大鼠诱导性多能干细胞向神经前体细胞诱导分化研究

Differentiation of rat induced pluripotent stem cells into neural progenitor cells in vitro

目的探讨大鼠来源的诱导性多能干细胞(iPS细胞)向神经前体细胞(NPC)定向诱导分化的方法。方法在大鼠iPS细胞培养到悬浮类胚体阶段加入维甲酸(RA)诱导分化,对分化获得的细胞,分别通过免疫荧光染色和实时定量PCR方法检测NPC标志物、用BrdU免疫荧光染色检测其增殖能力、并通过大鼠视网膜下腔移植对其存活、整合情况及分化能力进行检测。结果经诱导分化获得的细胞能够在贴壁培养条件下形成Rosette结构、表达NPC标志物并且绝大多数呈BrdU阳性,移植到大鼠视网膜下腔后能分化为神经元和胶质细胞。结论大鼠iPS细胞能够通过悬浮EB加RA诱导的方法分化为NPC,有可能作为细胞移植治疗视网膜退行性疾病的供体细胞。

Objective To investigate the differentiation of rat induced pluripotent stem cells（iPSCs） to neural progenitor cells（NPC）.Methods Rat iPSCs were cultured on mouse embryonic fibroblasts（MEFs）feeder layers for 2d and subsequently deprived of feeder cells to form embryoid bodies,which were incubated in nonadhesive Petri dishes with Retinoic acid（RA） for 4 days.NPC like cells derived from rat iPS cells were confirmed by immunofluorescence and qPCR for gene expression and were examined with BrdU incorporation assay for their proliferation capacity.The survival,integration and differentiation capacity of these iPSCs-derived NPCs were also tested after the cells were subretinally transplanted into RCS rats.Results The rat iPSCs-derived NPCs formed neural rosettes in adherent culture.Results of immunofluorescence and qPCR analysis showed that these cells expressed NPC markers and positive for BrdU staining.Some of the NPC donor cells,after subretinal transplantation well survived,were integrated into retina and showed markers of retinal neurons and glia cells.Conclusion Rat iPSCs can be induced to differentiate into NPCs by EB-RA method,which can differentiate into neurons and glia cells after subretinal transplantation.Potentially,iPSC-derived NPCs might be used as donor cells for retinal degeneration.