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Indumathi Mariappan, Savitri Maddileti, Praveen Joseph, Jamila H. Siamwala, Vasundhara Vauhini; Enriched Cultures of Retinal Cells From BJNhem20 Human Embryonic Stem Cell Line of Indian Origin. Invest. Ophthalmol. Vis. Sci. 2015;56(11):6714-6723. doi: 10.1167/iovs.15-17364.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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