Flk1+ and VE-Cadherin+ Endothelial Cells Derived from iPSCs Recapitulates Vascular Development during Differentiation and Display Similar Angiogenic Potential as ESC-Derived Cells

Rationale
Induced pluripotent stem (iPS) cells have emerged as a source of potentially unlimited supply of autologous endothelial cells (ECs) for vascularization. However, the regenerative function of these cells relative to adult ECs and ECs derived from embryonic stem (ES) cells is unknown. The objective was to define the differentiation characteristics and vascularization potential of Fetal liver kinase (Flk)1+ and Vascular Endothelial (VE)-cadherin+ ECs derived identically from mouse (m)ES and miPS cells.
Methods and Results
Naive mES and miPS cells cultured in type IV collagen (IV Col) in defined media for 5 days induced the formation of adherent cell populations, which demonstrated similar expression of Flk1 and VE-cadherin and the emergence of EC progenies. FACS purification resulted in 100% Flk1+ VE-cadherin+ cells from both mES and miPS cells. Emergence of Flk1+VE-cadherin+ cells entailed expression of the vascular developmental transcription factor Er71, which bound identically to Flk1, VE-cadherin, and CD31 promoters in both populations. Immunostaining with anti-VE-cadherin and anti-CD31 antibodies and microscopy demonstrated the endothelial nature of these cells. Each cell population (unlike mature ECs) organized into well-developed vascular structures invitro and incorporated into CD31+ neovessels in matrigel plugs implanted in nude mice invivo.
Conclusion
Thus, iPS cell-derived Flk1+VE-cadherin+ cells expressing the Er71 are as angiogenic as mES cell-derived cells and incorporate into CD31+ neovessels. Their vessel forming capacity highlights the potential of autologous iPS cells-derived EC progeny for therapeutic angiogenesis.

研究依据
诱导多能干细胞（iPS细胞）已成为获取用于血管生成的自体内皮细胞（EC）的潜在无限来源。然而，相较于成体内皮细胞及胚胎干细胞（ES细胞）来源的内皮细胞，此类细胞的再生功能尚不明确。本研究旨在明确以相同方式从小鼠（m）ES细胞及小鼠诱导多能干细胞（miPS细胞）中诱导得到的胎肝激酶1（Flk1）阳性、血管内皮钙粘蛋白（VE-钙粘蛋白）阳性内皮细胞的分化特征与血管生成潜能。

方法与结果
将未分化的小鼠ES细胞及miPS细胞在铺有IV型胶原（IV Col）的限定培养基中培养5天，可诱导形成贴壁细胞群，两类细胞均表现出Flk1与VE-钙粘蛋白的相似表达，并产生内皮细胞子代。通过荧光激活细胞分选（FACS）纯化后，从小鼠ES细胞及miPS细胞中均得到了纯度100%的Flk1阳性、VE-钙粘蛋白阳性细胞。Flk1阳性、VE-钙粘蛋白阳性细胞的出现伴随血管发育转录因子Er71的表达，该因子在两类细胞中均可特异性结合Flk1、VE-钙粘蛋白及CD31启动子。通过抗VE-钙粘蛋白与抗CD31抗体进行免疫染色并结合显微镜观察，证实了此类细胞的内皮细胞特性。与成熟内皮细胞不同，两类细胞群均可在体外形成发育良好的血管结构，并可在植入裸鼠体内的基质胶栓中整合进入CD31阳性新生血管。

结论
综上，表达Er71的iPS细胞来源的Flk1阳性、VE-钙粘蛋白阳性细胞，其成血管活性与小鼠ES细胞来源的同类细胞相当，且可整合进入CD31阳性新生血管。此类细胞的血管形成能力凸显了自体iPS细胞来源的内皮细胞子代在治疗性血管生成领域的应用潜力。