Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor

Abstract

Background hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. Methods Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other’s (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. Results A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. Conclusion The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.

Generating hematopoietic cells from human pluripotent stem cells: approaches, progress and challenges

“…At AGM, HE undergoes EHT to obtain specific lineage characteristics: upregulation of hematopoietic cell markers CD43 and CD45, while downregulation of endothelial makers, like CD144 and CD31, namely conduct a transition of endothelial cells to a hematopoietic cell fate to become HSCs (Bertrand et al 2010 ; Guibentif et al 2017 ; Velten et al 2017 ; Zhou et al 2016 ). Inspired by the EHT process, several groups have established approaches to differentiate HE from hPSCs to generate functional HSPCs and blood cells in vitro (Choi et al 2012 ; Ditadi et al 2015 ; Garcia-Alegria et al 2018 ; Kennedy et al 2012 ; Vargas-Valderrama et al 2022 ). Choi et al showed that definitive hematopoietic cells can be obtained from hPSC-derived HE progenitors and equipped with an enhanced myeloid and erythroid lineage potential.…”

Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins

“…Hematopoietic differentiation has been shown to require stages of mesodermal induction, hematoendothelial speci cation, vascular arterialization, endothelial-to-hematopoietic transition, and hematopoietic maturation (40,45). Both EB and MB approaches have previously been reported to recapitulate these steps to varying degrees (37,(46)(47)(48)(49)(50)(51)(52).…”

ScRNA-seq and spatial transcriptomics: exploring the occurrence and treatment of coronary-related diseases starting from development

“…Endothelial progenitor cells, which retain their differentiation potential in the adult heart, are one of the main sources of neovascularization after myocardial infarction ( 59 ). Li et al selected isolated mouse hearts for single-cell sequencing and found that endothelial progenitor cells in coronary resident endothelial cells after myocardial infarction could form new blood vessels via clonal proliferation ( 5 ).…”