Abstract
The mechanisms which regulate HSC regeneration following stress or injury remain poorly understood. Precise study of HSCs during regeneration has been impeded by the rarity of the HSC population and depletion of phenotypic HSCs early following genotoxic stresses, such as total body irradiation (TBI). We isolated bone marrow (BM) ckit+sca-1+lin- (KSL) cells, which are enriched for HSCs, from adult C57Bl6 mice before and at several time points following TBI, as a means to map the dynamic molecular response of HSC regeneration. Following 550cGy TBI, BM KSL cells were depleted by 7 days post-TBI, whereas KSL cell recovery was evident at day+14. We isolated BM KSL cells and myeloid progenitor cells (c-kit+sca-1-lin- cells) at day +14 and compared the gene expression profile of regenerating HSCs versus steady state HSCs (non-irradiated) and committed progenitor cells. We identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed in regenerating BM KSL cells compared to non-irradiated KSL cells (3.3 fold, p<0.0001).
Grb10 is a member of the family of imprinted genes which are predominately expressed in numerous stem cell populations, including embryonic stem cells, skin and muscle stem cells. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) in detected in 7 day culture (p=0.03 and p=0.002, respectively). Furthermore, mice which were competitively transplanted with Grb10-deficient KSL cells had 10-fold decreased donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed > 10-fold deficit in long-term repopulating capacity in Grb10 deficient KSL cells compared to Grb10 expressing KSL cells (p=0.006 for %CD45.1+ donor cells in secondary mice).
In order to examine the effect of Grb10-deficiency on HSC fate and hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8-week old Grb10m/+ (1 mutant allele, 1 wild type allele) were found to have 10-fold decreased Grb10 expression in BM lin- cells and had normal range complete blood counts. However, BM CFCs were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006) and competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (1/14, 7% vs. 5/14, 38% of mice with > 0.1% donor CD45.2+ cells). These results suggest that Grb10 regulates HSC self-renewal in vitro and in vivo.
Mechanistically, Grb10m/+ mice displayed no alterations in the cell cycle status or frequency of apoptotic cells within BM HSCs compared to Grb10+/+ mice. However, single cytokine functional screening suggested that Grb10 regulates SCF-mediated proliferation of HSCs. Grb10m/+ BM KSL cells generated significantly less CFCs in culture in response to SCF treatment compared to Grb10+/+ KSL cells (p=0.008). Commensurate with this, SCF-mediated activation of mTOR was significantly increased in Grb10m/+ KSL cells compared to that observed in Grb10+/+ KSL cells (p=0.006). These data suggest that cytokine-mediated induction of mTOR signaling, which has been shown to deplete functional HSCs, is antagonized by Grb10, and that Grb10 is necessary to block cytokine-mediated HSC differentiation in vitro and in vivo. Grb10 represents a novel regulator of HSC fate determination and a new mechanistic target to facilitate HSC self-renewal. Studies are underway to determine whether Grb10 is also necessary for HSC regeneration after TBI.
Disclosures
No relevant conflicts of interest to declare.
Platelet-derived growth factor receptor-alpha positive cardiac progenitor cells derived from multipotent germline stem cells are capable of cardiomyogenesis in vitro and in vivo
