title: Optimizing mesoderm progenitor selection and three-dimensional microniche culture allows highly efficient endothelial differentiation and ischemic tissue repair from human pluripotent stem cells.
abstract: 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.
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China.
authors.forename: Fengzhi
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, 100084, China.
authors.forename: Yaqian
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, 100084, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China.
authors.forename: Fuyu
authors.initials: F
authors.lastname: Duan
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China.
authors.forename: Rujin
authors.initials: R
authors.lastname: Huang
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Plastic Surgery, Beijing Tsinghua Changgung Hospital, Beijing, 102218, China.|School of Medicine, Tsinghua University, Beijing, 100084, China.
authors.forename: Sophia Chia-Ning
authors.initials: SC
authors.lastname: Chang
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, 100084, China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Du
authors.affiliation: Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China. jie.na@tsinghua.edu.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Na
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D005121:Extremities / Q000473:pathology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D055879:Gene Knock-In Techniques; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007511:Ischemia / Q000473:pathology / Q000628:therapy* / Q000662:veterinary; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018919:Neovascularization, Physiologic / Q000187:drug effects; D049349:Receptors, Lysosphingolipid / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D016222:Fibroblast Growth Factor 2; D049349:Receptors, Lysosphingolipid; D042461:Vascular Endothelial Growth Factor A; C000589015:MESP1 protein, human
keywords: 3D culture; Endothelial cells; Human pluripotent stem cells; MESP1; Vascularization