Abstract
BackgroundStrategies of generating functional blood cells from human pluripotent stem cells (hPSCs) remain largely unsuccessful due to the lack of a comprehensive understanding of hematopoietic development. Endothelial-to-hematopoietic transition (EHT) serves as the pivotal mechanism for the onset of hematopoiesis and is negatively regulated by TGF-β signaling. However, little is known about the underlying details of TGF-β signaling during EHT.MethodsIn this study, by applying genome-wide gene profiling, we identified muscle segment homeobox2 (MSX2) as a potential mediator of TGF-β signaling during EHT. We generated MSX2-deleted human embryonic stem cell (hESC) lines using the CRISPR/Cas9 technology and induced them to undergo hematopoietic differentiation. The role of MSX2 in hematopoiesis and functional regulation of TGFβ signaling in EHT was studied.ResultsWe identified MSX2 as a novel regulator of human hematopoiesis. MSX2 deletion promotes the production of hematopoietic cells from hESCs. Functional and bioinformatics studies further demonstrated that MSX2 deletion augments hematopoietic differentiation of hESCs by facilitating EHT. Mechanistically, MSX2 acts as a downstream target of TGFβ signaling to mediate its function during EHT.ConclusionsOur results not only improve the understanding of EHT, but may also provide novel insight into the efficient production of functional blood cells from hPSCs for regenerative medicine.
Highlights
Strategies of generating functional blood cells from human pluripotent stem cells remain largely unsuccessful due to the lack of a comprehensive understanding of hematopoietic development
muscle segment homeobox2 (MSX2) is suppressed upon inhibition of TGFβ signaling To explore the molecular mechanism by which TGFβ signaling regulates Endothelialto-hematopoietic transition (EHT) during hematopoietic differentiation of human embryonic stem cell (hESC), we added SB431542, a well-established TGFβ inhibitor, at the stage of EHT in a chemically defined hematopoietic differentiation system previously established by us [31], and subsequently performed RNA-seq by collecting the CD31+ cells at day 8 of differentiation (Fig. 1a)
Our findings revealed that MSX2 deletion augments hematopoietic differentiation of human pluripotent stem cells (hPSCs), no apparent defects in hematopoietic development has been observed in Msx2 knockout mice
Summary
Strategies of generating functional blood cells from human pluripotent stem cells (hPSCs) remain largely unsuccessful due to the lack of a comprehensive understanding of hematopoietic development. It remains a great challenge to generate HSCs with robust multilineage engraftment potential and infusion dosage levels of functional blood cells from hESCs, mainly because of the lack of understanding of the underlying mechanisms controlling hematopoietic development [4]. HPCs emerge from HEPs through the process of endothelial-to-hematopoietic transition (EHT), which serves as a vital mechanism for the initiation of hematopoiesis and is tightly controlled by several signaling pathways [6, 7]. Activation of TGFβ signaling completely abolishes the generation of HPCs from HEPs, while TGFβ inhibition promotes the transition—a response conserved in both mouse and human embryonic stem cells [15,16,17,18]. Despite its well documented vital role in EHT, little is known about how TGFβ signaling exerts its function during this process
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