Transgene-free haematopoietic stem and progenitor cells from human induced pluripotent stem cells

Despite considerable efforts, the generation of vector-free hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (hiPSCs) is not yet possible, preventing the development of cell therapies to cure hematologic diseases. Here, we report the generation of HSCs with multilineage and self-renewal potential from hiPSCs using a specific one-step procedure over a culture period of 17 days. After injection into immunocompromised mice, cells derived from hiPSCs settle in the bone marrow and form a robust multicellular hematopoietic population that can be serially transplanted. RNA sequencing of individual cells suggests that this repopulating activity likely relies on a hematopoietic population transcriptionally similar to HSCs derived from human embryonic aorta. Overall, our results should enable the generation of HSCs from hiPSC and will be instrumental to identify important regulators of HSC production during human ontogeny. Overall design: Embroid bodies (EB) derived from hiPS and cultivated until the chosen timepoints were dissociated in single cells and analysed by scRNAseq using the Chromium 10X v2 technology. Mouse bone marrow 20 weeks after engraftment of the EB cells was flushed and analysed by scRNAseq using the Chromium 10X v3.1 technology

尽管已有大量研究投入，但目前仍无法通过人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）制备无载体造血干细胞（vector-free hematopoietic stem cells, HSCs），这一技术瓶颈阻碍了用于治疗血液系统疾病的细胞疗法的开发。本研究报道了一种特异性一步法培养方案，在17天的培养周期内，可从hiPSCs中获得具备多系分化潜能与自我更新能力的造血干细胞。将此类细胞注射至免疫缺陷小鼠体内后，由hiPSCs分化得到的细胞可定植于骨髓，并形成稳定的多细胞造血群体，且可进行连续移植。单细胞RNA测序分析结果显示，具备造血重建能力的细胞群体，其转录特征与人类胚胎主动脉来源的造血干细胞高度相似。综上，本研究成果实现了从hiPSCs制备造血干细胞的技术路径，将有助于阐明人类个体发育过程中造血干细胞产生的关键调控因子。实验整体设计：将hiPSCs诱导生成的胚状体（Embryoid bodies，EB）培养至预设时间点后，解离为单细胞悬液，采用Chromium 10X v2技术进行单细胞RNA测序（single-cell RNA sequencing, scRNAseq）分析；将EB细胞移植入小鼠体内20周后，冲洗收集小鼠骨髓细胞，采用Chromium 10X v3.1技术进行单细胞RNA测序分析。