Background: Human pluripotent stem cell (hPSC)–derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel.

Methods: We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3β inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix–mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2–degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5⁺ cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription–polymerase chain reaction, and histological analysis.

Results: The resultant hPSC-derived CDH5⁺ cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS–, or human umbilical vein EC–injected groups. However, the group receiving the PA-RGDS–encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC–injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months.

Conclusions: We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS–mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs.

背景：人类多能干细胞（hPSC）衍生的内皮细胞（EC）由于分化系统中的成分不确定以及体内细胞存活率低而在临床上用途有限。在这里，我们旨在开发一个完全定义且临床上兼容的系统，以将hPSC区分为EC。此外，我们旨在通过用肽两亲性（PA）纳米基质凝胶封装hPSC-ECs来提高细胞存活率，血管形成和治疗潜力。

方法：我们通过用糖原合酶激酶3β抑制剂在胶原蛋白包被板上培养，诱导hPSCs分化为中胚层谱系。接下来，添加血管内皮生长因子，内皮生长因子和碱性成纤维细胞生长因子用于内皮谱系分化，然后进行CDH5（VE-钙黏着蛋白）分选。我们通过掺入细胞粘附配体Arg-Gly-Asp-Ser（RGDS）和基质金属蛋白酶2可降解序列，构建了模拟PA纳米基质凝胶（PA-RGDS）的细胞外基质。然后，我们评估了hPSC-CDH5 ⁺的封装是否 通过激光多普勒灌注成像，生物发光成像，实时逆转录-聚合酶链反应和组织学分析，PA-RGDS中的细胞可在后肢缺血模型中增强长期细胞存活和血管再生作用。

结果：产生的hPSC衍生的CDH5 ⁺细胞（hPSC-ECs）表现出高度丰富和真正的EC特性和促血管生成活性。与中，PA-RGDS-或人脐静脉EC-注射组相比，hPSC-ECs注射入缺血性后肢显示出更好的灌注恢复和更高的血管形成能力。但是，与单纯注射hPSC-EC的组相比，接受PA-RGDS封装的hPSC-EC的组显示出更好的灌注恢复，更强劲和更长的细胞存活时间（> 10个月），以及更高和更长的血管生成和血管整合能力。令人惊讶的是，嫁接的hPSC-ECs表现出以前未知的持续和动态的血管形成行为：初始血管周围浓度，新血管形成的指导作用以及在10个月内逐步并入血管。

结论：我们通过临床兼容系统产生了高度富集的hPSC-EC。此外，这项研究表明，生物相容的PA-RGDS纳米基质凝胶可通过延长的独特血管生成和血管形成特性，显着提高hPSC-EC在缺血环境中的长期存活率，并改善hPSC-EC的新血管形成作用。这种PA-RGDS介导的hPSC-EC移植可以作为基于细胞的治疗和hPSC-EC长期行为研究的新平台。