injury. MSCs can be isolated from adult human eye and induced to proliferate in vitro by HB-EGF stimulation. HB-EGF has been shown to initiate MSC derived retinal regeneration by activating the Notch and Wnt signalling pathways in the zebrafish. Since Notch inhibition in human MSC promotes their differentiation into retinal ganglion cells (RGCs) in vitro, and activation of the Wnt signalling pathway can promote MSC growth, it would be important to investigate whether HB-EGF activation of these cells may trigger interactive signals between the Notch and

Wnt signalling pathways that can control adult stem cell growth and differentiation in vitro

Methods: Müller stem cells were cultured on matrigel coated flasks in DMEM media containing 2% foetal calf serum in the presence or absence of HB-EGF and γ -secretase inhibitor RO4929097. mRNA and protein isolated from these cells were examined for



.

ARVO 2014 Annual Meeting Abstracts expression of molecules of the Notch and Wnt signalling pathways and RGC marker Brn3b using RT-PCR and Western-blot analysis.

Morphological changes were examined by phase contrast microscopy.

Results: Culture of Müller stem cells with exogenous HB-EGF, caused an increase in gene expression of the Notch downstream target Hes1 and the canonical Wnt signalling components Wnt2b and β -catenin. We found that the γ -secretase inhibitor RO4929097 induced down-regulation in HB-EGF gene expression, as well as in gene and protein expression of the Notch downstream target

Hes1. MSCs cultured with RO4929097 acquired a neuronal-like morphology, displaying a very thin elongated cytoplasm with bright phase cell body and the formation of multiple neurites. It also induced up-regulation of Brn3b gene expression, a marker of RGC.

The Wnt signalling ligand Wnt2b gene and protein expression were also down-regulated in these cells by this inhibitor.

Conclusions: The results showed that HB-EGF causes activation of both the Notch and Wnt signalling pathways in adult human

Müller stem cells. Since this appears to be an evolutionary conserved interaction observed during the spontaneous retinal regeneration observed in zebrafish, it would be important to investigate whether activation of these pathways is suppressed in the adult human eye and whether they can be modulated to induce endogenous retina regeneration in the adult human eye.

Commercial Relationships: Angshumonik Angbohang , None;

Karen Eastlake , None; Silke Becker , None; Na Wu , None; G

Astrid Limb , None

Support: UCL Biomedicine Grand Challenge Studentship


Presentation Time: 8:30 AM–10:15 AM microRNAs regulate periodontal ligament-derived stem cell retinal differentiation

Tsz Kin Ng 1, 2 , Kwong Wai Choy 3 , Hoi Kin Wong 3 , Chi Pui Pang 1 ,

Herman S. Cheung 2, 4 .

Kong;

1 Department of Ophthalmology & Visual

Sciences, The Chinese University of Hong Kong, Kowloon, Hong

2 Geriatric Research, Education and Clinical Center, Miami

Veterans Affairs Medical Center, Miami, FL; 3 Department of

Obstetrics & Gynaecology, The Chinese University of Hong Kong,

Shatin, Hong Kong; 4 Department of Biomedical Engineering,

University of Miami, Miami, FL .

Purpose: To determine the microRNA (miRNA) signature of human adult periodontal ligament stem cells (PDLSC) retinal differentiation.

Methods: Human adult PDLSC were induced to the retinal lineage using the noggin-Dkk1-IGF-1 approach. The miRNA expression was analyzed by the miRNA microarray technique and validated by

TaqMan assay. The predicted miRNA target genes were analyzed by gene ontology.

Results: A total of 71 human miRNAs were differentially expressed before and after retinal induction, which 44 of them were upregulated and 27 were downregulated. Of the 5 selected miRNAs, 4 miRNAs

(hsa-miR-132, hsa-miR-29b, hsa-miR-630 and hsa-miR-7) were validated. Gene ontology analysis of the predicted miRNA target genes confirmed the induction treatment closely related to neuronal differentiation processes.

Conclusions: The expression changes of miRNAs and their target genes during retinal induction process revealed the genetic and epigenetic regulatory mechanism for human adult stem cell differentiation.

Commercial Relationships: Tsz Kin Ng , None; Kwong Wai Choy ,

None; Hoi Kin Wong , None; Chi Pui Pang , None; Herman S.

Cheung , None

Support: The VA Merit Review Grant and the Senior VA Research

Career Scientist Award, Miami, and a block grant of the University

Grants Committee Hong Kong, Hong Kong



Modulation of the Notch and Wnt signalling by TGF- β in adult human Müller stem cells.

NA WU 1, 2 , Joseph Wiseman 1 , Yuan Lei 2 , Karen Eastlake 1 , Xinghuai

Sun 2 , G Astrid Limb 1 . 1 National Institute for Health Research

(NIHR) Biomedical Research Centre at Moorfields Eye Hospital

NHS Foundation Trust and UCL Institute of Ophthalmology, UCL,

London, United Kingdom; 2 Department of Ophthalmology and Visual

Science, Eye & ENT Hospital, Shanghai Medical College, Fudan

University, Fudan University, Shanghai, China .

Purpose: The transforming growth factor β (TGF- β ) and the Wnt,

Notch signalling pathways all play crucial roles in many biological processes, including embryonic development, cell fate differentiation and cell proliferation. There is evidence for a crosstalk between the

Notch and Wnt pathways during retinal development, but it is not known whether co-activation of these pathways may occur in adult human Müller stem cells (hMSCs) or whether these can be modulated by TGF- β .It was therefore the aim of this study to investigate the effect of this cytokine on the expression of molecules involved in these two pathways

Methods: Human Müller stem cells were cultured on matrigel using

DMEM containing foetal calf serum (FCS). hMSCs were cultured in the absence or presence of the γ -secretase inhibitor DAPT plus bFGF to induce their differentiation into RGCs. Exogenous TGF- β 1 or

TGF- β 3 were added to the differentiating or control cells which were cultured for up to 7 days. mRNA and protein extracted from these cells were examined by RT-PCR, western-blot and immuno-staining techniques for the expression of molecules of the Notch and Wnt signalling pathways. The hexosaminidase assay and Ki67 immunostaining were used to test the effect of TGF- β on cell proliferation.

Results: Notch inhibition by DAPT caused down-regulation in gene expression of Hes1 and Wnt 2b. Similarly, TGF- β 1 alone caused a decrease in gene and protein expression of the Notch downstream target Hes1 and the Wnt signalling ligand Wnt2b, while inducing an increase of the canonical Wnt signalling intracellular component

β -catenin. Down-regulation of these genes by DAPT was not modified by addition of TGF- β 1 or TGF- β 3 to the cultured cells.

It was also observed that both TGF- β 1 and TGF- β 3 had a similar effect on the inhibition of cell growth.

Conclusions: These results provide evidence that TGF- β directly induces down-regulation of the Notch target Hes1 and the Wnt signalling ligand Wnt2b, as well as upregulation of β -catenin.

Furthermore, down-regulation of Notch by DAPT results in downregulation of Wnt2b, suggesting a crosstalk between these two signalling pathways. However, TGF- β does not have additive effects on the down-regulation of Hes1 and Wnt2b by DAPT. The observations suggest that these two molecules may activate similar intracellular pathways mediating neural differentiation of Müller stem cells.

Commercial Relationships: NA WU , None; Joseph Wiseman ,

None; Yuan Lei , None; Karen Eastlake , None; Xinghuai Sun ,

None; G Astrid Limb , None

Support: MRC & National Natural Science Foundation of China



.




Comparison of viral and mRNA-reprogrammed human induced pluripotent stem cells for retinal differentiation

Jason S. Meyer 1, 2

Ohlemacher 1

Indianapolis, IN; 2

, Akshayalakshmi Sridhar 1 , Clara Iglesias 1 , Sarah

. 1 Biology, Indiana Univ- Purdue Univ Indianapolis,

Stark Neuroscience Research Institute, Indiana

University, Indianapolis, IN .

Purpose: The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risk of genomic integration or constitutive transgene expression due to viral delivery. The delivery of reprogramming factors by mRNA eliminates these risks and may provide a safer alternative, but the efficient retinal differentiation of such cells has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs for retinal differentiation.

Methods: Human fibroblasts were transfected daily with mRNAs encoding for reprogramming genes, whereas similar cultures of human fibroblasts were infected with retroviruses to deliver these genes in parallel. Pluripotency was confirmed by live cell staining for cell surface antigens and positive colonies were identified and manually isolated, yielding stable lines of hiPSCs. New lines of hiPSCs were differentiated to a retinal fate following established protocols, and the efficiency of retinal specification from hiPSCs was compared between the two systems at various stages of differentiation.

Results: Both mRNA and retroviral methods of reprogramming yielded stable lines of hiPSCs expressing numerous pluripotencyrelated characteristics as assessed by immunocytochemistry and

RT-PCR. Upon differentiation, no overt differences were found in the ability of these cells to adopt a retinal fate as assessed by the expression of retinal progenitor-associated genes. Within two months of differentiation, many types of retinal cells could be derived from both sources, including retinal pigment epithelium, photoreceptors, and retinal ganglion cells.

Conclusions: The data presented demonstrates the feasibility of utilizing mRNA-based reprogramming strategies to derive lines of patient-specific hiPSCs for purposes of retinal differentiation.

Differences in the differentiation capacity of these cells from both sources were not readily observed. Given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages because they lack the risks of genomic integration or constitutive expression, such methods likely represent a promising new approach for retinal stem cell research, particularly those for translational purposes.


Akshayalakshmi Sridhar

Ohlemacher

Support:

, None

Jason S. Meyer

, None;

, None;

Clara Iglesias , None; Sarah

BrightFocus Foundation Grant# G2012027, Fight for Sight



Differentiation and Characterization of Retinal Ganglion Cells

Derived from Human Pluripotent Stem Cells

Sarah Ohlemacher 1 , Jason S. Meyer 1, 2 . 1 Biology, IUPUI,

Indianapolis, IN; 2 Stark Neuroscience Research Institute, Indiana

University, Indianapolis, IN .

Purpose: Human pluripotent stem cells (hPSCs) possess the unique ability to readily differentiate into any cell type of the body. As such, they can serve as comprehensive and novel tools for drug screening, disease modeling, and cell replacement therapies. Although previous studies have demonstrated the ability to differentiate hPSCs to a retinal lineage, the ability to derive retinal ganglion cells (RGCs) from hPSCs has been largely overlooked to date. Establishing a method to acquire RGCs from hPSCs would serve as a novel system to study human retinogenesis as well as establishing a foundation for the development of patient specific therapies for diseases of retinal ganglion cells, such as glaucoma.

Methods: Following previously established protocols, hPSCs were induced to differentiate towards a retinal fate and RGCs were subsequently characterized by the RGC-associated transcription factors BRN3 and Islet-1. The developmental process underlying this

RGC differentiation was further analyzed by immunocytochemistry and RT-PCR analysis for the expression of expected RGC associated characteristics.

Results: Within the first 40 days of differentiation, RGCs were readily identifiable within differentiating cultures of hiPSCs based on their progression through a CHX10-positive retinal progenitor intermediary as well as their later adoption of the RGC-specific transcription factor BRN3. Analysis of these differentiating cultures at various timepoints by immunocytochemistry and RT-PCR analysis revealed that these cells expressed numerous characteristics of

RGCs, including the expression of MATH5 and PAX6, as well as morphological characteristics associated with RGCs. In addition, treatment with intrinsic and/or extrinsic factors were tested for their ability to modulate RGC specification.

Conclusions: The data presented within this study demonstrates the ability of hiPSCs to serve as a reliable source of patientderived RGCs, as seen by their ability to proceed through predicted developmental stages that yield a mature RGC population possessing many RGC-associated characteristics. This protocol will be advantageous for future studies into the normal and abnormal development of RGCs and as such, will be instrumental as a tool to study optic neuropathies that affect this cell population.


Meyer , WARF (P)

Support:

Sarah Ohlemacher , None; Jason S.

BrightFocus Foundation Grant #G2012027, Fight for Sight



Age-related Gene Expression Changes in the Murine Optic Nerve

Lamina

Yan Guo, Zara Meharabyan, Steven L. Bernstein. Ophthalmology,

Univ of Maryland Sch of Medicine, Baltimore, MD .

Purpose: The optic nerve lamina (ONL) is a unique optic nerve structure that borders the retina and optic nerve. The ONL is believed to play an important role in many optic nerve diseases, including nonarteritic anterior ischemic optic neuropathy (NAION) and open angle glaucoma (OAG). We recently demonstrated that some cells in the ONL possess the ability to develop into a number of different cell types, suggesting pluripotency. We utilized a panel of genespecific primers to examine both the pluripotency and types of cell differentiation in this region, and evaluated the relative changes in gene expression in different age group.

Methods: 10 wild type mice (C57BL/6J) each at 15 day, 6 months and >1year old were utilized for the analysis. The first two mm of the optic nerve head was utilized for the ONL, as well as tissue from the retina and posterior ON (posterior 3mm of optic nerve). mRNA was extracted using Qiagen RNaeasy Micro kit, and unbiased linear

RNA amplification using a single chimeric primer and isothermal amplification (Nugen) was performed, to enable sufficient material for multiple assays. Following cDNA generation, we performed quantitative PCR (qPCR). A variety of gene markers were used



.

ARVO 2014 Annual Meeting Abstracts including Nestin, Sox2, and Sox1 for progenitor cells, as well as a number of genes for each glial cell type and glial progenitor line.

Reactions were performed using SYBR green Super mix on an iCycler.

Results: qPCR revealed differential gene expression between the lamina and optic nerve, as well as between the different age groups.

Gli-1, Nestin and SOX-2 gene expression were considerably elevated in the lamina and ON, compared with the retina. In contrast, the highest levels of Olig-2 and MBP gene products, corresponding to mature oligodendrocytes, were found in the posterior ON region.

High levels of GFAP and s100 β expression were detected in posterior

ON, less in the lamina, and minimal levels expressed in the retina.

Conclusions: The ONL is a progenitor cell niche, whose capacity for self-renewal declines during aging. ONL gene expression is distinct from either the retina or ON. Our results suggest that the ONL lamina region possesses a multi-progenitor cell population that may give rise to the different glial cell lines, as well as possess some capacity for neuronal generation.


None; Steven L. Bernstein

Support: EY-015304

Yan Guo

, None

, None; Zara Meharabyan



Is Mesenchymal Stem Cell Homing To The Injured Retina

Required For Visual Preservation In The RCS Rat?

,

Benjamin Bakondi, YuChun Tsai, Bin Lu, Sergey Girman, Lin Shen,

Melissa K. Jones, Shaomei Wang. Regenerative Medicine Institute,

Cedars-Sinai, Los Angeles, CA .

Purpose: Retinal degeneration is slowed by intravenous (IV) injection of allogeneic bone marrow-derived mesenchymal stem cells (MSCs) prior to the onset of degeneration in the RCS rat.

However, the mechanism(s) for MSC-mediated retinal protection has yet been determined. It is unclear whether MSC homing to the retina is required for vision rescue and is the subject of the current investigation.

Methods: The predominant mechanism for MSC migration to tissue injury is mediated through the CXCR4 chemokine receptor, which was stimulated (SDF-1; 50ng/ml) or antagonized (AMD3100; 10uM) for 30 minutes prior to infusion of allogeneic MSCs (1.6 x 106 cells) into RCS rats on postnatal day 25. Optokinetic responses (OKR) and electroretinograms (ERG) were performed on postnatal day 60.

For cell tracking experiments, MSCs were labeled with a lipophilic fluorescent dye (PKH26 or PKH67; Sigma) prior to injection and cell distribution was determined 24 and 72 hours post-injection via flow cytometry and histological examination.

Results: SDF-1 pre-treatment enhanced the migration capacity of

MSCs in vitro, while AMD3100 pre-treatment reduced it, compared with control (saline) treatment. OKR and ERG recordings showed that injection of SDF-1 pre-treated MSCs preserved visual function in RCS rats compared with injection of saline or AMD3100-treated

MSCs. Fluorescent donor cells were not detected at or near the injury site at 24 or 72 hours, the typical timeframe for MSC chemotaxis.

MSCs were detected in the blood circulation, lungs, and bone marrow.

Conclusions: SDF-1 pre-treatment of MSCs enhanced the preservation of vision in the RCS rat. Further study is under way to determine whether MSC homing to the retina is required for visual preservation.

Commercial Relationships: Benjamin Bakondi , None; YuChun

Tsai , None; Bin Lu , None; Sergey Girman , None; Lin Shen , None;

Melissa K. Jones , None; Shaomei Wang , None

Support: NIH R01 EY020488-02, W81XWH-DOD, FFB, Fund from the Regenerative Medicine Institute at Cedars-Sinai Medical

Center



In vitro differentiation of human neuronal progenitor cells towards retinal pigment epithelium-like cells

Volker Enzmann 1, 2 , Luca Tamó 2, 3 , Carolyn Trepp 1, 2 , Sebastian Wolf 1 .

1 Ophthalmology, University of Bern, Bern, Switzerland; 2 Clinical

Pulmonary Research, University of Bern, Bern, Switzerland; 3

Medicine, University of Bern, Bern, Switzerland .

Purpose: Degeneration of the retinal pigment epithelium (RPE) is the main etiology of several retinal diseases. The use of stem/progenitor cells to replace the damaged tissue has been proposed recently. The aim of the study was to investigate whether immortalized human neuronal progenitor cells were able to differentiate towards RPE-like cells.

Methods: ReNcells, human cortical neuronal progenitor cells

(Millipore, Temecula, USA), were used for differentiation. The cells were incubated with RPE-conditioned medium (CM), pigment epithelium-derived factor (PEDF) or retinoic acid (RA) for up to 14 days. Gene and protein expression of stem (nestin), neuronal ( β IIItubulin), glial (GFAP) and RPE (RPE65, bestrophin) markers were analyzed by qRT-PCR and immunohistochemistry (IHC). Additional retinal genes (MITF, CRALBP, PAX-6, CHX-10) were investigated by qRT-PCR. RPE-related function after differentiation was tested by an in vitro phagocytosis assay.

Results: In comparison to undifferentiated ReNcells, nestin was still slightly upregulated in all differentiated cells on the gene level but downregulated on the protein level. On the other hand, β III-tubulin was upregulated on both levels under all treatment conditions. GFAP gene and protein levels were increased significantly in RA-treated cells only. RPE65 gene expression was upregulated in cells incubated with CM or RA. Gene expression of bestrophin was strongly upregulated under all conditions. IHC showed increased expression of these RPE markers in all differentiated cells. Gene expression of

CHX-10, MITF and CRALBP was upregulated in all differentiated cells, whereas PAX-6 was upregulated only in CM & RA-treated samples. Importantly, gene expression of RPE-related genes was frequently higher in differentiated cells than in the RPE controls. In the phagocytosis assay ReNcells cultured with CM or PEDF showed significantly higher phagocytosis rate than undifferentiated ReNcells.

Conclusions: The most efficient substance for differentiation towards RPE-like cells appeared to be RPE-CM. The results show that under the influence of growth factors neuronal progenitor cells can differentiate into RPE-like cells. Therefore, these cells might be a new source for regenerative treatment of degenerative retinal diseases.

Commercial Relationships: Volker Enzmann , None; Luca Tamó ,

None; Carolyn Trepp , None; Sebastian Wolf , None



Efficient generation of retinal pigment epithelium cells from human embryonic stem cells

Furong Gao 1 , Zongyi Li 1 , Weiye Li 2 , Lixia Lu 1 , Guotong

Xu 1 . 1 Shanghai Tenth’s People Hospital, Department of

Ophthamology,Tongji Eye Institute and Department of Regenerative

Medicine, Tongji University School of Medicine, Shanghai, China;

2 Department of Ophthalmology, Drexel University College of

Medicine, Philadelphia, PA .



.


Purpose: Retinal pigment epithelium (RPE) disorders usually cause problems with vision. RPE derived from human embryonic stem cells (hESCs) may become a promising therapeutic option for transplantation in these retinal diseases. However, induction of hESCs to RPE cells often takes several months with a low frequency.

The purpose of this study was to establish an improved method that takes a short time and with a high efficiency.

Methods: ShhES2 cells were induced to differentiate into RPE by the embryoid body (EB) formation method. We treated embryoid bodies with a combination of CKI-7, SB431542, and two other factors in N2B27 medium with or without PJ34. The EBs were cultured for 3 days and then transferred to matrigel-coated dishes for attached growth for two weeks. At last, the cells were maintained in hESCs medium without bFGF for an additional 2 weeks. Then, the cells were passaged for further differentiation. Assessment of differentiation was performed using pigmentation formation, mRNA and immunocytochemistry. The abilities of hESCs-derived RPE cells in rescuing retinal structure and visual function of RCS rats after subretinal transplantation were evaluated by electroretinogram

(ERG), histology (HE staining) and immunohistology (TUNEL assay).

Results: Compared to the four factors group, PJ34, in combination with the four factors increased the expression of transcripts of the

RPE cell markers MITF and RPE65 after 4 weeks of differentiation, but decreased photoreceptor markers Rx and CHX10. In addition, in the PJ34 group, pigmented areas with a cobblestone appearance began to appear within the differentiating clusters after 4 weeks, and by 6 weeks, almost all the ShhES2 differentiating cells contained pigment granules and had a cobblestone appearance. In the absence of PJ34 group, much less pigmented areas were observed.

Furthermore, the transplanted hESCs-derived RPE cell can improve the retinal structure and function of RCS rats.

Conclusions: differentiation of hESCs toward an RPE fate at a high efficiency in a short time. The subretinal transplantation of hESC-derived RPE appears to improve the structure and function of degenerative retina of RCS rats. These findings indicate a new short-term and efficient protocol for differentiation of hESCs to RPE cells and thus may be useful in the treatment of retinal degenerations.


Weiye Li

This study demonstrated that PJ34 can promote the

, None; Lixia Lu

Furong Gao

, None;

, None;

Guotong Xu

Zongyi Li

, None

, None;



Molecular and Functional comparison of limbal iPS cell- and retina-derived RGCs

Sharada Paudel Kattel, Sowmya Parameswaran, Iqbal Ahmad.

University of Nebraska Medical Center, Omaha, NE .

Purpose: The induced pluripotent stem (iPS) cells represent a viable source of retinal progenitors for regenerative medicine for the retina

(Parameswaran et al., 2010, stem cells, 28:695-703). To address the barrier of insertional mutagenesis associated with nuclieic-acidderived iPS cells, we reprogrammed limbal cells to pluripotency by non-nucleic acid means (Balasubramanian et al., 2009, Stem Cells,

27:3053-3062), which is a robust source of retinal progenitors for generating RGCs. The therapeutic utility of limbal iPS cell-derived

RGCs will depend upon its acquisition of properties of RGCs present in the retina. Here we have compared the properties of limbal iPS cell-derived RGCs with that of RGCs present in the adult rat retina.

Methods: Rat limbal iPS cells generation and their subsequent neural differentiation were achieved as previously described

(Balasubramanian et al., 2009). Limbal iPS cell-derived retinal progenitors were cultured in the presence of rat E14 retinal cell conditioned medium (E14CM) to generate RGCs. RGCs from the adult retina were obtained by the standard magnetic bead enrichment method using Thy1 antibody. The two RGC populations were subjected to immunocytochemical, Q-PCR and microarray analysis.

In addition, to evaluate their target specificity they were co-cultured with superior/inferior colliculus (SC/IC) explants.

Results: The limbal iPS cell-derived RGCs, like their retinal counterparts expressed regulators and markers of RGCs. In addition, they expressed molecules necessary for axonal pathfinding. However, their morphology was not as elaborate as RGCs enriched from the adult retina. Their processes like that of RGCs from the adult retina, could discriminate between SC and IC cells in establishing contacts. Preliminary comparative microarray analyses revealed reprogramming of the genome that approximated that of RGC obtained from the adult retina.

Conclusions: Our preliminary comparative analysis suggests that limbal iPS cell-derived RGCs embodies properties similar to that of

RGCs derived from the adult retina and therefore may also function similarly.


Sowmya Parameswaran

Support:

Sharada Paudel Kattel

, None; Iqbal Ahmad

NEI, Research to Prevent Blindness

, None

, None;



Elucidating the role of FGF signaling using an iPS cell model of retinal development

Eric Clark 1 , Ruchira Singh 1, 3 , Molly Wilson 1 , Jackie Meyer 1 , David

Kuai 1 , Joe Phillips 1 , David M. Gamm 2, 3 . 1 Waisman Center, University of Wisconsin, Madison, WI; 2 Dept. of Ophthalmology and Visual

Sciences, University of Wisconsin, Madison, WI; 3 McPherson Eye

Research Institute, University of Wisconsin, Madison, WI .

Purpose: Studies in animal models have implicated a regulatory loop between FGF signaling and the transcription factor Visual System

Homeobox 2 (VSX2) in the maintenance of neural retinal progenitor cell (NRPC) identity and regulation of retinogenesis. However, the specific roles of FGF family members in these processes remain to be elucidated. We used a human induced pluripotent stem cell (hiPSC) model of retinogenesis to examine the expression and function of specific FGFs during neural retina (NR) development.

Methods: hiPSCs derived from a patient with a functional null mutation in VSX2 (R200Q) and an unaffected sibling underwent targeted differentiation to obtain optic vesicle structures (hiPSC-

OVs). qRT-PCR was used to evaluate transcript levels of specific

FGFs and FGF receptors over time. qRT-PCR, Western blots, and

ICC were employed to evaluate the effect of exogenous FGF and neutralizing antibody treatment on the expression of selected genes and proteins involved in retinal development. Lastly, phosphorylation assays were performed to determine the effect of FGFs on ERK1/2 activation.

Results: Time course studies revealed biphasic expression profiles of FGF3, 8, and 9 in differentiating hiPSC-OVs, with peaks corresponding to the formation of the eye field and OV. Furthermore, expression levels of FGF3, 9, 19 were higher in WT vs. (R200Q)

VSX2 hiPSC-OVs. Continuous application of exogenous FGF9 to (R200Q)VSX2 hiPSC-OVs from d20-d90 led to increased expression of NR genes and decreased expression of RPE markers.

Treatment of (R200Q)VSX2 hiPSC-OVs with exogenous FGF during specific developmental time windows revealed a role for

FGF9 in proliferation and differentiation of NRPCs. In contrast, treatment of WT hiPSC-OVs with neutralizing antibody did not have a significant effect on the expression of NRPC markers, suggesting that FGF9 deficiency is tolerated in the presence of functional



.


VSX2. Phosphorylation assays indicated that FGF9-mediated effects on (R200Q)VSX2 hiPSC-OVs are likely mediated by ERK 1/2 activation.

Conclusions: Our results demonstrate that both VSX2 and FGF9 play roles in the proliferation and differentiation of NRPCs, and that functional loss of VSX2 can be overcome by FGF9. These insights increase our understanding of human retinogenesis and may prove useful in the production of retinal cell types for therapeutic purposes.


None;

None;

Molly Wilson

Joe Phillips

Support:

, None;

, None;

Eric Clark , None;

Jackie Meyer , None;

David M. Gamm

Ruchira Singh

, None

David Kuai ,

NIH R01 EY021218, NIH P30 HD03352, Foundation

Fighting Blindness Wynn-Gund Translational Research Award,

Retina Research Foundation

,



Subpopulations of human umbilical cord mesenchymal stem cells exhibit differential rescusing functions on retinal degeneration in

RCS rats

Li Wang 1, 2 , Haibin Tian 1, 2 , Peng Li 1, 2 , Zongyi Li 1, 2 , Chunpin Lian 1, 2 ,

Qingjian Ou 1, 2 , Lixia Lu 1, 2 , Weiye Li 1, 3 , Guotong Xu 1, 2 . 1 Department of Ophthalmology of Shanghai Tenth Hospital, and Tongji Eye

2 institute, Tongji University School of Medicine, Shanghai, China;

Department of Regenerative Medicine and Stem Cell Research

Center, Tongji University School of Medicine, Shanghai, China;

3 Department of Ophthalmology, Drexel University College of


Purpose: To provides more information about the heterogeneity of human umbilical cord mesenchymal stem cells (UCMSCs) and a new strategy for UCMSCs treatment of retinal degeneration (RD) by choosing appropriate subpopulations.

Methods: Two subsets of human umbilical cord mesenchymal stem cells (UCMSCs), termed as UCMSC1 and UCMSC2, were isolated by single cell cloning, specific surface markers were analyzed by flow cytometry. Proliferating rate and gene expression were carried out by MTT and RNA sequencing analysis. The gene levels of growth factors and cytokines were measured by Real-time PCR.

Their differentiation abilities were confirmed by culturing UCMSC in adipogenic, osteogenic and chondrogenic differentiation media.

UCMSC1 and UCMSC2 were transplanted into subretinal space of

RCS rats, their therapeutic functions were confirmed by analyzing retinal nuclear layer thickness, apoptotic photoreceptors and electroretinogram.

Results: Both the two subpopulations share similar membrane marker phenotypes. However, UCMSC1 showed stronger ability than UCMSC2 regarding proliferating rate, colony forming ability, adipogenic and osteogenic potential, whereas the latter exhibited increased chondrogenic ability. RNA sequencing analysis further showed the differential gene levels relative to proliferation, differentiation and immunoregulation in UCMSC1 and UCMSC2.

After transplanted into subretinal space of RCS rats, UCMSC1 had stronger vision rescue function compared to UCMSC2, including increased b ¬ -wave amplitude, retinal nuclear layer thickness, and decreased apoptotic photoreceptors. Furthermore, When subjected to interleukin-6 treatment in vitro, mimicking the transplanted MSCs under inflammation condition in RD, UCMSC1 expressed much higher levels of growth factors than UCMSC2, such as bFGF, CNTF,

EGF, HGF, PEDF, which indicates that the differential therapeutic capacities of UCMSC subpopulations involve distinct paracrine functions.

Conclusions: Two subpopulations with distinct morphology, proliferation, differentiation potentials, marker expression and gene expression were derived from human UCMSCs. In addition, the different subpopulations of human UCMSCs had distinct therapeutic functions on RD.


Peng Li

Ou

, None;

, None;

Zongyi Li

Lixia Lu

Li Wang

, None;

, None;

, None; Haibin Tian

Chunpin Lian

Weiye Li , None;

, None;

, None;

Qingjian

Guotong Xu , None



MicroRNA Signatures Associated with the Differentiation of

Human Müller Glia with Stem Cell Characteristics towards a

Retinal Ganglion Cell Fate

Hari Jayaram 1, 2 , Megan F. Jones 2, 1 , Dan Frampton 4 , Karen

Eastlake 2, 1 , Silke Becker 2, 1 , Karl Matter 3 , G Astrid Limb 2, 1 . 1 National

Institute for Health Research (NIHR) Biomedical Research Centre for

Ophthalmology at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, United Kingdom;

2 Ocular Biology & Therapeutics, UCL Institute of Ophthalmology,

London, United Kingdom; 3 Cell Biology, UCL Institute of

Ophthalmology, London, United Kingdom; 4 Department of Infection

& Immunity, University College London, London, United Kingdom .

Purpose: MicroRNAs (miRNAs) are small, endogenous noncoding RNAs involved in the post-transcriptional silencing of gene expression during development and disease. The purpose of this study was to determine the miRNA signature associated with Notch pathway downregulation and in vitro differentiation of Human Müller

Glia with Stem Cell Characteristics (hMSCs) towards a Retinal

Ganglion Cell (RGC) fate.

Methods: hMSCs were differentiated in vitro towards a retinal ganglion cell fate using established protocols. Total cellular RNA was isolated from both differentiated and undifferentiated cells

(Four replicates per condition) and the expression profiles were interrogated using the Agilent miRNA microarray platform. Partek

Genomics Suite software was used to normalise the expression data. Subsequent analysis was performed using ANOVA in order to identify differentially expressed miRNAs using a 5% False Discovery

Rate (adjusted P-values < 0.05). Validation of the microarray results was achieved by performing quantitative PCR.

Results: Analysis of the microarray data identified nineteen differentially expressed miRNAs showing upregulation in differentiated cells when compared to undifferentiated controls

(Fold Change >= 0.5, p<0.05). These included miR-199b, miR-151, miR-204 and let-7i whose target genes include components of the

NOTCH pathway, and miR-222 that is known to be associated with the development of neurite outgrowth.

Conclusions: The miRNA signature associated with differentiation of hMSCs towards retinal ganglion cells is characterised by the upregulation of miRNAs known to be involved with the NOTCH pathway and neurite outgrowth. The identification of these targets and their future experimental modulation may provide opportunity for future research into the induction of in vivo retinal ganglion cell differentiation by hMSCs.

Commercial Relationships: Hari Jayaram , None; Megan F. Jones ,

None; Dan Frampton , None; Karen Eastlake , None; Silke Becker ,

None; Karl Matter , None; G Astrid Limb , None

Support: Fight For Sight New Lecturers Award, United Kingdom



.




Adipose derived stem cell therapeutic potential for treatment of diabetic retinopathy is modulated by both oxygen tension and diabetic status of donor cells

Thomas A. Mendel 1, 2 , Stephen Cronk 3 , Jaymes Beech 3 , Alexander M.

2 . 1 Pathology, University of Guendel 2 , Shayn Peirce

Charlottesville, VA; 3

3 , Paul A. Yates

Virginia, Charlottesville, VA; 2 Ophthalmology, University of Virginia,

Biomedical Engineering, University of Virginia,

Charlottesville, VA .

Purpose: Adipose derived stem cells (ASCs) stabilize the retinal microvasculature in the Akimba model of diabetic retinopathy (DR) through both direct contact and paracrine effects. Factors influencing the therapeutic efficacy are unknown, but critical to elucidate towards translational application. In this study, we examine the impact of oxygen tension and diabetic status of ASC donor on the secretome

ASC-derived-pericytes in vitro and how they alter therapeutic efficacy in vivo.

Methods: ASCs were isolated from both diabetic Akimba (dASC) and normo-glycemic wildtype mice (nASC) at 9 weeks of age. DiIlabeled passage 4 dASC and nASCs-pericytes were then injected intravitreally in 6 week old Akimba mice, with eyes harvested 4 weeks later for analysis of ASC incorporation and impact on the microvasculature. Supernatant from in vitro cultured dASCs and nASCs-pericytes was collected and evaluated by multiplex ELISA with respect to angiogenic secretome. In parallel, normoxic passage

5 human ASCs were subjected to hypoxic conditions (1% oxygen) or left in normoxic conditions, with supernatant collected 24 hours later and analyzed by multiplex ELISA.

Results: Pericytes derived from diabetic ASCs, in contrast to normo-glycemic ASCs, were unable to prevent damage to the retinal microvasculature that occurs in the Akimba DR mouse (Fig 1A). A number of pro-angiogenic factors were found to be diminished in the supernatant of dASCs as compared to nASCs, including IGFBP3 (Fig

1B). In vitro assays of proliferation and apoptosis were also altered in dASCs as compared to nASCs. Similarly, hypoxia diminished

IBFBP3 expression in cultured hASCs (Fig 2).

Conclusions: Exogenous diabetic ASC-pericytes show diminished ability to protect retinal microvasculature in a murine model of DR.

Diabetes impairs ASC-pericyte viability and alters the pro-angiogenic secretome. Hypoxia similarly impacts ASC-pericyte paracrine activity, reducing IGFBP3, a factor known to ameliorate oxygeninduced retinopathy. These findings may explain the inability of native retinal pericytes to rescue the microvasculature in a diabetic environment, and suggest autologous stem cell approaches to treating

DR may be problematic. It may be possible to enrich environmental conditions as well as establish viability assays that can both predict and enhance ASC therapeutic efficacy.



.


Commercial Relationships: Thomas A. Mendel , U.S. Provisional

Patent Application Serial No. 61/684,375 (P); Stephen Cronk , None;

Jaymes Beech , None; Alexander M. Guendel , None; Shayn Peirce ,

U.S. Provisional Patent Application Serial No. 61/684,375 (P); Paul

A. Yates , Genentech/Roche (C), Owner RetiVue LLC (I), RetiVue

LLC (E), U.S. Provisional Patent Application Serial No. 61/684,375

(P)

Support: NIH T32GM08715 and NIH EY022063-01



The optic nerve lamina is a neural progenitor cell niche

Steven L. Bernstein 1 , Zara Mehrabyan 1 , Candace Kerr 2 , Sally

Temple 3 , Jeffrey Stern 3 , Yan Guo 1 . 1 Ophthalmology and Visual

Sciences, Univ of Maryland Sch of Medicine, Baltimore, MD;

2 Department of Biochemistry and Molecular Biology, University of

Neural Stem Cell Institute, Maryland-Baltimore, Baltimore, MD; 3

Rensselaer, NY .

Purpose: The optic nerve (ON) is a myelinated CNS tract connecting the eye and brain, comprised of retinal ganglion cell (RGC) axons.

RGC axons are unmyelinated in the retina but are myelinated after traversing the optic nerve lamina (ONL). The reason for this discontinuity is unknown but suggests that the ONL may facilitate this process. One possibility is that the ONL is a source of myelinating progenitor cells. This would be of great significance to a number of ON diseases, including glaucoma, which may originate from problems in the ONL. The ONL has an unusual vascularization distinct from the rest of the ON, in that the ONL receives circulation from the retina, underlying choroid, and intrinsic ON vasculature.

Thus, the ONL is uniquely poised in this area to provide a progenitor cell niche. We evaluated the ONL as a potential source for progenitor cells.

Methods: Rodent ONL vasculature was mapped using two photon microscopy, and analyzed by gene expression and immunohistochemically for nestin and other neural progenitor genes, myelin components, oligodendrocyte and astrocyte precursors (Olig-

1, PDGFR α , NG2), and neuronal proteins. We generated ONL-cell cultures and also utilized two mouse strains (ER-Cre-Nestin and

-PDGFR α ), crossing them with ROSA26/LoxP mice with enhanced yellow fluorescent protein (EYFP) to localize after progenitor development. Human donor tissues of a variety of ages were analyzed for nestin expression.

Results: Nestin expression strongly localizes to the ONL in the young adult animal, with more differentiated, but still immature

NG2+ glial precursors directly behind the ONL. EDU-mitotic labeling reveals mitotic nestin (+) cells with slow turnover. ER-Cre-

PDGFR X ROSA double heterozygous mice demonstrated EYFP oligodendrocytes banding directly under the ONL, in the anterior

ON. The ONL expresses genes for early neuronal, as well as glial function. ONL-nestin expression declines during both human and rodent aging, with a near absence in humans over 60y of age.

Conclusions: The ONL is a progenitor cell niche. This niche may aid directionality of myelination/myelin barrier function, function for necessary replacement of glial cells in the highly stressful environment of the eye-nerve junction, and may generate postembryonic retinal neurons. The loss of the ONL progenitor niche may play a key role in the progression of intrinsic ON diseases such as glaucoma, where current treatment options are of limited usefulness.

Commercial Relationships: Steven L. Bernstein , None; Zara

Mehrabyan , None; Candace Kerr , None; Sally Temple , None;

Jeffrey Stern , None; Yan Guo , None

Support: NIH grants RO1EY015304 and EY019529



Generation of ciliary epithelium from mouse ES and iPS cells

Hirofumi Kinoshita

Mandai 2

1 , Kiyoshi Suzuma 1 , Jun Kaneko

, Takashi Kitaoka 1 , Masayo Takahashi 2 . 1

2 , Michiko

Department of

Ophthalmology, Nagasaki Univ School of Medicine, Nagasaki,

Japan; 2 Laboratory for Retinal Regeneration, RIKEN Center for

Developmental Biology, Kobe, Japan .

Purpose: Recently, Eiraku et al reported a protocol for selforganizing optic-cup morphogenesis in three-dimensional culture.

With this procedure, we could differentiate mouse embryonic stem

(ES) cells or mouse induced pluripotent stem (iPS) cells into neural retina, retinal pigment epithelium (RPE), as well as tissue derived from neural ectoderm in the three-dimensional structure. Here, we report a possibility that ciliary epithelium, which is essential for production of aqueous humor, could be differentiated from mouse

ES/iPS cells.

Methods: Mouse ES/iPS cells (Rx knock-in GFP ES line mESRx+;

Nrlp-eGFP transgenic iPS line) were maintained in maintenance culture as it might lead to undesirable differentiation of cells. Cells were differentiated by using modified Eiraku’s differentiation protocol, induced optic-cup-like structures were investigated morphologically, or immunohistochemically. E16.5 and P5 of

C57BL/6J mice were evaluated as control.

Results: We could show autonomous formation of the optic-cuplike structure from a three-dimensional culture of mouse ES/iPS cell aggregates by using modified Eiraku’s differentiation protocol.

To evaluate whether there is a development of ciliary epithelium in this optic-cup-like structure, we identified immunohistochemistry for ciliary marker. Prospective ciliary epitheliums, bilayer of pigmented and non-pigmented epithelium (PE and NPE), were seen in peripheral region of inner and outer epithelium in E16.5 mouse eye. Aquaporin1-positive staining was seen in the NPE.

Connexin43-positive staining was seen in the ciliary epitheliums (PE and NPE), RPE and gap junction, which is structure between PE and

NPE. In P5 mouse eye, neural retina and ciliary epithelium became morphologically distinguishable more clearly. Aquaporin1-positive staining was seen in the NPE, especially in the anterior pars plicata.

Aquaporin4-positive staining was seen in the NPE, connexin43positive staining was seen in the ciliary epitheliums (PE and NPE),

RPE and gap junction. Differentiated structures derived from mouse ES cells were Rx-GFP positive. In immunohistochemistry of aggregates derived from mouse iPS cells, expression of aquaporin1, connexin43 were positive in ciliary epithelium like structures.

Conclusions: We could differentiate the ciliary epithelium like structures from mouse ES/iPS cells. These complexes showed similarity of morphology and immunostaining of ciliary epitheliums in vivo.

Commercial Relationships: Hirofumi Kinoshita , None; Kiyoshi

Suzuma , None; Jun Kaneko , None; Michiko Mandai , None;

Takashi Kitaoka , None; Masayo Takahashi , None



.




Subpopulations of monkey bone marrow mesenchymal stem cells exhibit similar therapeutic functions on retinal degeneration in

RCS rats

Haibin Tian 1, 2 , Peng Li 1, 2 , Li Wang 1, 2 , Zongyi Li 1, 2 , Chunpin Lian 1,

2 , Qingjian Ou 1, 2 , Lixia Lu 1, 2 , Weiye Li 1, 3 , Guo-Tong Xu 1, 2 . 1 Tongji

University Eye institute, Tongji University Medical school, Shanghai,

China; 2 epartment of Regenerative Medicine and Stem Cell Research

3

Center, Tongji University School of Medicine, Shanghai, China;

Department of Ophthalmology, Drexel University College of


Purpose: To provide more information about the heterogeneity of monkey bone marrow mesenchymal stem cells (BMSCs) and to confirm whether different subpopulations could be acquired by cell membrane marker CD90, and their therapeutic functions on retinal degeneration (RD) will be investigated.

Methods: Two subsets of monkey BMSCs, CD90+ and CD90-, were isolated by flow cytometry, specific surface markers were further confirmed by flow cytometry. Proliferating rate and the gene levels of growth factors and cytokines were measured by MTT method and real-time PCR. Their differentiation abilities were confirmed by culturing BMSCs in adipogenic, osteogenic and chondrogenic differentiation media. CD90+ BMSCs and CD90- BMSCs were transplanted into subretinal spaces of RCS rats, their therapeutic functions were confirmed by analyzing retinal nuclear layer thickness, apoptotic photoreceptors and electroretinogram.

Results: Both the two CD90+ and CD90- subpopulations share similar membrane marker phenotypes, both are positive for CD73,

CD44, CD29, and negative for CD45, CD34, MHCII. However, differential expression of CD90 did not influence the characteristics of the two subpopulations, regarding proliferating rate, colony forming ability, growth factor generation, adipogenic osteogenic and chondrogenic potentials. After transplanted into subretinal space of

RCS rats, both CD90+ and CD90- subpopulations showed similar therapeutic functions, including increased b ¬ -wave amplitude, retinal nuclear layer thickness, and decreased apoptotic photoreceptors.

Furthermore, when passaged in vitro, CD90- cells gradually acquired membrane molecule CD90 after two passages, which suggested that

CD90- subpopulation could switch into CD90+ subpopulation when cultured in vitro.

Conclusions: Subpopulations could be distinguished by cell membrane markers. However, subpopulations may not always show distinct characteristics, and membrane markers are not stable when cells are cultured in vitro, which may account for their similar therapeutic functions.

Commercial Relationships: Haibin Tian , None; Peng Li , None; Li

Wang , None; Zongyi Li , None; Chunpin Lian , None; Qingjian Ou ,

None; Lixia Lu , None; Weiye Li , None; Guo-Tong Xu , None ciliary epithelium derived retinal stem cells.We hypothesize that absence of ephrin-A2 and -A3 also increases Müller cell proliferation and neurogenic potential in the adult.

Methods: Expression of ephrin-A2 and -A3 and their receptor

EphA4 in the retina and Müller cells was assessed by immunostaining and real-time PCR.To label proliferating cells,purified Müller cells of both wild-type and A2-/-A3-/- mice were treated with 0.5

m M

BrdU for 24 hours in culture.Percentage of BrdU+ cells was then recorded,and expression of retinal progenitor markers was evaluated with real-time PCR.In another series of experiments,purified Müller cells were induced to differentiate in the defined medium for 14 days and stained with primary antibodies against a photoreceptor marker recoverin or retinal ganglion cell marker β -III-tublin for evaluation of their potential of trans-differentiation into retinal neurons.

Results: Expression of ephrin-A2/A3 and their receptor EphA4 was detected in both the retinas and purified Müller cell cultures.

Using double-immunolabeling of EphA4 and CRALBP,a marker of Müller cells,in retinal sections we further demonstrate Müller cell expression of EphA4. Moreover,results of real-time PCR confirmed that ephrin-A3 and EphA4 expression is particularly enriched in Müller cells.Expression of neural progenitor cell markers

Pax6,Chx10,Ngn2,Sox2 were significantly increased in Müller cells derived from A2-/-A3-/- mice as compared to those of wild-type mice.The percentage of proliferating Müller cells was significantly higher in cultures derived from A2-/-A3-/- mice than that from wild-type mice.Induction of neuron trans-differentiation also induced significantly higher percentage of recoverin+ and β -III-tublin+ cells in Müller cell cultures derived from A2-/-A3-/- mice.These data suggest enhanced neurogenic potential of Müller cells in the absence of ephrin-A2 and-A3.

Conclusions: Our results indicate that the adult mouse retina expresses negative regulators for retinal stem cell growth,ephrin-A2/

A3,and their receptor EphA4 on Müller cells.The Müller cells derived from A2-/-A3-/- mice show a higher proliferation and neurogenic potentials in culture than those from wild-type mice.Thus,ephrin-A2 and -A3 contribute negatively to the regenerative behavior of Müller cells in adult mice.

Commercial Relationships: Ruilin Zhu , None; Kin-Sang Cho ,

None; Yuan Fang , None; Liu Yang , None; Dongfeng F. Chen , None

Support: Department of Veterans Affairs (1I01RX000110),

Department of Defense (W81XWH-09-2-0091), Lion’s Foundation

Grants to D.F.C. and K.S.C. National Natural Science Foundation of

China (No.81170837; NSFC81100667 )



Enhanced Neurogenic Potential of Müller Cells in the Absence of

Ephrin-A2/A3

Ruilin Zhu 1, 2 , Kin-Sang Cho 2 , Yuan Fang 3 , Liu Yang 1 , Dongfeng F.

Chen 2, 4 . 1 Ophthalmology, Peking University First Hospital, Key

Laboratory of Vision Loss and Restoration, Ministry of Education,

Beijing, China; 2 Schepens Eye Research Institute, Massachusetts

Eye and Ear, Harvard Medical School, Boston, MA; hospital, Fudan University, Shanghai, China; 4

3 Eye and ENT

Boston VA Healthcare

System, Boson, MA .

Purpose: We showed previously that ephrin-A2 and -A3 are negative regulators for the growth of neural progenitor cells in the brain and



.

ARVO 2014 Annual Meeting Abstracts 218 Stem Cell I Monday, May

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