Abstract
Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.
Highlights
Single cell transplantation of fresh wild type hematopoietic stem cells (HSC) can successfully repopulate multi-lineage hematopoiesis in myeloid-ablated mice [1,2,3]
Unlike induced pluripotent stem cells, which can be reported by the OCT4 gene reporter, currently, there is no single gene acting as a reporter that can robustly mark bona fide HSC in vitro
These results show that NA10hdLSL/+ and Vav-Cre (NA10hd) confered a competitive advantage on hematopoietic stem cells (HSC) in the transplantation setting
Summary
Single cell transplantation of fresh wild type hematopoietic stem cells (HSC) can successfully repopulate multi-lineage hematopoiesis in myeloid-ablated mice [1,2,3]. The success rates of engraftment remained low in recipients when using 1% donor cell contribution as a readout of successful engraftment. Unlike induced pluripotent stem cells, which can be reported by the OCT4 gene reporter, currently, there is no single gene acting as a reporter that can robustly mark bona fide HSC in vitro. Various functional tests for HSC have been developed [3]. It is still necessary to produce genetically enhanced HSC for a more sensitive stemness-readout of HSC in vitro.
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