Acta Biomaterialia ( IF 9.4 ) Pub Date : 2020-06-27 , DOI: 10.1016/j.actbio.2020.06.034 Thomas Richardson 1 , Connor Wiegand 1 , Fatimah Adisa 1 , K Ravikumar 1 , Joe Candiello 1 , Prashant Kumta 2 , Ipsita Banerjee 2
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Human pluripotent stem cells (hPSCs) have enormous potential to alleviate cell needs for regenerative medicine, however these cells require expansion in cell colonies to maintain cell-cell contact, thus limiting the scalability needed to meet the demands of cell therapy. While the use of a Rho-associated protein kinase (ROCK) inhibitor will allow for culture of single cell hPSCs, typically only 50% of cells are recovered after dissociation. When hPSCs lose cell-cell contact through E-cadherin, dissociation induced apoptosis occurs. In this study, we hypothesized that the extracellular E-cadherin domain of hPSCs will bind to synthetic E-cadherin peptides presented on a hydrogel substrate, mimicking the required cell-cell contact and thereby retaining single-cell viability and clonogenicity. Hence, the objective of this study was to functionalize alginate hydrogels with synthetic peptides mimicking E-cadherin and evaluate peptide performance in promoting cell attachment, viability, maintaining pluripotency, and differentiation potential. We observed that alginate conjugated with synthetic E-cadherin peptides not only supported initial cell attachment with high viability, but also supported hPSC propagation and high fold expansion. hPSCs propagated on the peptide modified substrates maintained the hPSC characteristic pluripotency and differentiation potential, characterized by both spontaneous and directed differentiation.
Statement of Significance
Human pluripotent stem cells (hPSCs) have enormous potential to alleviate cell needs for regenerative medicine and cell therapy. However, scalable culture of hPSCs is challenged by its need for maintenance of cell-cell contact, dissociation of which triggers the apoptotic pathway. Hence hPSCs are commonly maintained as colonies over Matrigel coated culture plates. Furthermore, use of xenogenic and undefined Matrigel compromises the translational potential of hPSCs. In this work we have developed a completely defined substrate to enable adherent culture of hPSCs as single cells. This substrate prevents apoptosis of the single cells and allows significant fold expansion of hPSCs while maintaining pluripotency and differentiation potential. The developed substrate is expected to be a cost-effective and translatable alternative to Matrigel.
中文翻译：
工程化肽修饰的水凝胶平台，可用于人多能干细胞的繁殖。
人类多能干细胞（hPSC）具有缓解再生医学所需细胞的巨大潜力，然而，这些细胞需要在细胞集落中扩增以维持细胞与细胞的接触，从而限制了满足细胞疗法需求所需的可扩展性。尽管使用Rho相关蛋白激酶（ROCK）抑制剂可培养单细胞hPSC，但解离后通常仅回收50％的细胞。当hPSC通过E-cadherin失去细胞与细胞的接触时，发生解离诱导的凋亡。在这项研究中，我们假设hPSC的细胞外E-钙粘蛋白结构域将与水凝胶基质上呈现的合成E-钙粘蛋白肽结合，从而模仿所需的细胞与细胞之间的接触，从而保持单细胞的活力和克隆形成能力。因此，这项研究的目的是用模拟E-钙粘蛋白的合成肽功能化藻酸盐水凝胶，并评估肽在促进细胞附着，生存力，维持多能性和分化潜能方面的性能。我们观察到藻酸盐与合成的E-钙粘蛋白肽缀合不仅支持具有高生存力的初始细胞附着，而且还支持hPSC繁殖和高倍数扩增。在肽修饰的底物上繁殖的hPSC保持了hPSC的特征性多能性和分化潜能，以自发分化和定向分化为特征。我们观察到藻酸盐与合成的E-钙粘蛋白肽缀合不仅支持具有高生存力的初始细胞附着，而且还支持hPSC繁殖和高倍数扩增。在肽修饰的底物上繁殖的hPSC保持了hPSC的特征性多能性和分化潜能，以自发分化和定向分化为特征。我们观察到藻酸盐与合成的E-钙粘蛋白肽缀合不仅支持具有高生存力的初始细胞附着，而且还支持hPSC繁殖和高倍数扩增。在肽修饰的底物上繁殖的hPSC保持了hPSC的特征性多能性和分化潜能，以自发分化和定向分化为特征。
重要声明
人类多能干细胞（hPSC）具有巨大的潜力，可减轻细胞对再生医学和细胞疗法的需求。然而，hPSC的可扩展培养受到维持细胞间接触的需要的挑战，其解离触发细胞凋亡途径。因此，hPSC通常作为基质在Matrigel包被的培养板上保留。此外，使用异种和不确定的基质胶会损害hPSC的翻译潜力。在这项工作中，我们开发了一个完全定义的底物，以使hPSC的粘附培养成为单个细胞。该底物阻止了单细胞的凋亡，并允许hPSC的显着倍数扩张，同时保持了多能性和分化潜能。预计开发的基材将是Matrigel的一种经济高效且可翻译的替代品。
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