State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.
Stem Cell Rev Rep. 2014 Aug;10(4):526-38. doi: 10.1007/s12015-014-9511-6.
Recently, physical factors in the local cellular microenvironment have been confirmed with strong influences on regulating stem cell fate. Despite the recent identification of the rotary cell culture system (RCCS) as a bioreactor for culturing stem cells, the underlying biological role provided by RCCS in the lineage differentiation of embryonic stem cells (ESCs) remains largely undefined. Here, we explored the embryoid body (EB) formation and subsequent differentiation of mouse ESCs in RCCS. We demonstrated that EBs formed in RCCS were more homogeneous and bigger in size compared with those in the static condition. Further, we determined that mesendoderm differentiation was prominently enhanced, while neuroectodermal differentiation was significantly suppressed in RCCS. Surprisingly, we found that Wnt/β-catenin signaling was greatly enhanced mainly due to the increased expression of Wnt3 during ESC differentiation in RCCS. Inhibition of Wnt/β-catenin signaling by DKK1 decreased the expression of Brachyury and attenuated mesendoderm differentiation in RCCS. Intriguingly, Wnt3a markedly increased Brachyury expression under static condition rather than in RCCS. Taken together, our findings uncover a new role of rotary suspension culture in initializing the early differentiation of ESCs.
最近，局部细胞微环境中的物理因素被证实对调节干细胞命运具有强烈影响。尽管旋转细胞培养系统（RCCS）最近被确定为培养干细胞的生物反应器，但 RCCS 在胚胎干细胞（ESCs）谱系分化中的潜在生物学作用在很大程度上仍未得到定义。在这里，我们探索了 RCCS 中胚胎体（EB）的形成和随后的小鼠 ESCs 分化。我们证明，与静态条件相比，RCCS 中形成的 EB 更加均匀且体积更大。此外，我们确定中胚层分化明显增强，而神经外胚层分化在 RCCS 中受到显著抑制。令人惊讶的是，我们发现 Wnt/β-catenin 信号通路显著增强，主要是由于 RCCS 中 ESC 分化过程中 Wnt3 的表达增加。通过 DKK1 抑制 Wnt/β-catenin 信号通路会降低 RCCS 中的 Brachyury 表达并减弱中胚层分化。有趣的是，Wnt3a 在静态条件下而非 RCCS 中显著增加了 Brachyury 的表达。总之，我们的研究结果揭示了旋转悬浮培养在启动 ESCs 早期分化中的新作用。
