Enriched Cultures of Retinal Cells From BJNhem20 Human Embryonic Stem Cell Line of Indian Origin

Abstract

Purpose: To test the retinal differentiation potential and to establish an optimized protocol for enriching retinal cells from an Indian origin, human embryonic stem cell (hESC) line, BJNhem20.

Methods: The BJNhem20 cells were cultured and expanded under feeder-free culture conditions. Differentiation was initiated by embryoid body (EB) formation and were cultured on Matrigel in neural induction medium (NIM) for 1 week and further maintained in retinal differentiation medium (RDM). After 1 month, the neuro-retinal progenitor clusters located at the center of pigmented retinal patches were picked and cultured as suspended neurospheres in RDM for 3 days and subsequently on Matrigel in neuro-retinal medium. The mildly pigmented, immature retinal pigmented epithelial (RPE) cells were picked separately and cultured on Matrigel in RPE medium (RPEM). After 1 week, the confluent neuro-retinal and RPE cultures were maintained in RDM for 2 to 3 months and characterized by immunofluorescence and RT-PCR.

Results: The BJNhem20 cells efficiently differentiated into both neuro-retinal and RPE cells. The early retinal progenitors expressed Nestin, GFAP, Pax6, Rx, MitfA, Chx10, and Otx2. Neuro-retinal cells expressed the neural markers, Map2, β-III tubulin, acetylated tubulin and photoreceptor-specific markers, Crx, rhodopsin, recoverin, calbindin, PKC, NeuroD1, RLBP1, rhodopsin kinase, PDE6A, and PDE6C. Mature RPE cells developed intense pigmentation within 3 months and showed ZO-1 and Phalloidin staining at cell–cell junctions and expressed RPE65, tyrosinase, bestrophin1, Mertk, and displayed phagocytic activity.

Conclusions: This study confirms the retinal differentiation potential of BJNhem20 cells and describes an optimized protocol to generate enriched populations of neuro-retinal and RPE cells.

Retinitis pigmentosa is a progressive genetic disorder resulting in gradual degeneration of photoreceptors and RPE cells of the retina, leading to night blindness and gradual loss of vision, which later progresses to complete visual impairment and blindness. Mutations in several genes associated with the phototransduction pathway, retina-specific transporters and transcription factors, and vitamin A metabolism are linked to this disease. Therefore, gene therapy and cell replacement therapy offer great promise in the treatment of such conditions. However, the absence of stem cells in an adult retina has initiated a search for alternate sources that can generate retinal cell types suitable for regenerative applications. 

Among the adult ocular stem cell sources, a minor population of the cells of the ciliary margin was shown to have retinal stem cell properties.¹ However, they have very limited potential to generate neurospheres (0.1%) and also lack the ability to differentiate to all cell types of the retina. Therefore, pluripotent cells, such as the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have become valuable stem cell sources for generating retinal cell types suitable for regenerative applications. Earlier studies have shown that it is possible to derive neuro-retinal (NR) progenitors, mature photoreceptors, and RPE cells from human ESCs.^2–11 Embryonic stem cell–derived retinal cell types also have been shown to rescue disease phenotype to some extent when transplanted in small and large animal models.^4,12–16 In an ongoing phase I/II clinical trial approved by the Food and Drug Administration (FDA), allogeneic human ESC (hESC)-derived RPE cells are being used in the treatment of patients with Stargardt's macular dystrophy and dry AMD.^17,18 

Given this interest in generating retinal cell types for clinical applications, a well-characterized hESC line with a propensity to differentiate into retinal lineages is of great value. To this end, we describe an efficient protocol to derive retinal cell types from a well-characterized Indian origin, hESC line, BJNhem20.^19–21 This line has been submitted to the UK Stem Cell Bank (UKSCB accession No. R-08-021) and listed in the European Human Pluripotent Stem Cell Registry (hPSC^reg) (JNCSRe002-A [BJNhem20]), International Stem Cell Registry, and the National Institutes of Health (NIH) Human Embryonic Stem Cell Registry (NIH Approval No. NIHhESC-10-0084). The International Stem Cell Initiative has conducted a genetic screen on ethnically diverse hESC lines and reported various genetic alterations and a common candidate on chromosome 20 that drives culture adaptation in more than 20% of the lines tested. The BJNhem20 line was part of this study and was found to be normal without any genetic abnormalities both at the early (sample code: TT-20-003-K-E-P19) and late passages (sample code: TT-20-004-K-L-P87) tested.²² This line is also available for research use and projects involving its use are eligible for NIH funding consideration. The Table provides the details of all reported, Indian origin, hESC lines listed in the International Stem Cell Registry. 

Table

 

Details of Indian-Origin hESC Lines Listed in the International Stem Cell Registry

 

Materials and Methods

The study was reviewed and approved by the institutional review board of the LV Prasad Eye Institute, Hyderabad, India, and the research followed the tenets of the Declaration of Helsinki. 

Human Embryonic Stem Cell Culture and Maintenance

The hESC line, BJNhem20 cells are cultured on irradiated or mitomycin C–treated mouse embryonic fibroblast feeders using the standard human ES medium (KnockOut Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% KnockOut serum replacement [KOSR], 10% fetal bovine serum, 2 mM Glutamax, 0.1 mM nonessential amino acids [NEAA], 0.1 mM β-mercaptoethanol, 50 U/mL penicillin, 50 μg/mL streptomycin, and 10 ng/mL basic FGF [bFGF; Life Technologies, Carlsbad, CA, USA]). The cells are also adapted to feeder-free culture conditions on Matrigel (Corning, Inc., Corning, NY, USA) coated plates using mTeSR1 culture medium (STEMCELL Technologies, Vancouver, Canada). The cultures are passaged manually by cutting individual colonies using the bent tips of flame-pulled glass Pasteur pipettes, followed by gentle trituration to generate small 5- to 10-cell clusters. Alternatively, the cells are also passaged using 0.5 mM EDTA in calcium/magnesium-free PBS as described elsewhere.²⁹ Split ratios of approximately 1:6 to 1:10 were followed and the cultures were split after every 3 to 4 days before they reached 80% to 90% confluence. 

Retinal Differentiation of hES Cells

The BJNhem20 cells were cultured under feeder-free conditions as described above. Differentiation was initiated by embryoid body (EB) formation in suspension cultures for 3 days in nonadherent dishes using differentiation medium (DM) that contains DMEM/F12, 4% KOSR, 0.1 mM NEAA, 2 mM Glutamax, 50 U/mL penicillin, and 50 μg/mL streptomycin (Life Technologies). After 3 days, the EBs are grown as adherent cultures on Matrigel-coated dishes, in neural induction medium (NIM), which contains DM supplemented with 1% N2 supplement (Life Technologies) and 100 ng/mL Noggin (R&D Systems, Minneapolis, MN, USA) for 1 week and further continued for 1 month in retinal differentiation medium (RDM), which contains DM and 2% B27 supplement (Life Technologies). Pigmented retinal progenitor cell clusters with neural progenitor rosettes and surrounding pigmented RPE patches were observed at this stage. 

Subculture and Enrichment of Retinal Cells

The neural rosettes from pigmented cell clusters were isolated and further cultured as suspended neurospheres in RDM for 2 to 3 days. The resulting neurospheres containing retinal progenitors were further plated on Matrigel-coated dishes or chamber slides and maintained as adherent cultures in NR medium (NRM) containing DM supplemented with 1% N2, 5 ng/mL bFGF, and 10 ng/mL DKK1. The mildly pigmented and proliferating RPE patches surrounding the central NR islands were then manually picked using flame-pulled glass Pasteur pipettes and cultured on collagen I matrix or Matrigel-coated dishes or chamber slides (Corning, Inc.) or on Transwell cell culture inserts (Corning, Inc.) in RPE medium (RPEM) containing DM supplemented with 2% B27, 10 ng/mL ActivinA (R&D Systems), and 10 μM Y-27632 (Sigma-Aldrich Corp., St. Louis, MO, USA). Supplementary Figure S1 shows the schematic representation of the step-wise retinal differentiation protocol adapted in the study. After 1 week, the NR and RPE cultures reached confluence and were continuously maintained in RDM, with media changes on every third day for up to 2 to 3 months. The differentiation protocol was repeated several times in independent experiments, n = 7. The differentiated cells were characterized for NR and RPE-specific marker expression by immunofluorescence and RT-PCR analysis. 

Immunocytochemistry

Growing hES cultures or the differentiated retinal cells were seeded and cultured on glass coverslips or on chamber slides. When the cells were ready for immunostaining, they were briefly washed with 1X PBS, fixed for 10 minutes using freshly prepared 3.5% formaldehyde in PBS and permeabilized for 10 minutes with 0.5% Triton X-100 in PBS, with three saline washes after each step. The cells were then blocked with 1% BSA at room temperature for 1 hour and then sequentially incubated with specific primary and fluorescent dye–conjugated secondary antibodies at appropriate dilutions for 1 hour each, with three saline washes after each incubation step. Supplementary Table S1 summarizes the details of antibodies used in the study. Propidium iodide (PI) or 4′,6-diamidino-2-phenylindole (DAPI) were used as counter stains. Intact cell sheets grown on collagen matrix were directly processed for immunohistochemistry by standard procedures and the tissue sections were examined by hematoxylin and eosin staining. Alkaline phosphatase staining was carried out using an assay kit by following the manufacturer's instruction (Merck Millipore, Darmstadt, Germany). The cells are finally washed, mounted on a glass slide, and imaged using an epifluorescence microscope (Olympus IX71; Olympus, Shinjuku, Tokyo, Japan) or a confocal microscope, LSM 510 (Carl Zeiss Microscopy GmbH, Jena, Germany). The images were analyzed using the Image-Pro Express Version 6.0 Imaging Software (Media Cybernetics, Inc., Rockville, MD, USA) and LSM 510 Meta, Version 3.2 software (Carl Zeiss Microscopy GmbH), respectively, and the composites were prepared using Adobe Photoshop CS (Adobe Systems, Inc., San Jose, CA, USA). 

Florescence-Activated Cell Sorting (FACS) Quantification of Cultured Cells

Enriched passage one NR and RPE cell suspensions were prepared and processed for immune labeling as described above. The final cell suspensions were analyzed using FACS Aria I cell sorter and the data analysis was carried out using FACS Diva software (BD Biosciences, San Jose, CA, USA). 

Secreted Protein Quantification by ELISA

Culture supernatants of enriched cultures of BJNhem20-derived RPE cells grown in six-well plates were collected (1.5 mL/well) and aliquots of 100 μL were used to analyze and quantify the secreted VEGF and pigmented epithelium-derived factor (PEDF) levels by sandwich ELISA method as per the manufacturer's instructions (R&D Systems). Culture supernatants of ARPE-19 and HEK 293T cells were used as controls. 

Gene Expression by RT-PCR

Total RNA was isolated from cell samples by Trizol method and cDNAs were prepared by reverse transcription using SuperScript II RT kit (Life Technologies) as per the manufacturer's instruction. Polymerase chain reactions were performed for all the genes tested using the cDNAs as reaction templates. The starting template concentrations were normalized for all the samples tested based on the expression levels of the housekeeping gene, eEF1α. Supplementary Table S2 summarizes the primer details of various genes analyzed in the study, n = 3. The amplicons were resolved on 1% (wt/vol) agarose gels, stained with ethidium bromide, imaged under UV light, and documented using Gel Doc XR+ System (BioRad, Hercules, CA, USA). 

Phagocytosis Assay

The BJNhem20-derived RPE cells were grown on Matrigel-coated glass chamber slides (BD Biosciences) and incubated with 1.0-μm sized green fluorescence latex beads (Sigma-Aldrich Corp.) at a concentration of 1 × 10⁶ beads/mL for 6 hours at 37°C. The cells were then washed thoroughly with PBS for five times to remove extracellular, free-floating beads and processed for immunocytochemistry and imaging as described above. 

Statistical Analysis

Test values were reported as mean values ± SD. Group means were compared using the Student's unpaired t-test; P < 0.05 was considered statistically significant (*) and P > 0.05 was considered statistically insignificant (#). 

Results

Stemness and Pluripotent Properties of BJNhem20

The Indian origin, hESC line, BJNhem20, used in the study was originally derived, characterized, and reported by Inamdar et al.¹⁹ Growing cultures of this hESC line maintained the typical round and flat colony morphology with distinct boundaries and the cells maintained a high nuclear to cytoplasmic ratio as shown in Figures 1A and 1B. The cells expressed the pluripotent stem cell markers, Oct4, SSEA4, Nanog, and alkaline phosphatase as shown in Figures 1D through 1G. It was also reported to express TRA 1-60, TRA 1-81, Sox2, DNMT3B, Rex1, LeftyA, and others.¹⁹ On suspension culture, they differentiated and readily formed circular EBs within 2 to 3 days, as shown in Figure 1C, and gave rise to cell types of ectoderm, mesoderm, and endoderm lineages.¹⁹ The cells were also shown to be capable of teratoma formation when injected subcutaneously into nude mice.¹⁹ The karyotype of this female line was reported to be normal and was shown to have the propensity to generate beating cardiomyocytes.¹⁹ 

Figure 1

 

Morphology and stem cell marker expression in BJNhem20 cells. Phase images of growing cultures of BJNhem20 cells in a feeder-free culture condition on Matrigel (A, B) and floating cultures of embryoid bodies at day 3 of differentiation (C). Scale bar: 100 μM (A–C). Expression of pluripotent stem cell markers, Oct3/4 (D), SSEA4 (E), Nanog (F), and alkaline phosphatase (G) in growing cultures. Scale bars: 20 μM (D, E), 100 μM (F, G).

Here, we report the retinal differentiation potential of BJNhem20 and describe a stepwise differentiation protocol for deriving retinal progenitors and also to establish enriched cultures of RPE cells and NR cells. 

Retinal Differentiation of BJNhem20

Retinal differentiation was carried out as described in the methods section. Briefly, 2- to 3-day-old EBs were grown as adherent cultures on Matrigel-coated plates in NIM for 1 week. The addition of recombinant human noggin protein in NIM inhibits BMP signaling, and in the presence of N2 supplement, it promotes early neuro-ectodermal commitment and differentiation. The cells were further cultured under retinal differentiation-promoting conditions in RDM containing 2% B27 supplement for a further 1 month. After 1 month of retinal differentiation, visibly pigmented retinal clusters consisting of very early retinal progenitors were observed (Figs. 2A, 2B). The neural-like cell rosettes within the pigmented clusters were manually picked and grown separately as neurospheres in suspension for 3 days in RDM to enrich for NR progenitors (Fig. 2C). 

Figure 2

 

Retinal differentiation and enrichment of BJNhem20-derived retinal cells. Phase images of retinal differentiation cultures. (A) Full dish (60-mm diameter) view of a 1-month-old differentiation culture, wherein small clusters of retinal progenitors begin to form and appear as pigmented patches (arrows). (B) Zoomed in view of a pigmented cluster. Proliferating RPE cells are seen all around a central cluster of NR progenitors. (C) Suspension culture of the central island of NR progenitors to generate enriched cultures of growing neurospheres. (D) Adherent cultures of manually isolated patches of pigmented RPE cell clusters on matrigel. On adhesion, the RPE cells loose the pigments, become fibroblastic, and proliferate to expand in culture. (E) Adherent cultures of neurospheres containing NR progenitors to generate mixed populations of NR cells. (F) Enriched and confluent cultures resulting in sheets of mildly pigmented and uniformly hexagonal-shaped RPE cells at 2 months, which further matures and becomes intensely pigmented by 3 months. Scale bars: 100 μM.

It is well established that the ocular surface ectoderm- derived bFGF and the periocular mesenchyme-derived TGFβ family of proteins, such as activin A, play an important role in NR and RPE fate commitment, respectively.³⁰ Also, it is known that inhibition of wnt signaling is necessary to promote terminal differentiation and maturation of NR progenitors. Therefore, the neurospheres are further grown as adherent cultures in NRM containing N2 supplement (1%), bFGF (5 ng/mL), and DKK1 (10 ng/mL). Highly proliferating cells with typical neuron-like morphology are seen migrating out of the adhered neurospheres within 1 day (Fig. 2E) to generate enriched cultures of mixed populations of various NR cells within 2 months. 

Both the mildly pigmented or nonpigmented immature RPE cells that proliferated and migrated out of the central pigmented retinal clusters were manually picked and maintained separately as adherent cultures in RPEM containing B27 supplement (2%), activin A (10 ng/mL), and Rho-associated protein kinase (ROCK) inhibitor, Y-27632 (10 μM), to allow RPE cell proliferation and expansion. The proliferating and migrating RPE cells initially assumed a fibroblastic morphology (Fig. 2D). As the adherent cultures became confluent, they regained the typical cobble-stone morphology. Addition of ROCK inhibitor to RPE cultures during maintenance and passaging has been reported to promote cell proliferation, reduce cell death, prevent epithelial-mesenchymal transition (EMT),³¹ and help in the establishment of a uniform monolayer of hexagonal-shaped, highly pigmented, mature RPE cells within 2 to 3 months (Fig. 2F). 

Characterization of BJNhem20-Derived Retinal Cells

Retinal cells derived at different stages of differentiation were analyzed by immunocytochemistry. The pigmented retinal clusters (Fig. 2A) that emerged at 1 month after differentiation confirmed the presence of retinal progenitors that expressed the neural progenitor marker, Nestin; glial progenitor marker, glial fibrillary acidic protein (GFAP); early eye-field commitment markers, Pax6 and Rx; RPE lineage commitment marker, MitfA; and the NR lineage commitment marker, Chx10 (Fig. 3). It is important to note that the selective isolation of retinal progenitors based on pigmentation and neural rosette morphology at early stages of differentiation helps in subsequent enrichment of mature retinal cells. 

Figure 3

 

Expression of early commitment markers by BJNhem20-derived retinal progenitor cells. Early retinal progenitors present within the pigmented clusters at 1 month of differentiation expressed Nestin (A), GFAP (B), Pax6 (C), Rx (D), MitfA (E), and Chx10 (F). Scale bars: 20 μM.

Immunocytochemistry of 3-month-old NR cultures confirmed the expression of general neuron-specific, Map2, β-III Tubulin, and acetylated Tubulin; rod-cone progenitor-specific, Crx and mature photoreceptor-specific, rhodopsin and recoverin (Figs. 4A–F). Although the photoreceptor cells are not morphologically mature, they expressed rhodopsin protein in an asymmetrical pattern within the cytosol, whereas its expression is normally localized to the outer segments of mature rod photoreceptor cells. The NR cells also coexpressed some of the progenitor and mature cell markers, such as the Pax6, Crx, recoverin, and rhodopsin (Figs. 4G–I). 

Figure 4

 

Expression of mature NR markers. Adherent cultures of enriched NR spheres at 2 to 3 months after differentiation consisted of mixed populations of possibly all types of retinal neurons and expressed the general neural markers, Map2 (A), β-III tubulin (B), acetylated tubulin (C), and the photoreceptor-specific markers Crx (D), rhodopsin (E), and recoverin (F) (all in green). Arrows mark the asymmetrically localized rhodopsin. The cells are counterstained with PI in (A, B, D, E) to mark the nuclei (in red). Retinal cells coexpressing the markers such as Pax6 (green) and Crx (red) (G), Recoverin (green) and Pax6 (red) (H), rhodopsin (green) and Crx (red) (I). Arrows indicate the dual positive cells and arrowheads mark the cells expressing only a single marker. Scale bars: 20 μM.

Enriched cultures of RPE cells resulted in the formation of uniformly pigmented sheets of matured epithelial monolayer at 3 months, as shown in Figures 5A and 5B. Immunocytochemistry of the compact epithelial sheets confirmed the expression of tight junction protein, ZO-1 (Fig. 5C), and an orderly arrangement of actin cytoskeletal bundles as shown by Phalloidin staining (Fig. 5F). The cells also expressed the mature RPE-specific marker, RPE65. This gene codes for an enzyme, isomerohydrolase that converts all-trans retinol to 11-cis retinal during phototransduction and plays an important role in the visual cycle. This enzyme exists in both the membrane-bound and cytosolic forms, as shown in Figure 5D. The RPE cells play an important role in the clearance of subretinal cellular debris, and this function is mediated by active phagocytosis. The BJNhem20-derived RPE cells also displayed phagocytosis, as shown by the internalization of fluorescently labeled latex beads (Fig. 5C). Intact monolayer sheets of RPE cells cultured on collagen I matrix appeared to be highly polarized cells with tight cell-cell junctions, basally positioned nuclei, numerous apical microvilli structures, and pigmented melanosomes in the cytosol (Figs. 5G, 5H). The BJNhem20-derived RPE cells also secreted significant levels of both VEGF and PEDF, when compared with the ARPE-19 and HEK 293T cells, respectively (Figs. 5I, 5J), and the levels were comparable with those reported for PSC-derived RPE cells in other studies.^32,33 

Figure 5

 

Expression of RPE markers. Morphology of enriched sheets of mature RPE cells at 3 months after differentiation. Note the compact arrangement of hexagonal-shaped cells with mild to intense pigmentation (A, B). Intact tight junctions are marked by ZO-1 staining in red (C). Both the cytosolic and membrane-bound forms of RPE65 protein were detected in mature, 3-month-old cultures (in green) with PI staining to mark the nuclei (in red) (D, E). The organized actin cytoskeletal bundles are marked by Phalloidin staining in green with DAPI staining to mark the nuclei (F). The green fluorescent latex beads that were phagocytosed by the cells are seen as green dots within the cytoplasm of intact cells (C). Hematoxylin and eosin–stained intact RPE cell sheets on collagen I matrix (G, H). Note the polarized epithelium with tight cell–cell junctions, basally-positioned nuclei (arrowhead), numerous melanosomes and apical microvilli structures (arrow). Scale bars: 100 μM (A), 20 μM (B–H). Bar graphs represent the average levels of secreted VEGF and PEDF proteins in the culture supernatants of ARPE-19, HEK 293T, and hESC-derived RPE cells, n = 3. Error bars: represent the SD.

The FACS quantification of NR and RPE cultures confirmed cell enrichment within a single passage. Relatively lower levels of Pax6-positive cells (62%) indicated that the retinal stem cells had undergone lineage commitment and differentiation (Fig. 6B). High percentage of Chx10 (87%) and Mitf (94%) positive cells confirmed the enrichment of lineage committed progenitors (Figs. 6C, 6F). Also, the proportion of mature retinal cells expressing the markers such as Nrl and RPE65 were 31% and 34%, respectively (Figs. 6D, 6G). 

Figure 6

 

Florescence-activated cell sorting quantification of BJNhem20 derived retinal cells. Florescence-activated cell sorting plots of passage 1 NR and RPE cells labeled with isotype control antibody (A, E), anti-Pax6 antibody (B), anti-Chx10 antibody (C), anti-Nrl antibody (D), anti-Mitf antibody (F), and anti-RPE65 antibody (G). Summary of the mean percent positive cells ± SD (H), n = 3.

Further, we compared the gene expression patterns of different stem cell and retinal markers at days 0, 30, and 90 after differentiation by RT-PCR and representative gel images of various genes tested are shown in Figure 7. In brief, the pluripotent stem cell markers, Oct3/4, Nanog, and hTERT were downregulated as the cells entered into the differentiation program. This coincided with the stepwise activation of retinal progenitor markers, such as Otx2, MitfA, Chx10, and Rx at d30. Similarly, the expression of NR markers, such as NeuroD1, Crx, recoverin, calbindin, PDE6A, PDE6C, RLBP1, PKC-β, rhodopsin kinase, and Opsin-MW, and RPE markers, such as tyrosinase, RPE65, Bestrophin1, and Mertk, were upregulated at d30 and d90 (Fig. 7). 

Figure 7

 

Reverse transcription PCR profiling of NR and RPE-specific genes. Agarose gel pictures of RT-PCR products of various pluripotent stem cell– and retina-specific genes expressed by the undifferentiated cells (D0) and by retinal differentiation cultures at 1 month (D30) and 3 months (D90) after differentiation. The cDNAs of different test samples were normalized using eEF1α as the loading control.

Discussion

A large body of literature evidence has confirmed that adult mammalian and primate retina are amenable for cell replacement therapy. Transplantations of human fetal retinal progenitors^34,35 or photoreceptor precursors¹² are shown to preserve or improve visual functions in animal models of retinal dystrophy. However, a reliable and renewable source of donor cells such as the hESCs and iPSCs are being intensely explored for adopting them in cell replacement therapies. As mentioned earlier, many reports have shown that retinal cells derived from hESCs are amenable for scaling up and are effective in delaying disease progression and in improving visual functions in preclinical animal studies. 

The results of the present study confirm the retinal differentiation potential of the hESC line, BJNhem20. Using the protocol described here, it is possible to efficiently differentiate and enrich RPE cells and NR progenitors and expand them enough for further downstream applications. Although complex cocktails of culture components have been reported to drive retinal differentiation, our protocol involves an initial EB formation and short-term culture in the presence of Noggin and N2 supplement to drive neuro-ectodermal differentiation. Subsequent differentiation in B27-supplemented conditions promoted eye-field specification within 1 month. Manual isolation of these mildly pigmented eye-field–specified clusters ensures early enrichment of retinal progenitors. These early-stage retinal progenitors expressed Nestin, GFAP, Pax6, Chx10, Rx, and Mitf. Adherent culture of isolated retinal progenitors gave rise to both RPE and NR patches. Expansion of isolated RPE cell patches, their maturation, pigmentation, and maintenance of proper cell morphology was enabled by culturing them in the presence of ActivinA and ROCK inhibitor. The mature RPE cells were highly pigmented, polarized cells with apical microvilli and hexagonal morphology, and expressed RPE65, ZO-1, VEGF, and PEDF proteins at appropriate levels. Similarly, bFGF and DKK1 treatment enabled the expansion and maturation of NR cells that expressed Crx, recoverin, rhodopsin, cone opsin, and other NR markers. 

Recent reports on the clinical outcomes of FDA-approved, phase I/II trials have established the safety and tolerability of subretinal transplantations of hESC-derived RPE cells in the treatment of patients with Stargardt's macular dystrophy and dry AMD.^17,18,36 It is well known that the eye is an immune-privileged site, being established by the barrier functions of RPE cells and the endothelial cells of retinal vasculatures. The early observations and outcomes of this trial confirm that in spite of being an allogeneic cell source, the hESC-derived RPE cells transplanted in the subretinal space of the patient's eye did not elicit any significant immunologic response. The reports also revealed that there were no signs of hyperproliferation, tumorigenicity, or ectopic tissue formation. Fundus photographs and optical coherence tomography images of transplantation sites of the retina in treated eyes have confirmed that the RPE grafts survived, proliferated/expanded in vivo for up to 37 months post transplantation, and contributed to marginal improvements in visual parameters. These developments were encouraging to consider BJNhem20-derived retinal cells for possible preclinical and clinical applications. 

As a next step toward clinical translation, it is important to adapt early-passage cells into feeder-free and xeno-free culture conditions under a current good manufacturing practice (cGMP) work flow. Also, the retinal differentiation steps described in the study could be easily adapted to GMP requirements using clinical grade, xeno-free reagents. To ensure the safety of hESCs and their derivatives, it is important to reconfirm their pathogen-free status (bacteria, fungi, mycoplasma, and viruses, such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and syphilis) using appropriate GMP-compliant testing methods. It is also important to ensure that the enriched RPE cells are free of undifferentiated cell contaminants by testing them for the absence of tumor formation when transplanted in immune-compromised mice models. Finally, a preclinical safety and efficacy study in retinal dystrophic rodent models would ensure the biological activity and functional relevance of ESC-derived retinal cells. 

Although these are clear and obvious requirements for future clinical considerations, this report highlights the retinal differentiation potential of the hESC line, BJNhem20. The retinal derivatives and their progenies could serve as in vitro models in basic research and in pharmacologic drug screening and testing. A cGMP-compliant cell preparation could serve as valuable allogeneic donor cells in the treatment of patients with various forms of retinal dystrophies. 

Acknowledgments

The authors thank Maneesha Inamdar, PhD, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, for providing the hESC line, BJNhem20, and also for providing technical guidance related to hESC culture; Sreedhar Rao Boyenpally, BSc, and Dilip Kumar Mishra, MD, Ophthalmic Pathology Laboratory, L V Prasad Eye Institute, for their expert guidance with immunohistochemistry experiments; and Dorairajan Balasubramanian, PhD, for his critical review and useful suggestions in finalizing the manuscript. 

Supported by grants from the Department of Biotechnology, Government of India, Champalimaud Foundation, Portugal, and the Hyderabad Eye Research Foundation (IM). The authors alone are responsible for the content and writing of the paper. 

Disclosure: I. Mariappan, None; S. Maddileti, None; P. Joseph, None; J.H. Siamwala, None; V. Vauhini, None