Lakins Johnathon N, Chin Andrew R, Weaver Valerie M
Department of Surgery, Center for Bioengineering and Tissue Regeneration, University of California, San Francisco, San Francisco, CA, USA.
Methods Mol Biol. 2012;916:317-50. doi: 10.1007/978-1-61779-980-8_24.
Human embryonic stem cell (hESc) lines are likely the in vitro equivalent of the pluripotent epiblast. hESc express high levels of the extracellular matrix (ECM) laminin integrin receptor α6β1 and consequently can adhere robustly and be propagated in an undifferentiated state on tissue culture plastic coated with the laminin rich basement membrane preparation, Matrigel, even in the absence of supporting fibroblasts. Such cultures represent a critical step in the development of more defined feeder free cultures of hESc; a goal deemed necessary for regenerative medical applications and have been used as the starting point in some differentiation protocols. However, on standard non-deformable tissue culture plastic hESc either fail or inadequately develop the structural/morphological organization of the epiblast in vivo. By contrast, growth of hESc on appropriately defined mechanically deformable polyacrylamide substrates permits recapitulation of many of these in vivo features. These likely herald differences in the precise nature of the integration of signal transduction pathways from soluble morphogens and represent an unexplored variable in hESc (fate) state space. In this chapter we describe how to establish viable hESc colonies on these functionalized polyacrylamide gels. We suggest this strategy as a prospective in vitro model of the genetics, biochemistry, and cell biology of pre- and early-gastrulation stage human embryos and the permissive and instructive roles that cellular and substrate mechanics might play in early embryonic cell fate decisions. Such knowledge should inform regenerative medical applications aimed at enabling or improving the differentiation of specific cell types from embryonic or induced embryonic stem cells.
人胚胎干细胞（hESc）系可能在体外等同于多能性上胚层。hESc表达高水平的细胞外基质（ECM）层粘连蛋白整合素受体α6β1，因此即使在没有支持性成纤维细胞的情况下，也能在涂有富含层粘连蛋白的基底膜制剂基质胶的组织培养塑料上牢固黏附并以未分化状态增殖。这种培养代表了hESc更明确的无饲养层培养发展中的关键一步；这一目标被认为是再生医学应用所必需的，并且已被用作一些分化方案的起点。然而，在标准的不可变形组织培养塑料上，hESc要么无法发育，要么不能充分发育体内上胚层的结构/形态组织。相比之下，hESc在适当定义的可机械变形的聚丙烯酰胺底物上生长，可以重现许多这些体内特征。这些可能预示着来自可溶性形态发生素的信号转导途径整合的精确性质存在差异，并代表了hESc（命运）状态空间中一个未被探索的变量。在本章中，我们描述了如何在这些功能化的聚丙烯酰胺凝胶上建立可行的hESc集落。我们建议将此策略作为一种前瞻性的体外模型，用于研究原肠胚形成前期和早期人类胚胎的遗传学、生物化学和细胞生物学，以及细胞和底物力学在早期胚胎细胞命运决定中可能发挥的允许和指导作用。这些知识应为旨在促进或改善从胚胎或诱导胚胎干细胞分化特定细胞类型的再生医学应用提供信息。
