Niche regulation of engraftable HSCs from iPSCs

About This Grant

Summary: Hematopoietic stem cells (HSCs) are vital for sustaining blood lineages and homeostasis in adulthood. While HSC transplantation is a highly effective treatment for bone marrow disorders and blood cancers, its availability is often limited by the shortage of HLA-matched donors. To address this limitation, extensive efforts have been dedicated to developing differentiation strategies for the de novo generation of engraftable HSCs from human pluripotent stem cells (hPSCs), including induced pluripotent stem cells (iPSCs). However, hematopoietic cells derived from hPSC often show limited engraftment capability and sometimes skewed lineage potential, suggesting that essential cues for full functional HSC specification are missing during critical developmental windows. During hematopoietic development, definitive HSCs and HSC-independent progenitors arise primarily in the aortic-gonad-mesonephros (AGM) region. These hematopoietic stem/progenitor cells (HSPCs) originate from hemogenic endothelial cells (HEs) through a process of endothelial-to-hematopoietic transition (EHT), within arterial endothelial cell (aEC) enriched environment. While the requirement of the arterial program in AGM-like HSPC development is recognized, the cellular niche signals guiding cell fate acquisition and HSPC generation remain elusive, especially in humans. This project aims to investigate the role of arterial vascular niche in regulating AGM-like HSPC emergence utilizing autologous endothelial cells derived from iPSCs. We hypothesize that autologous hPSC-derived aECs provide critical niche cues to direct HE fate, promote EHT, and enhance engraftable HSPC generation. We developed a chemically defined, feeder-free monolayer system that recapitulates two distinct waves of primitive and definitive hematopoiesis. In Aim 1, we will characterize the niche effect of autologous aECs on definitive hematopoiesis by investigating whether the niche function requires cell-cell contact, elucidate the molecular mechanism underlying aEC niche function by single-cell RNA sequencing, and develop a new WAS-RUNX1-GFP reporter system to track HE fate, EHT, and HSPC emergence in real time. In Aim 2, we will determine whether aEC- primed HSPCs contain long-term engraftable HSCs by transplantation in immunodeficient mice, and evaluate their self-renewal and multilineage reconstitution capacities. In Aim 3, we will investigate the role of ephrin B2 (EFNB2), a specific gene for arterial specification and Notch signaling target, in aEC niche function by siRNA knockdown, overexpression, and inhibition with specific peptides. Our study will provide mechanistic insights into how the aEC niche regulates human AGM-like HSPC development from iPSCs and offer an in vitro model for the broader understanding of arterial niche signaling in human hematopoiesis.