Gata2 haploinsufficiency promotes proliferation and functional decline of hematopoietic stem cells with myeloid bias during aging

Abstract

During aging, hematopoietic stem cell (HSC) function wanes with important biological and clinical implications for benign and malignant hematology, and other comorbidities, such as cardiovascular disease. However, the molecular mechanisms regulating HSC aging remain incompletely defined. GATA2 haploinsufficiency driven clinical syndromes initially result in primary immunodeficiencies and routinely evolve into hematologic malignancies on acquisition of further epigenetic mutations in both young and older patients. Using a conditional mouse model of Gata2 haploinsufficiency, we discover that during aging Gata2 promotes HSC proliferation, monocytosis, and loss of the common lymphoid progenitor. Aging of Gata2 haploinsufficient mice also offsets enhanced HSC apoptosis and decreased granulocyte-macrophage progenitor number normally observed in young Gata2 haploinsufficient mice. Transplantation of elderly Gata2 haploinsufficient HSCs impairs HSC function with evidence of myeloid bias. Our data demonstrate that Gata2 regulates HSC aging and suggest the mechanisms by which Gata2 mediated HSC aging has an impact on the evolution of malignancies in GATA2 haploinsufficiency syndromes.

Figure 1.

Long-term Gata2 haploinsufficiency enhances HSC proliferation, decreases HSC abundance, and causes a reduction in common lymphoid progenitors. (A) Relative expression of Gata2 mRNA in purified hematopoietic compartments, including HSCs (LSK_CD150⁺CD48⁻), HPC1 (LSK_CD150⁻CD48⁺), HPC2 (LSK_CD150⁺CD48⁺), LMPP (LSK_CD34⁺CD135^hi), CMP (LK_CD34⁺CD16/32⁻), GMP (LK_CD34⁺CD16/32⁺), MEP (LK_CD34⁻CD16/32⁻), CLP (Lin⁻c-kit^loSca1^loCD127⁺CD135⁺), myeloid cells (Mac1⁺Gr1⁺), T cells (CD3_ε), and B cells (B220). n = 2 replicates per population. (B) Experimental design for analysis of long-term and short-term Gata2 haploinsufficiency. Gata2^+/fl; Vav-iCre⁻ (control) and Gata2^+/fl; Vav-iCre⁺ mice were analyzed at 18 to 20 months and at 8 to 12 weeks old. (C-D) Representative immunophenotypic HSPC analysis and gating scheme for Gata2 ^+/fl; Vav-iCre⁻ (control) and Gata2^+/fl; Vav-iCre⁺ mice at 18 to 20 months (C) and absolute cell count of primitive and committed hematopoietic populations (D) from control (n = 9) and Gata2^+/fl; Vav-iCre⁺ (n = 7) mice. MPPs are LSK_CD150⁻CD48⁻. Percentages represent a frequency of live nucleated BM cells. Data from 3 independent experiments. (E) Representative flow cytometry plots for cell cycle analysis of BM HSCs from Gata2^+/fl; Vav-iCre⁻ (control) and Gata2^+/fl; Vav-iCre⁺ mice at 18 to 20 months using Ki-67/4′,6-diamidino-2-phenylindole (DAPI). Bivariate plots showing the frequency of HSCs in G0 (Ki-67⁻DAPI⁻), G1 (Ki-67⁺DAPI⁻), and S/G2/M (Ki-67⁺DAPI⁺) phases from control (n = 9) and Gata2^+/fl; Vav-iCre⁺ (n = 7) mice from 3 independent experiments. (F) The percentage of BM HSCs in G0, G1, and S/G2/M cell cycle phases from aged mice (n = 9 control and 7 Gata2^+/fl; Vav-iCre⁺) and young mice (n = 6 for each genotype) from 3 independent experiments for each condition. Data presented as mean ± standard error of the mean (SEM). Statistical analysis is performed using Mann-Whitney U test. Significant data: *P < .05; **P < .01; ***P < .001.

Figure 2.

Elderly Gata2 haploinsufficient HSCs have a functional defect in reconstitution of multilineage hematopoietic compartments with a myeloid bias. (A) Schematic representation of competitive HSC transplantation experiment. Three hundred HSCs from aged control or Gata2^+/fl; Vav-iCre⁺ mice (CD45.2⁺) together with 2 × 10⁵ unfractionated BM competitor cells (CD45.1⁺) were transplanted into lethally irradiated (9.5 Gy) recipient mice (CD45.1⁺). Four independent biological replicates were used for each genotype. Donor chimerism in PB was tested every 4 weeks until week 16 after transplant. (B) Proportion of CD45.2 donor-derived cells in PB after transplantation of donor cells from control (n = 8 recipients) or Gata2^+/fl; Vav-iCre⁺ mice (n = 8 recipients). (C-E) Percentages of CD45.2 donor-derived cells contribution to mature cells in PB (C) BM HSPCs (D) and BM committed myeloid/lymphoid progenitors (E) at week 16 after transplantation of control or Gata2^+/fl; Vav-iCre⁺ donor cells. n = 8 recipients for each genotype from 2 independent experiments. (F) Fold change ratios of CD45.2 donor-derived cell contribution to mature PB cells in aged mice (n = 8 control and 8 Gata2^+/fl; Vav-iCre⁺) in relation to young mice (n = 8 control and 8 Gata2^+/fl; Vav-iCre⁺) donor cells after normalizing to their control counterparts. 2 to 3 independent experiments were performed for each condition. Data is presented as mean ± SEM. Statistical analysis is performed using the Mann-Whitney U test. Significant data: *P < .05; **P < .01; ***P < .001.