小鼠胚胎干细胞单层分化为神经细胞的PROTOCAL

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Pachernik, J., et al. (2002). "Neural differentiation of mouse embryonic stem cells grown in monolayer." Reproduction Nutrition Development 42(4): 317-326.3 S6 c. ?: v' X5 {
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目前将小鼠胚胎干细胞分化为神经细胞的方法均需要形成拟胚体，并使用全反式视黄酸。本文探讨是否可以通过简单的单层分化将小鼠胚胎干细胞分化神经细胞。首先，mES细胞在添加或不添加aRA的含血清的DMEM培养基中培养2天，然后将陪夜换成含血清的DMEM 或不含血清但添加了ITSF(胰岛素、转铁蛋白、硒、纤连蛋白)的DMEM/F12培养基继续培养。检测细胞形态的变化和分子标志物的表达。最终来说，mES细胞在两天含血清DMEM 培养后换成添加ITSF的DMEM/F12继续培养的方法可以在表观上产生大量的神经细胞。总之，体外条件下不形成EB及不添加aRA也可将mES分化为神经细胞。
( r! H: |" g( gTo drive neural differentiation of mouse embryonic stem (ES) cells, various culture protocols have been previously developed that all require the formation of embryoid bodies, usually combined with a treatment by all-trans retinoic acid (aRA). Here, we investigated whether or not neural differentiation can also occur in a simplified monolayer culture. Mouse ES cells were plated in serum-containing DMEM media with and without aRA and grown under these conditions for 2 days. Then, the cells were transferred to fresh serum-containing DMEM media and/or to serum-free DMEM/F12 media supplemented with a mixture of insulin, transferrin, selenium, and fibronectin (ITSF) for further culture. The changes in cell morphology and in the expression of selected molecular markers were monitored. Generally, in contrast to all the others, the protocol consisting of a 2-day culture in serum-containing DMEM followed by continuous exposure to the ITSF supplement in DMEM/F12 drove a vast majority of ES cells to generate phenotypic signs of neural lineage. Altogether, neural differentiation can be achieved in vitro without the step involving the formation of embryoid bodies as well as the treatment by aRA.
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