Siriraj Center for Regenerative Medicine, Research Department, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Stem Cell Res Ther. 2020 Nov 11;11(1):481. doi: 10.1186/s13287-020-01997-w.
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.
人类诱导多能干细胞（hiPSC）为基于细胞的治疗、疾病建模和药物筛选提供了造血细胞的可再生来源。然而，当前的无血清/饲养层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，或者产生胚状体（EBs），这是劳动密集型的，并且在分化过程中会引起异质性。在这里，我们报告了一种简单的无饲养层和无血清单层方案，用于高效生成 iPSC 衍生的多能造血内皮祖细胞（HEP），它可以进一步分化为内皮细胞和造血细胞，包括红细胞和 T 谱系。
HEP 是从 iPSC 中通过抑制 GSK3 信号通路 2 天，然后添加 VEGF 和 FGF2 3 天开始形成的。HEP 进一步在血管生成条件下诱导成熟内皮细胞（EC），并通过与 OP9-DL1 饲养细胞共培养诱导 T 细胞。通过补充 TGF-β 信号抑制剂进一步促进 HEP 的内皮向造血转变（EHT）。通过在三阶段红细胞液体培养系统中培养造血干细胞/祖细胞（HSPC）进行红细胞分化。
我们的方案显著增加了分化第 5 天 KDR CD34 CD31 HEP 的数量。HEP 在血管生成条件下进一步培养促进了成熟 EC 的形成，成熟 EC 表达 CD34、CD31、CD144、vWF 和 ICAM-1，并能形成血管样网络和乙酰化低密度脂蛋白（Ac-LDL）摄取。此外，HEP 分化为 CD8 T 淋巴细胞，在 TCR 刺激下可扩增 34 倍。HEP 阶段 TGF-β 信号的抑制促进了 EHT，并产生了大量表达 CD34 和 CD43 的 HSPC。进行红细胞分化时，这些 HSPC 可扩增 40 倍，并在红细胞发育的各个阶段呈现形态变化。
该方案提供了一种高效简单的方法来生成多能 HEP，并可用于大量生成所需的血细胞，应用于基础研究，包括发育研究、疾病建模和药物筛选以及再生医学。
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