Supplementary Material for: Robust Differentiation of Human Pluripotent Stem Cells into Lymphatic Endothelial Cells Using Transcription Factors

Introduction: Generating new lymphatic vessels has been postulated as an innovative therapeutic strategy for various disease phenotypes, including neurodegenerative diseases, metabolic syndrome, cardiovascular disease, and lymphedema. Yet, compared to the blood vascular system, protocols to differentiate human induced pluripotent stem cells (hiPSCs) into lymphatic endothelial cells (LECs) are still lacking.

Methods: Transcription factors, ETS2 and ETV2 are key regulators of embryonic vascular development, including lymphatic specification. While ETV2 has been shown to efficiently generate blood endothelial cells, little is known about ETS2 and its role in lymphatic differentiation. Here, we describe a method for rapid and efficient generation of LECs using transcription factors, ETS2 and ETV2.

Results: This approach reproducibly differentiates four diverse hiPSCs into LECs with exceedingly high efficiency. Timely activation of ETS2 was critical, to enable its interaction with Prox1, a master lymphatic regulator. Differentiated LECs express key lymphatic markers, VEGFR-3, LYVE-1, and Podoplanin, in comparable levels to mature LECs. The differentiated LECs are able to assemble into stable lymphatic vascular networks in vitro, and secrete key lymphangiocrine, reelin.

Conclusion: Overall, our protocol has broad applications for basic study of lymphatic biology, as well as toward various approaches in lymphatic regeneration and personalized medicine.

引言：生成新生淋巴管被认为是针对多种疾病表型的创新治疗策略，涵盖神经退行性疾病、代谢综合征、心血管疾病及淋巴水肿。然而，相较于血管系统，目前仍缺乏将人类诱导多能干细胞（human induced pluripotent stem cells，hiPSCs）定向分化为淋巴管内皮细胞（lymphatic endothelial cells，LECs）的成熟方案。
方法：转录因子ETS2与ETV2是胚胎血管发育（包括淋巴谱系特化）的关键调控因子。既往研究证实ETV2可高效诱导生成血管内皮细胞，但目前对ETS2及其在淋巴分化中的功能仍知之甚少。本研究报道了一种利用转录因子ETS2和ETV2快速、高效获取LECs的方法。
结果：该方法可稳定地将四种不同来源的hiPSCs以极高效率定向分化为LECs。适时激活ETS2是其与淋巴管主控调控因子Prox1相互作用的关键。分化得到的LECs可表达淋巴管核心标志物VEGFR-3、LYVE-1及Podoplanin，其表达水平与成熟LECs相当。体外实验显示，分化所得LECs可组装形成稳定的淋巴管网络，并能分泌关键的淋巴管内分泌因子reelin。
结论：综上，本研究建立的方案可为淋巴管生物学基础研究、淋巴管再生相关的多种研究策略以及个性化医疗提供广阔的应用前景。