Deterministic role of sonic hedgehog signalling pathway in specification of hemogenic versus endocardiogenic endothelium from differentiated human embryonic stem cells.

Abstract

Embryonic stem cells (ESCs) have been shown to have an ability to form a large number of functional endothelial cells in vitro, but generating organ-specific endothelial cells remains a challenge. Sonic hedgehog (SHH) pathway is one of the crucial developmental pathways that control differentiation of many embryonic cell types such as neuroectodermal, primitive gut tube and developing limb buds; SHH pathway is important for functioning of adult cell of skin, bone, liver as well as it regulates haematopoiesis. Misregulation of SHH pathway leads to cancers such as hepatic, pancreatic, basal cell carcinoma, medulloblastoma, etc. However, its role in differentiation of human ESCs into endothelial cells has not been completely elucidated. Here, we examined the role of SHH signalling pathway in endothelial differentiation of hESCs by growing them in the presence of an SHH agonist (purmorphamine) and an SHH antagonist (SANT-1) for a period of 6days. Interestingly, we found that activation of SHH pathway led to a higher expression of set of transcription factors such as BRACHYURY, GATA2 and RUNX1, thus favouring hemogenic endothelium; whereas inhibition of SHH pathway led to a reduced expression of set of markers such as RUNX1 and BRACHURY, and an increased expression of set of markers - NFATC1, c-KIT, GATA4, CD31 & CD34, thus favouring endocardiogenic endothelium. The results of this study have revealed the previously unreported deterministic role of SHH pathway in specification of endothelial cells differentiated from human ESCs into hemogenic vs. endocardiogenic lineage; this finding could have major implications for clinical applications.