A streamlined method to generate endothelial cells from human pluripotent stem cells via transient doxycycline-inducible ETV2 activation.

IF 9.2 1区 医学 Q1 PERIPHERAL VASCULAR DISEASE Angiogenesis Pub Date : 2024-07-05 DOI:10.1007/s10456-024-09937-5
Allen Chilun Luo, Jiuhai Wang, Kai Wang, Yonglin Zhu, Liyan Gong, Umji Lee, Xiang Li, Daniel M Tremmel, Ruei-Zeng Lin, Donald E Ingber, James Gorman, Juan M Melero-Martin
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Abstract

The development of reliable methods for producing functional endothelial cells (ECs) is crucial for progress in vascular biology and regenerative medicine. In this study, we present a streamlined and efficient methodology for the differentiation of human induced pluripotent stem cells (iPSCs) into induced ECs (iECs) that maintain the ability to undergo vasculogenesis in vitro and in vivo using a doxycycline-inducible system for the transient expression of the ETV2 transcription factor. This approach mitigates the limitations of direct transfection methods, such as mRNA-mediated differentiation, by simplifying the protocol and enhancing reproducibility across different stem cell lines. We detail the generation of iPSCs engineered for doxycycline-induced ETV2 expression and their subsequent differentiation into iECs, achieving over 90% efficiency within four days. Through both in vitro and in vivo assays, the functionality and phenotypic stability of the derived iECs were rigorously validated. Notably, these cells exhibit key endothelial markers and capabilities, including the formation of vascular networks in a microphysiological platform in vitro and in a subcutaneous mouse model. Furthermore, our results reveal a close transcriptional and proteomic alignment between the iECs generated via our method and primary ECs, confirming the biological relevance of the differentiated cells. The high efficiency and effectiveness of our induction methodology pave the way for broader application and accessibility of iPSC-derived ECs in scientific research, offering a valuable tool for investigating endothelial biology and for the development of EC-based therapies.

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通过瞬时强力霉素诱导的 ETV2 激活,从人多能干细胞生成内皮细胞的简化方法。
开发生产功能性内皮细胞(ECs)的可靠方法对于血管生物学和再生医学的发展至关重要。在本研究中,我们提出了一种简化、高效的方法,利用强力霉素诱导系统瞬时表达ETV2转录因子,将人类诱导多能干细胞(iPSC)分化为诱导EC(iEC),并在体外和体内保持血管生成能力。这种方法简化了方案,提高了不同干细胞系之间的可重复性,从而减轻了直接转染方法(如mRNA介导的分化)的局限性。我们详细介绍了多西环素诱导 ETV2 表达的 iPSCs 的生成及其随后向 iECs 的分化,在四天内达到 90% 以上的效率。通过体外和体内试验,衍生的 iECs 的功能和表型稳定性得到了严格验证。值得注意的是,这些细胞表现出关键的内皮标志物和能力,包括在体外微生理平台和小鼠皮下模型中形成血管网络。此外,我们的研究结果表明,通过我们的方法生成的 iECs 与原代 ECs 在转录和蛋白质组方面非常接近,这证实了分化细胞的生物学相关性。我们的诱导方法的高效性和有效性为 iPSC 衍生 ECs 在科学研究中的更广泛应用和可及性铺平了道路,为研究内皮生物学和开发基于 EC 的疗法提供了宝贵的工具。
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来源期刊
Angiogenesis
Angiogenesis PERIPHERAL VASCULAR DISEASE-
CiteScore
21.90
自引率
8.20%
发文量
37
审稿时长
6-12 weeks
期刊介绍: Angiogenesis, a renowned international journal, seeks to publish high-quality original articles and reviews on the cellular and molecular mechanisms governing angiogenesis in both normal and pathological conditions. By serving as a primary platform for swift communication within the field of angiogenesis research, this multidisciplinary journal showcases pioneering experimental studies utilizing molecular techniques, in vitro methods, animal models, and clinical investigations into angiogenic diseases. Furthermore, Angiogenesis sheds light on cutting-edge therapeutic strategies for promoting or inhibiting angiogenesis, while also highlighting fresh markers and techniques for disease diagnosis and prognosis.
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