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.