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목적 : We aimed to establish an efficient method for retinal ganglion cell (RGC) differentiation from human pluripotent stem cells (hPSCs) using defined factors.
방법 : To define the contribution of specific signal pathways to RGC development and optimize the differentiation of hPSCs toward RGCs, we examined RGC differentiation in three stages: (1) eye field progenitors expressing the eye field transcription factors (EFTFs), (2) RGC progenitors expressing MATH5, and (3) RGCs expressing BRN3B and ISLET1. By monitoring the condition that elicited the highest yield of cells expressing stage-specific markers, we determined the optimal concentrations and combinations of signaling pathways required for efficient generation of RGCs from hPSCs.
결과 : Precise modulation of signaling pathways, including Wnt, IGF-1 and FGF, and mechanical isolation of neural rosette cell significantly enriched RX+/ PAX6+ eye field progenitors from hPSCs by day 12. Notch inhibition facilitated differentiation into MATH5+ progenitors at 90% efficiency by day 20, and these cells further differentiated to BRN3B+/ ISLET1+ RGCs at 45% efficiency by day 40. Differentiated RGCs were functional as exemplified by their ability to generate action potentials, express microfilament components on neuronal processes, and exhibit axonal transportation of mitochondria.
결론 : This protocol offers highly defined culture conditions for RGC differentiation from hPSCs and in vitro disease model and cell source for transplantation for diseases related to RGCs.
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