Figure 2
From: Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line
Retinal development is recapitulated in differentiating stem cells.
(a–c) Temporal qPCR analysis of differentiation. Gene expression was first normalized to GAPDH and CREBBP and then normalized to the value of undifferentiated A81-H7 hESCs. Error bars represent SEM. (a) Expression of eye field transcription factors. (b) Expression of optic vesicle, neural retina markers and neuronal markers. (c) Expression of RGC-associated markers. (d) qPCR profile for presence of retinal cell types on day 40 of differentiation. Expression was normalized to GAPDH and CREBBP, A81-H7 d0 cells (black) were compared to differentiated cultures (red). Error bars represent SEM. Cell type markers: neurons – VGLUT1, MAP2; Müller glia-GFAP, GS; RGCs - BRN3B; amacrine cells - GAD1, SLC6A9; bipolar cells - CABP5, PRKCA; horizontal cells - LHX1, PROX1; photoreceptors - CRX, RHO, RCVRN; RPE - MITF, RPE65. (e) Immunostaining of day 49 cultures shows remnants of retinal organization. CRX+ photoreceptor progenitors in green and mCherry+ RGCs in magenta. CRX+ cells – white arrows - appear to segregate from mCherry+ axons, suggesting division between the outer nuclear layer and the nerve fiber layer. Scale bar = 500 μm.
