(A) study of regulators of endothelial cell differentiation

Abstract

The early regulators involved in initiating endothelial cell transcription factors expression remains to be elucidated. Here, I profiled the expression dynamics of transcription factors involved in endothelial cell specification from human embryonic stem cells. Through intensive mRNA-Seq, the expression characteristics of several classes of immediate early genes, such as FOS, JUN, EGR family, NR4A family and NPAS family at an early stage of endothelial cell differentiation was visualized. Our results showed that NPAS4, a homolog of npas4l exhibit immediate response upon VEGF-A stimuli. Through CRISPR/Cas9 genome editing and Tet-3G Cas9 inducible expression system, NPAS4 knockout and inducible Cas9 hESCs lines was generated, aiming to study role and mechanism of hHLH-PAS family members in endothelial cell specification under VEGF-A signaling. The results showed that NPAS4 depletion does not affect endothelial cell differentiation. HIF1A and AHR inducible knockout was not efficient. The role of Hippo pathway in tissue hemostasis has been well stated, its function in the onset developmental stage of vascular system has not been studied. In vitro self-assembled vascular organoid derived from hESCs provide opportunity to study the role of Hippo pathway either in early development stage or in pathologic condition. Through dual-gRNA mediated CRISPR/Cas9 system, YAP1 inducible knockout hESCs line was generated. Vascular organoid with vasculature structure was generated. These resources can be used for further mechanism study related to Hippo signaling pathway.