Abstract
SummaryAdvances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs.
Highlights
The blood-forming system is sustained by a rare subset of hematopoietic stem cells (HSCs) with the potential to differentiate into all mature blood cell types and to create potent daughter HSCs to maintain tissue homeostasis (Doulatov et al, 2012; Eaves, 2015; Ganuza et al, 2020; Laurenti and Gottgens, 2018)
Single long-term HSC (LT-HSC) can retain multipotency in vitro under minimal cytokine stimulation Previous studies suggested that stem cell factor (SCF) and thrombopoietin (TPO) are essential for HSC self-renewal and proliferation, but potentially dispensable for stem
Twenty-seven genes were commonly differentially expressed (13 up and 14 down) upon addition of SCF with an expected activation of ATP generation and nucleotide metabolism alongside a number of positive cell-cycle mediators (Mcm2, Mcm4, Mcm10, Rad51, and Rad51ap1) and a reduction in developmental and MAPK-mediated signaling (Figures 6B and S4A). In addition to these expected changes, we identified SCF targets induced in HSCs (Figure S4A) and show that expression of Mif (Ohta et al, 2012) and Txn1 (Schenk et al, 1994) are directly promoted upon SCF addition to functional HSCs
Summary
The blood-forming system is sustained by a rare subset of hematopoietic stem cells (HSCs) with the potential to differentiate into all mature blood cell types and to create potent daughter HSCs to maintain tissue homeostasis (Doulatov et al, 2012; Eaves, 2015; Ganuza et al, 2020; Laurenti and Gottgens, 2018). In the absence of robust purification strategies for functional HSCs in culture, it becomes virtually impossible to study the molecular profile of HSCs removed from their in vivo microenvironment. Previous studies have highlighted the potential for retaining long-term HSC (LT-HSC) function in cultures with low amounts of proliferation in the absence of excessive cytokine-induced signaling (Kobayashi et al, 2019; Yamazaki et al, 2006, 2009), these cultures were still predominantly non-HSCs. An in vitro system that could retain highly purified single HSCs would offer the potential to molecularly profile niche-independent HSCs and to resolve the essential components of self-renewal in vitro
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