Dependence of cell fate potential and cadherin switching on primitive streak coordinate during differentiation of human pluripotent stem cells

During gastrulation, the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT),which is marked by cadherin switching, where E-Cadherin is downregulated, and N-Cadherin is upregulated.To understand the relationships between differentiation, EMT, and cadherin switching, we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols, as well as variants in which signaling through key pathways including Activin, BMP, and Wnt were modulated.We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus, cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently, E-Cadherin knockout or overexpression did not alter cell fates outcomes during differentiation. Overall, although all processes are regulated by the same set of signaling pathways, the extent of cadherin switching and EMT can vary substantially within cells adopting the same cell fate. Overall design: RNA-seq of human pluripotent stem cells (ESI017) directed for 24 h into four primitive streak populations - Definitive Endoderm(day1) (DE(a)), Anterior PS (APS), Mid PS (MPS), Posterior PS (PPS) - and of matched cultures further differentiated for an additional 24 h toward Definitive Endoderm(day2) (DE(b)), Paraxial Mesoderm (PM), or Lateral Mesoderm (LM). Each condition was collected in 2 biological replicates.

在原肠胚形成过程中，原条（primitive streak, PS）形成并开始向中内胚层亚型分化。该过程涉及上皮间质转化（epithelial-mesenchymal transition, EMT），其特征为钙粘蛋白转换：E-钙粘蛋白表达下调，而N-钙粘蛋白表达上调。为解析分化、上皮间质转化与钙粘蛋白转换之间的关联，我们借助已建立的实验方案，对人类多能干细胞（human pluripotent stem cells, hPSCs）向原条分化、继而向中内胚层亚型分化过程中的上述进程进行了检测；同时还设置了调控激活素、骨形成蛋白（BMP）、Wnt等关键通路信号的变体实验组。研究发现，通过信号扰动使细胞获得从前到后不同位置的原条身份，对细胞后续的分化潜能影响极小，但会显著影响钙粘蛋白转换的程度。E-钙粘蛋白下调程度与N-钙粘蛋白上调程度并无关联，且二者对信号通路的依赖机制存在差异。不过，整个原条区域细胞普遍具备的分化潜能存在一个例外：中到后位原条身份的细胞丧失了定型内胚层（definitive endoderm）分化潜能。因此，被诱导至不同位置原条的细胞，在中胚层内具备相似的分化潜能，但钙粘蛋白转换程度存在差异。与此一致的是，在分化过程中，E-钙粘蛋白基因敲除或过表达均不会改变细胞命运走向。总体而言，尽管上述所有进程均受同一组信号通路调控，但在获得相同细胞命运的细胞中，钙粘蛋白转换与上皮间质转化的程度仍存在显著差异。整体实验设计：对经既定方案定向诱导24小时的人类多能干细胞（ESI017）进行RNA测序，诱导得到4种原条群体——定型内胚层（第1天，DE(a)）、前位原条（APS）、中位原条（MPS）、后位原条（PPS）；同时对匹配的培养物继续诱导分化24小时，得到定型内胚层（第2天，DE(b)）、轴旁中胚层（PM）或侧中胚层（LM）。每个条件设置2次生物学重复。