McGaugh Emily C, Nostro M Cristina
Toronto General Research Institute, University Health Network; McEwen Centre for Regenerative Medicine, University Health Network; Department of Physiology, University of Toronto.
Toronto General Research Institute, University Health Network; McEwen Centre for Regenerative Medicine, University Health Network; Department of Physiology, University of Toronto;
J Vis Exp. 2017 Mar 7(121):55265. doi: 10.3791/55265.
Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1/NKX6-1 cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1/NKX6-1 pancreatic progenitors at the end of stage 4 are capable of generating mature β cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1/NKX6-1 pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to β cells that could eventually be used for the treatment of diabetes.
多能干细胞具有自我更新和分化为多种谱系的能力，这使其成为生成胰腺祖细胞的有吸引力的来源，这些胰腺祖细胞可用于糖尿病的研究和未来治疗。本文概述了一种四阶段分化方案，旨在从人类胚胎干细胞（hESCs）中生成胰腺祖细胞。该方案可应用于多种人类多能干细胞（hPSC）系。生成胰腺祖细胞所采用的方法是将hESCs分化以准确模拟胰腺发育的关键阶段。这始于确定内胚层的诱导，这是通过在激活素A、碱性成纤维细胞生长因子（bFGF）和CHIR990210存在的情况下培养细胞来实现的。用成纤维细胞生长因子10（FGF10）和多穗柯碱进一步分化和模式化可产生类似于后肠前部的细胞。添加视黄酸、NOGGIN、SANT - 1和FGF10可将后肠前部细胞分化为胰腺内胚层特征性的细胞。最后，表皮生长因子（EGF）、烟酰胺和NOGGIN的组合可有效生成PDX1/NKX6 - 1细胞。进行流式细胞术以确认胰腺发育关键阶段特定标志物的表达。第4阶段结束时的PDX1/NKX6 - 1胰腺祖细胞在移植到免疫缺陷小鼠后能够生成成熟的β细胞，并且在体外可进一步分化以产生胰岛素分泌细胞。因此，如本方案所示，高效生成PDX1/NKX6 - 1胰腺祖细胞非常重要，因为它提供了一个在体外研究人类胰腺发育的平台，并提供了具有分化为β细胞潜力的细胞来源，最终可用于治疗糖尿病。
