Li Yongqin, Ding Jianyi, Araki Daisuke, Zou Jizhong, Larochelle Andre
Cellular and Molecular Therapeutics Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
iPSC Core Facility, NHLBI, NIH, Bethesda, MD 20892, USA.
bioRxiv. 2023 Feb 21:2023.02.21.529379. doi: 10.1101/2023.02.21.529379.
Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal and MAPK signaling pathways by stage-specific addition of small molecule regulators CHIR99021, SB431542 and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable generation of human HSPCs with functionality .
The ability to produce functional HSPCs by differentiation of human iPSCs holds enormous potential for cellular therapy of human blood disorders. However, obstacles still thwart translation of this approach to the clinic. In keeping with the prevailing arterial-specification model, we demonstrate that concurrent modulation of WNT, Activin/Nodal and MAPK signaling pathways by stage-specific addition of small molecules during human iPSC differentiation provides a synergy sufficient to promote arterialization of HE and production of HSPCs with features of definitive hematopoiesis. This simple differentiation scheme provides a unique tool for disease modeling, in vitro drug screening and eventual cell therapies.
有几种分化方案可使人诱导多能干细胞（iPSC）分化为造血干细胞和祖细胞（HSPC），但目前仍缺乏能促进具有自我更新、多谱系分化及植入潜力的HSPC发育的优化方案。为改进人iPSC分化方法，我们分别通过阶段特异性添加小分子调节剂CHIR99021、SB431542和LY294002来调节WNT、激活素/节点蛋白（Activin/Nodal）和丝裂原活化蛋白激酶（MAPK）信号通路，并检测其对培养物中造血内皮形成的影响。对这些信号通路的调控产生了足够的协同作用，相对于对照培养条件，可增强动脉血源性内皮（HE）的形成。重要的是，这种方法显著增加了具有自我更新和多谱系分化特性的人HSPC的产量，以及培养物中逐步成熟的表型和分子证据。总之，这些发现为人iPSC分化方案提供了逐步改进，并为操纵内在细胞信号以生成具有功能的人HSPC提供了一个框架。
通过人iPSC分化产生功能性HSPC的能力在人类血液疾病的细胞治疗中具有巨大潜力。然而，这一方法向临床转化仍面临障碍。根据普遍的动脉特化模型，我们证明在人iPSC分化过程中通过阶段特异性添加小分子同时调节WNT、激活素/节点蛋白和MAPK信号通路，可产生足够的协同作用，促进HE的动脉化并产生具有定型造血特征的HSPC。这种简单的分化方案为疾病建模、体外药物筛选及最终的细胞治疗提供了一个独特工具。
