Lee Shin-Jeong, Sohn Young-Doug, Andukuri Adinarayana, Kim Sangsung, Byun Jaemin, Han Ji Woong, Park In-Hyun, Jun Ho-Wook, Yoon Young-Sup
Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA (S.-J.L., Y.-D.S., A.A., S.K., J.B., J.W.H., Y.-S.Y.).
Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea (S.-J.L., Y.-S.Y.).
Circulation. 2017 Nov 14;136(20):1939-1954. doi: 10.1161/CIRCULATIONAHA.116.026329. Epub 2017 Sep 29.
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.
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.
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.
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.
人多能干细胞（hPSC）来源的内皮细胞（EC）在临床应用中存在局限性，原因是分化系统中的成分不明确以及体内细胞存活率低。在此，我们旨在开发一种完全明确且临床兼容的系统，将hPSC分化为EC。此外，我们旨在通过用肽两亲物（PA）纳米基质凝胶包裹hPSC-EC来提高细胞存活率、血管形成能力和治疗潜力。
我们通过在涂有胶原蛋白的平板上培养并添加糖原合酶激酶3β抑制剂，将hPSC诱导分化为中胚层谱系。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以促进内皮谱系分化，随后对CDH5（血管内皮钙黏蛋白）进行分选。我们通过掺入细胞黏附配体精氨酸-甘氨酸-天冬氨酸-丝氨酸（RGDS）和基质金属蛋白酶-2可降解序列，构建了一种模拟细胞外基质的PA纳米基质凝胶（PA-RGDS）。然后，我们使用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，评估在PA-RGDS中包裹hPSC-CDH5细胞是否能增强后肢缺血模型中的长期细胞存活率和血管再生效果。
所得的hPSC来源的CDH5细胞（hPSC-EC）表现出高度富集且真实的EC特征和促血管生成活性。与注射培养基、PA-RGDS或人脐静脉EC的组相比，将hPSC-EC注射到缺血后肢时，显示出更好的灌注恢复和更高的血管形成能力。然而，接受PA-RGDS包裹的hPSC-EC的组比单纯注射hPSC-EC的组表现出更好的灌注恢复、更强且更持久的细胞存活（>10个月）以及更高且更持久的血管生成和血管整合能力。令人惊讶的是，移植的hPSC-EC表现出先前未知的持续且动态的血管形成行为：最初在血管周围聚集，对新血管形成起引导作用，并在10个月内逐渐整合到血管中。
我们通过临床兼容的系统生成了高度富集的hPSC-EC。此外，本研究表明，生物相容性PA-RGDS纳米基质凝胶可显著提高hPSC-EC在缺血环境中的长期存活率，并通过延长且独特的血管生成和血管形成特性改善hPSC-EC的新生血管形成效果。这种PA-RGDS介导的hPSC-EC移植可作为基于细胞的治疗以及研究hPSC-EC长期行为的新型平台。
