Chow Theresa, Wong Frances T M, Monetti Claudio, Nagy Andras, Cox Brian, Rogers Ian M
1Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON Canada.
2Department of Physiology, University of Toronto, Toronto, ON Canada.
NPJ Regen Med. 2020 Apr 20;5:7. doi: 10.1038/s41536-020-0092-5. eCollection 2020.
In order to harness the potential of pluripotent stem cells, we need to understand how to differentiate them to our target cell types. Here, we developed a protocol to differentiate mouse embryonic stem cells (ESCs) to renal progenitors in a step-wise manner. Microarrays were used to track the transcriptional changes at each stage of differentiation and we observed that genes associated with metanephros, ureteric bud, and blood vessel development were significantly upregulated as the cells differentiated towards renal progenitors. Priming the ESCs and optimizing seeding cell density and growth factor concentrations helped improve differentiation efficiency. Organoids were used to determine the developmental potential of the renal progenitor cells. Aggregated renal progenitors gave rise to organoids consisting of LTL+/E-cadherin+ proximal tubules, cytokeratin+ ureteric bud-derived tubules, and extracellular matrix proteins secreted by the cells themselves. Over-expression of key kidney developmental genes, , , , and paralogs, during differentiation did not improve differentiation efficiency. Altogether, we developed a protocol to differentiate mouse ESCs in a manner that recapitulates embryonic kidney development and showed that precise gene regulation is essential for proper differentiation to occur.
为了利用多能干细胞的潜能,我们需要了解如何将它们分化为我们的目标细胞类型。在此,我们开发了一种方案,以逐步方式将小鼠胚胎干细胞(ESCs)分化为肾祖细胞。利用微阵列追踪分化各阶段的转录变化,我们观察到,随着细胞向肾祖细胞分化,与后肾、输尿管芽和血管发育相关的基因显著上调。预处理胚胎干细胞并优化接种细胞密度和生长因子浓度有助于提高分化效率。利用类器官确定肾祖细胞的发育潜能。聚集的肾祖细胞产生了由LTL+/E-钙黏蛋白+近端小管、细胞角蛋白+输尿管芽衍生小管以及细胞自身分泌的细胞外基质蛋白组成的类器官。在分化过程中过表达关键肾脏发育基因、、、和旁系同源物并未提高分化效率。总之,我们开发了一种以模拟胚胎肾脏发育的方式分化小鼠胚胎干细胞的方案,并表明精确的基因调控对于正确分化的发生至关重要。