BACKGROUND:A main obstacle in current valvular heart disease research is the lack of high-quality homogeneous functional heart valve cells. Human induced pluripotent stem cells (hiPSCs)-derived heart valve cells may help with this dilemma. However, there are no well-established protocols to induce hiPSCs to differentiate into functional heart valve cells, and the networks that mediate the differentiation have not been fully elucidated.METHODS:To generate heart valve cells from hiPSCs, we sequentially activated the Wnt, BMP4, VEGF (vascular endothelial growth factor), and NFATc1 signaling pathways using CHIR-99021, BMP4, VEGF-165, and forskolin, respectively. The transcriptional and functional similarity of hiPSC-derived heart valve cells compared with primary heart valve cells were characterized. Longitudinal single-cell RNA sequencing was used to uncover the trajectory, switch genes, pathways, and transcription factors of the differentiation.RESULTS:An efficient protocol was developed to induce hiPSCs to differentiate into functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells. After 6-day differentiation and CD144 magnetic bead sorting, ≈70% CD144⁺ cells and 30% CD144^– cells were obtained. On the basis of single-cell RNA sequencing data, the CD144⁺ cells and CD144^– cells were found to be highly similar to primary heart valve endothelial cells and primary heart valve interstitial cells in gene expression profile. Furthermore, CD144⁺ cells had the typical function of primary heart valve endothelial cells, including tube formation, uptake of low-density lipoprotein, generation of endothelial nitric oxide synthase, and response to shear stress. Meanwhile, CD144^– cells could secret collagen and matrix metalloproteinases, and differentiate into osteogenic or adipogenic lineages like primary heart valve interstitial cells. Therefore, we identified CD144⁺ cells and CD144^– cells as hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells, respectively. Using single-cell RNA sequencing analysis, we demonstrated that the trajectory of heart valve cell differentiation was consistent with embryonic valve development. We identified the main switch genes (NOTCH1, HEY1, and MEF2C), signaling pathways (TGF-β, Wnt, and NOTCH), and transcription factors (MSX1, SP5, and MECOM) that mediated the differentiation. Finally, we found that hiPSC-derived valve interstitial-like cells might derive from hiPSC-derived valve endothelial-like cells undergoing endocardial-mesenchymal transition.CONCLUSIONS:In summary, this is the first study to report an efficient strategy to generate functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells from hiPSCs, as well as to elucidate the differentiation trajectory and transcriptional dynamics of hiPSCs differentiated into heart valve cells.

中文翻译：

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人诱导多能干细胞定向分化为心脏瓣膜细胞

背景：目前瓣膜性心脏病研究的一个主要障碍是缺乏高质量均质的功能性心脏瓣膜细胞。人类诱导多能干细胞（hiPSC）衍生的心脏瓣膜细胞可能有助于解决这一困境。然而，目前还没有成熟的方案来诱导 hiPSC 分化为功能性心脏瓣膜细胞，并且介导分化的网络尚未完全阐明。 方法：为了从 hiPSC 生成心脏瓣膜细胞，我们依次激活 Wnt、BMP4 、 VEGF（血管内皮生长因子）和 NFATc1 信号通路分别使用 CHIR-99021、BMP4、VEGF-165 和 forskolin。与原代心脏瓣膜细胞相比，对 hiPSC 衍生的心脏瓣膜细胞的转录和功能相似性进行了表征。使用纵向单细胞 RNA 测序来揭示分化的轨迹、开关基因、途径和转录因子。 结果：开发了一种有效的方案来诱导 hiPSC 分化为功能性 hiPSC 衍生的瓣膜内皮样细胞和 hiPSC-衍生瓣膜间质样细胞。经过6天的分化和CD144磁珠分选，获得约70%的CD144 ⁺细胞和30%的CD144-^细胞。基于单细胞RNA测序数据，发现CD144 ⁺细胞和CD144-^细胞的基因表达谱与原代心脏瓣膜内皮细胞和原代心脏瓣膜间质细胞高度相似。此外，CD144 ⁺细胞具有原代心脏瓣膜内皮细胞的典型功能，包括管形成、低密度脂蛋白的摄取、内皮一氧化氮合酶的产生以及对剪切应力的反应。同时，CD144^细胞可以分泌胶原蛋白和基质金属蛋白酶，并分化成成骨或脂肪形成谱系，如原代心脏瓣膜间质细胞。因此，我们鉴定了 CD144 ⁺细胞和 CD144 ^–细胞分别为 hiPSC 衍生的瓣膜内皮样细胞和 hiPSC 衍生的瓣膜间质样细胞。使用单细胞RNA测序分析，我们证明心脏瓣膜细胞分化的轨迹与胚胎瓣膜发育一致。我们鉴定了介导分化的主要开关基因（NOTCH1、HEY1 和 MEF2C）、信号通路（TGF-β、Wnt 和 NOTCH）以及转录因子（MSX1、SP5 和 MECOM）。最后，我们发现 hiPSC 衍生的瓣膜间质样细胞可能源自经历心内膜-间质转化的 hiPSC 衍生的瓣膜内皮样细胞。结论：总而言之，这是第一项报告生成功能性 hiPSC 的有效策略的研究。研究人员利用 hiPSC 衍生的瓣膜内皮样细胞和 hiPSC 衍生的瓣膜间质样细胞，并阐明 hiPSC 分化为心脏瓣膜细胞的分化轨迹和转录动力学。