Induction of mouse neural crest cells from induced pluripotent stem cells in vitro
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摘要Neural crest cells (NCCs) are a group of embryonic cells which migrate long distances from the neural tube (i.e. developing central nervous system) to different locations of the embryos to form a multitude of derivatives. Based on their migration ability and multipotency, NCCs could be a potential cell source for cell therapy for birth defects involving abnormal NCC development such as Waardenburg syndrome and Hirschsprung’s disease. However, the limited availability of NCCs directly from embryos or primary cultures is one of the main difficulties in developing this cell type into a useful cell source for cell therapy. With the discovery of induced pluripotent stem cells (iPSCs) which possess an unlimited ability of self-renewal, studies began to focus on the in vitro induction of NCCs from iPSCs. In the present study, we sought to develop a robust and efficient protocol to induce NCC formation from mouse iPSCs in vitro. We first induced mouse iPSCs to form crestospheres by a combination of growth factors FGF2, EGF and BMP4. Six days after induction on a non-adhesive surface, round and compact crestospheres derived from iPSCs were observed. Cells within the spheres strongly expressed NCC markers Sox10, AP-2a, p75, Slug, Snail, id2 and id3., and flow cytometric analyses showed that more than half of the cells were p75 immunoreactive. Next, cells were induced to emigrate from the crestospheres on a laminin-coated culture surface in a culture medium containing FGF2 and EGF. Emigrated cells were immunoreactive to NCC markers Sox10, Ap2a, nestin and FoxD3. The final step was to keep the induced cells proliferative and immunoreactive to the NCC markers. This was achieved by culturing them in a medium containing GDNF, in addition to other components. The induced NCCs were then assessed for their migration and differentiation by transplanting them to an embryonic gut explant culture ex vivo. Four days after transplantation, induced NCCs migrated long distances from the transplantation site and differentiated into TuJ1+ neuronal cells. Hence, our results demonstrated that with a three-step protocol involving the formation of crestospheres, iPSCs could be effectively induced into NCCs which were able to undergo extensive migration and differentiation into early neurons.

Funding Source: The work was supported by grants from RGC General Research Fund (CUHK14102214), Theme-based Research Scheme (T12C-714/14-R) and Health and Medical Research Fund (01120456).
著者NIU Yuzhe, FENG Bo, ZHAO Hui, SHAM Mai Har, CHAN Wood Yee
會議名稱2017 ISSCR Annual Meeting
會議地點Boston Convention and Exhibition Center (BCEC)

上次更新時間 2018-20-01 於 18:38