Bauwens Céline Liu, Peerani Raheem, Niebruegge Sylvia, Woodhouse Kimberly A, Kumacheva Eugenia, Husain Mansoor, Zandstra Peter W
Department of Chemical Engineering and Applied Chemistry, University Health Network and Heart & Stroke Richard Lewar Centre of Excellence, University of Toronto,Toronto, Ontario, Canada.
Stem Cells. 2008 Sep;26(9):2300-10. doi: 10.1634/stemcells.2008-0183. Epub 2008 Jun 26.
To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation, a common approach to mimic some of the spatial and temporal aspects of in vivo development, are affected by three factors: input hESC composition, input hESC colony size, and EB size. Using a microcontact printing approach, size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently, size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover, upon forming EBs from these MP-hESCs, we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs, higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely, neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size, typical in most differentiation strategies, produces subsets of appropriate conditions for differentiation into specific cell types. Moreover, our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories.
为了更好地理解影响多能细胞分化的内源性参数，我们使用人类胚胎干细胞（hESCs）作为模型系统。我们证明，在模拟体内发育的一些时空方面的常用方法——聚集体（胚状体[EB]）诱导分化中，分化轨迹受三个因素影响：输入的hESC组成、输入的hESC集落大小和EB大小。使用微接触印刷方法，通过将单细胞悬液接种到微图案化（MP）细胞外基质岛上来形成大小特定的hESC集落。随后，通过将整个集落转移到悬浮培养中形成大小可控的EB，从而能够独立研究集落和聚集体大小对分化诱导的影响。对MP-hESC群体的基因和蛋白质表达分析表明，随着集落大小的减小，内胚层相关标志物Gata6与神经相关标志物Pax6的表达比值增加。此外，从这些MP-hESCs形成EB后，我们观察到分化轨迹受集落和EB大小影响的参数共同作用。在内胚层偏向（高Gata6/Pax6）的输入hESCs产生的MP-EBs中，在较大的EB大小下观察到更高的中胚层和心脏诱导。相反，神经偏向（低Gata6/Pax6）的输入hESCs产生的MP-EBs在较小的EB中表现出更高的心脏诱导。我们的分析表明，在大多数分化策略中典型的hESC集落和聚集体大小的异质性产生了分化为特定细胞类型的合适条件子集。此外，我们的研究结果表明，局部微环境调节可用于影响多能细胞分化轨迹的内源性参数。
