Zhang Fengzhi, Wang Lin, Li Yaqian, Liu Wei, Duan Fuyu, Huang Rujin, Chen Xi, Chang Sophia Chia-Ning, Du Yanan, Na Jie
Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China.
Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, 100084, China.
Stem Cell Res Ther. 2017 Jan 23;8(1):6. doi: 10.1186/s13287-016-0455-4.
Generation of large quantities of endothelial cells is highly desirable for vascular research, for the treatment of ischemia diseases, and for tissue regeneration. To achieve this goal, we developed a simple, chemically defined culture system to efficiently and rapidly differentiate endothelial cells from human pluripotent stem cells by going through an MESP1 mesoderm progenitor stage.
Mesp1 is a key transcription factor that regulates the development of early cardiovascular tissue. Using an MESP1-mTomato knock-in reporter human embryonic stem cell line, we compared the gene expression profiles of MESP1 and MESP1 cells and identified new signaling pathways that may promote endothelial differentiation. We also used a 3D scaffold to mimic the in vivo microenvironment to further improve the efficiency of endothelial cell generation. Finally, we performed cell transplantation into a critical limb ischemia mouse model to test the repairing potential of endothelial-primed MESP1 cells.
MESP1 mesoderm progenitors, but not MESP1 cells, have strong endothelial differentiation potential. Global gene expression analysis revealed that transcription factors essential for early endothelial differentiation were enriched in MESP1 cells. Interestingly, MESP1 cells highly expressed Sphingosine-1-phosphate (S1P) receptor and the addition of S1P significantly increased the endothelial differentiation efficiency. Upon seeding in a novel 3D microniche and priming with VEGF and bFGF, MESP1 cells markedly upregulated genes related to vessel development and regeneration. 3D microniches also enabled long-term endothelial differentiation and proliferation from MESP1 cells with minimal medium supplements. Finally, we showed that transplanting a small number of endothelial-primed MESP1 cells in 3D microniches was sufficient to mediate rapid repair of a mouse model of critical limb ischemia.
Our study demonstrates that combining MESP1 mesoderm progenitor cells with tissue-engineered 3D microniche and a chemically defined endothelial induction medium is a promising route to maximizing the production of endothelial cells in vitro and augment their regenerative power in vivo.
大量生成内皮细胞对于血管研究、缺血性疾病治疗及组织再生而言极为必要。为实现这一目标，我们开发了一种简单的、化学成分明确的培养系统，通过中胚层后心脏中胚层祖细胞阶段，高效且快速地将人多能干细胞分化为内皮细胞。
Mesp1是调控早期心血管组织发育的关键转录因子。利用MESP1 - mTomato敲入报告基因的人胚胎干细胞系，我们比较了MESP1和MESP1细胞的基因表达谱，并鉴定出可能促进内皮分化的新信号通路。我们还使用3D支架模拟体内微环境，以进一步提高内皮细胞生成效率。最后，我们将细胞移植到严重肢体缺血小鼠模型中，以测试经内皮诱导的MESP1细胞的修复潜力。
MESP1中胚层祖细胞而非MESP1细胞具有强大的内皮分化潜力。全基因表达分析表明，早期内皮分化所必需的转录因子在MESP1细胞中富集。有趣的是，MESP1细胞高表达鞘氨醇 - 1 - 磷酸（S1P）受体，添加S1P可显著提高内皮分化效率。接种到新型3D微环境中并用VEGF和bFGF预处理后，MESP1细胞显著上调了与血管发育和再生相关的基因。3D微环境还能在最少培养基补充的情况下，使MESP1细胞实现长期内皮分化和增殖。最后，我们表明在3D微环境中移植少量经内皮诱导的MESP1细胞足以介导严重肢体缺血小鼠模型的快速修复。
我们的研究表明，将MESP1中胚层祖细胞与组织工程3D微环境及化学成分明确的内皮诱导培养基相结合，是在体外最大化生成内皮细胞并增强其体内再生能力的一条有前景的途径。
