Department of Cell Biology, University of Virginia; Cardiovascular Research Center, University of Virginia.
Department of Medicine, Yale University School of Medicine; Department of Genetics, Yale University School of Medicine.
J Vis Exp. 2021 Mar 31(169). doi: 10.3791/62391.
Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3β inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.
血管广泛分布于人体所有组织中，具有多种功能。因此，从人类多能干细胞中分化出成熟的血管内皮细胞对于多种组织工程和再生应用至关重要。在体内，原始内皮细胞来源于中胚层谱系，并向特定的亚型分化，包括动脉、静脉、毛细血管、造血和淋巴管。造血内皮细胞特别有趣，因为在发育过程中，它们产生造血干细胞和祖细胞，然后在整个生命过程中产生所有的血液谱系。因此，创建一种在体外生成造血内皮细胞的系统将提供一个研究内皮细胞向造血细胞转化的机会，并可能导致体外产生人类血液产品，减少对人类供体的依赖。虽然已经有几种从人类胚胎干细胞中获得祖细胞和原始内皮细胞的方案，但尚未描述从人类干细胞中生成特征明确的造血内皮细胞的方法。这里介绍了一种从人类胚胎干细胞中大约在 1 周内获得造血内皮细胞的方法：一种分化方案，通过 GSK3β抑制剂（CHIR99021）诱导形成原始条纹细胞，然后通过 bFGF 介导中胚层谱系诱导，接着通过 BMP4 和 VEGF-A 促进原始内皮细胞发育，最后通过维甲酸诱导造血内皮细胞特化。该方案产生了一种明确的造血内皮细胞群体，可用于进一步了解其分子调控和内皮细胞向造血细胞的转化，这有可能应用于下游的治疗应用。
