Department of Internal Medicine I, Ulm University Hospital, 89081 Ulm, Germany.
Institute of Medical Systems Biology, Ulm University, 89069 Ulm, Germany.
Cells. 2022 Feb 8;11(3):582. doi: 10.3390/cells11030582.
Human pluripotent stem cells, with their ability to proliferate indefinitely and to differentiate into virtually all cell types of the human body, provide a novel resource to study human development and to implement relevant disease models. Here, we employed a human pancreatic differentiation platform complemented with an shRNA screen in human pluripotent stem cells (PSCs) to identify potential drivers of early endoderm and pancreatic development. Deep sequencing followed by abundancy ranking pinpointed six top hit genes potentially associated with either improved or impaired endodermal differentiation, which were selected for functional validation in CRISPR-Cas9 mediated knockout (KO) lines. Upon endoderm differentiation (DE), particularly the loss of SLC22A1 and DSC2 led to impaired differentiation efficiency into CXCR4/KIT-positive DE cells. qPCR analysis also revealed changes in differentiation markers CXCR4, FOXA2, SOX17, and GATA6. Further differentiation of PSCs to the pancreatic progenitor (PP) stage resulted in a decreased proportion of PDX1/NKX6-1-positive cells in SLC22A1 KO lines, and in DSC2 KO lines when differentiated under specific culture conditions. Taken together, our study reveals novel genes with potential roles in early endodermal development.
人类多能干细胞具有无限增殖和分化为人体几乎所有细胞类型的能力，为研究人类发育和建立相关疾病模型提供了新的资源。在这里，我们利用人类胰腺分化平台和人类多能干细胞（PSCs）中的 shRNA 筛选，鉴定早期内胚层和胰腺发育的潜在驱动因素。深度测序和丰度排序后，确定了六个与内胚层分化改善或受损相关的潜在候选基因，这些基因被选为 CRISPR-Cas9 介导的敲除（KO）实验的功能验证。在内胚层分化（DE）过程中，特别是 SLC22A1 和 DSC2 的缺失导致 CXCR4/KIT 阳性 DE 细胞的分化效率降低。qPCR 分析还显示了分化标志物 CXCR4、FOXA2、SOX17 和 GATA6 的变化。进一步将 PSCs 分化为胰腺祖细胞（PP）阶段，在 SLC22A1 KO 系中，PDX1/NKX6-1 阳性细胞的比例降低，在特定培养条件下分化时，DSC2 KO 系中也降低。总之，我们的研究揭示了在早期内胚层发育中具有潜在作用的新基因。
