Vitronectin-Activated Alpha V Beta 3 And Alpha V Beta 5 Integrin Signalling Specifies Haematopoietic Fate In Human Pluripotent Stem Cells

Objectives Vitronectin (VTN) has been widely used for the maintenance and expansion of human pluripotent stem cells (hPSCs) as feeder-free conditions. However, the effect of VTN on hPSC differentiation remains unclear. Here, we investigated the role of VTN in early haematopoietic development of hPSCs.Materials and Methods A chemically defined monolayer system was applied to study the role of different matrix or basement membrane proteins in haematopoietic development of hPSCs. The role of integrin signalling in VTN-mediated haematopoietic differentiation was investigated by integrin antagonists. Finally, small interfering RNA was used to knock down integrin gene expression in differentiated cells.Results We found that the haematopoietic differentiation of hPSCs on VTN was far more efficient than that on Matrigel that is also often used for hPSC culture. VTN promoted the fate determination of endothelial-haematopoietic lineage during mesoderm development to generate haemogenic endothelium (HE). Moreover, we demonstrated that the signals through alpha v beta 3 and alpha v beta 5 integrins were required for VTN-promoted haematopoietic differentiation. Blocking alpha v beta 3 and alpha v beta 5 integrins by the integrin antagonists impaired the development of HE, but not endothelial-to-haematopoietic transition (EHT). Finally, both alpha v beta 3 and alpha v beta 5 were confirmed acting synergistically for early haematopoietic differentiation by knockdown the expression of alpha v, beta 3 or beta 5.Conclusion The established VTN-based monolayer system of haematopoietic differentiation of hPSCs presents a valuable platform for further investigating niche signals involved in human haematopoietic development.