Human induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages. Formation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system. Our protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development. This protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

中文翻译：

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人源性多能干细胞来源的血内皮祖细胞的多向分化潜能

人类诱导的多能干细胞（hiPSC）提供了可再生的细胞来源，用于生成造血细胞，用于基于细胞的治疗，疾病建模和药物筛选。然而，当前的无血清/无饲养者的分化方案依赖于各种细胞因子的使用，这使得该过程非常昂贵或产生胚状体（EB），这是劳动密集型的并且可以在分化过程中引起异质性。在这里，我们报告一个简单的饲养者和无血清单层协议，以有效生成iPSC衍生的多能性血内皮祖细胞（HEPs），它可以进一步分化为包括红系和T谱系的内皮细胞和造血细胞。通过抑制GSK3信号传导2天，然后添加VEGF和FGF2 3天，开始从iPSC形成HEP。通过与OP9-DL1饲养细胞共培养，进一步将HEP诱导成血管生成状态的成熟内皮细胞（EC）和T细胞。补充TGF-β信号抑制剂可进一步促进HEP的内皮细胞向造血细胞的过渡。通过在三阶段红系液体培养系统中培养造血干/祖细胞（HSPC）进行红系分化。在分化的第5天，我们的方案显着提高了KDR + CD34 + CD31 + HEP的数量。HEPs在血管生成条件下的进一步培养促进了成熟EC的形成，表达CD34，CD31，CD144，vWF和ICAM-1，并且可能表现出血管样网络和乙酰化低密度脂蛋白（Ac-LDL）的形成。摄取。此外，HEP分化为CD8 + T淋巴细胞，经TCR刺激可扩增至34倍。在HEP阶段抑制TGF-β信号传导可促进EHT，并产生大量表达CD34和CD43的HSPC。在红系分化后，这些HSPCs扩增至40倍，并在红系发育阶段之后显示出形态变化。该方案为生成多能性HEP提供了一种有效而简单的方法，可用于大量生成所需的血细胞，用于基础研究，包括发育研究，疾病建模和药物筛选以及再生医学。在HEP阶段抑制TGF-β信号传导可促进EHT，并产生大量表达CD34和CD43的HSPC。在红系分化后，这些HSPCs扩增至40倍，并在红系发育阶段之后显示出形态变化。该方案为生成多能性HEP提供了一种有效而简单的方法，可用于大量生成所需的血细胞，用于基础研究，包括发育研究，疾病建模和药物筛选以及再生医学。在HEP阶段抑制TGF-β信号传导可促进EHT，并产生大量表达CD34和CD43的HSPC。在红系分化后，这些HSPCs扩增至40倍，并在红系发育阶段之后显示出形态变化。该方案为生成多能性HEP提供了一种有效而简单的方法，可用于大量生成所需的血细胞，用于基础研究，包括发育研究，疾病建模和药物筛选以及再生医学。