Deletion of the Imprinted Gene Grb10 Promotes Hematopoietic Stem Cell Self-Renewal and Regeneration

Abstract

Imprinted genes are differentially expressed by adult stem cells, but their functions in regulating adult stem cell fate are incompletely understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an imprinted gene, regulates hematopoietic stem cell (HSC) self-renewal and regeneration. Deletion of the maternal allele of Grb10 in mice (Grb10^m/+ mice) substantially increased HSC long-term repopulating capacity, as compared to that of Grb10^+/+ mice. After total body irradiation (TBI), Grb10^m/+ mice demonstrated accelerated HSC regeneration and hematopoietic reconstitution, as compared to Grb10^+/+ mice. Grb10-deficient HSCs displayed increased proliferation after competitive transplantation or TBI, commensurate with upregulation of CDK4 and Cyclin E. Furthermore, the enhanced HSC regeneration observed in Grb10-deficient mice was dependent on activation of the Akt/mTORC1 pathway. This study reveals a function for the imprinted gene Grb10 in regulating HSC self-renewal and regeneration and suggests that the inhibition of Grb10 can promote hematopoietic regeneration in vivo.

Keywords: adaptor protein; hematopoietic stem cells; imprinted gene; regeneration; self-renewal.

Figure 1. Grb10 expression is increased in regenerating BM HSCs

(A) At left, representative flow cytometric analysis of BM KSL cells in non-irradiated, adult C57Bl6 mice and at day +7 and day +14 following 550 cGy TBI. At right, mean numbers of BM KSL cells/femur are shown over time following TBI (n=8/group, means ± SEM). (B) The heat map shows the genes whose expression was most highly up- or down-regulated following 550 cGy TBI (n = 6 mice/sample, 6 samples/group. Red=increased expression; green=decreased expression). (C) Mean expression of Grb10 by qRT-PCR analysis of BM KSL cells or c-kit⁺sca-1^-lin^- progenitor cells in non-irradiated mice and at day +14 following 550cGy TBI (n = 6/group, ns=not significant). (D) Mean expression of Grb10 in BM CD34^-KSL HSCs, KSL stem/progenitors and other committed hematopoietic populations by qRT-PCR. WBM=whole bone marrow cells (n=6-10 mice/group). (E) Expression of Grb10, STAT5b and LMX1a in BM CD34^-KSL cells in steady state and at day +10 following 550cGy TBI (n = 6/group). (F) Expression of STAT5b (left) and Grb10 (right) in BM CD34^-KSL cells at day +3 following treatment with siRNA-STAT5b or scramble siRNA (n = 6/group)(all panels, means ± SEM). See also Table S1.

Figure 2. Maternal deletion of Grb10 increases HSC repopulating capacity

(A) Scatter plots of PB white blood counts (WBC), hemoglobin (Hb) and platelet counts (PLT) in 8 week old Grb10 ^m/+ mice and Grb10 ^+/+ mice (n=8–10 mice/group). (B) Mean percentages of erythroid progenitors (EPs) and megakaryocyte progenitors (MkPs) in Grb10 ^m/+ mice and Grb10 ^+/+ mice (n=8/group). (C) Mean BM cell counts, KSL cells, SLAM⁺KSL cells and CFCs in Grb10 ^m/+ mice and Grb10 ^+/+ mice (n=6-12/group; GEMM=colony forming unit-granulocyte erythroid monocyte megakaryocyte; BFU-E=burst forming unit-erythroid; GM=colony forming unit-granulocyte macrophage. (D) Donor (CD45.2⁺) cell engraftment over time in recipient CD45.1⁺ mice transplanted with 5 × 10⁴ BM cells from Grb10 ^m/+ mice or Grb10 ^+/+ mice, together with 2 × 10⁵ CD45.1⁺ competitor BM cells (n=8/ group. P=0.007, P=0.001, P=0.003, P=0.006 for 8, 12, 16, and 20 weeks, respectively, Mann-Whitney test). (E) Left, mean total donor CD45.2⁺ cells in the PB of recipient CD45.1⁺ mice at 20 weeks following competitive transplantation in each group; at right, mean donor-derived HSCs (SLAM⁺KSL cells) in the BM of recipient mice following competitive transplantation (n=8/group, Mann-Whitney test). (F) Mean donor-derived MPPs, CMPs, GMPs and MEPs in the BM of recipient mice following competitive transplantation (n=8/group, Mann-Whitney test). (G) Mean total donor CD45.2⁺ cells in secondary recipient mice at 12 weeks following competitive transplantation (3 × 10⁶ donor BM cells, 2 × 10⁵ competitor cells, n = 10-12/group; Mann-Whitney test)(all panels, means ± SEM). See also Figure S1.

Figure 3. Maternal deletion of Grb10 promotes HSC regeneration following irradiation

(A) Representative H & E stained femurs from Grb10 ^m/+ mice and Grb10 ^+/+ mice at day +10 following 550 cGy TBI and mean BM cell counts in each group (n=8 mice/group; 5x magnification, scale bar=500 μm). (B) Mean BM CFCs in Grb10 ^m/+ mice and Grb10 ^+/+ mice at day +10 and day +14 following 550 cGy TBI (n=6/group). (C) A representative flow cytometric plot of BM KSL cells and SLAM⁺KSL cells and numbers of BM KSL cells and SLAM⁺KSL cells are shown in Grb10 ^+/+ mice and Grb10 ^m/+ mice at day +10 following 550 cGy TBI (n=12 – 14 mice/group; percentages of KSL and SLAM⁺KSL cells are shown in the gates. Mean values represented by horizontal lines). (D) Mean percentages of erythroid progenitors (EPs) and megakaryocyte progenitors (MkPs) in Grb10 ^m/+ mice and Grb10 ^+/+ mice at day +10 following 550 cGy TBI (n=5/group). (E) Donor (CD45.2⁺) cell engraftment over time in the PB of recipient CD45.1⁺ mice transplanted with 5×10⁵ BM cells from Grb10 ^m/+ or Grb10 ^+/+ mice at day +10 following 550 cGy TBI, together with 1×10⁵ CD45.1⁺ competitor BM cells (n = 8 - 10/group, P=0.002, P=0.01, P=0.001, P=0.008, and P=0.03, for 4, 8, 12, 16 and 20 weeks, respectively; Mann-Whitney test). (F) Scatter plots show the percentage donor CD45.2⁺ cell, KSL cell, Mac-1/Gr-1⁺, B220⁺ and CD3⁺ engraftment in the BM at 20 weeks following competitive transplantation of irradiated donor BM cells from each group into recipient CD45.1⁺ mice (n = 8 - 10/group, Mann-Whitney test)(all panels, means ± SEM).

Figure 4. Grb10 deletion promotes HSC regeneration via induction of Akt/mTORC1 signaling

(A) At left, representative flow cytometric plots are shown of BrdU⁺ donor CD45.2⁺ BM cells at 48 hours following transplantation of BM cells from Grb10 ^+/+ mice or Grb10 ^m/+ mice into CD45.1⁺ recipients. At right, the scatter plot shows the mean percentage of BrdU⁺ BM cells in each group (n=6/group). (B) Mean percentages of Grb10 ^+/+ and Grb10 ^m/+ KSL cells in G₀, G₁ and G₂/S/M phase at 3 hours after 300 cGy in vitro irradiation (n = 6/group). (C) Mean percentages of Annexin V⁺ cells in BM KSL cells from Grb10 ^+/+ and Grb10 ^m/+ mice, at 3 hours after 300 cGy in vitro irradiation (n = 8/group). (D) Mean numbers of CFCs from 500 BM KSL cells isolated from Grb10 ^+/+ and Grb10 ^m/+ mice at day +3 following 300 cGy in vitro (n = 6/group). (E) Expression of cyclin dependent kinases and cyclin proteins in BM KSL cells from Grb10 ^+/+ mice and Grb10 ^m/+ mice at 3 hours after 300 cGy (n = 6/group). (F) Mean percentages of senescence associated-β-galactosidase (SA-β-Gal) positive CD34^-KSL cells in Grb10 ^+/+ and Grb10 ^m/+ mice at 24h following 700cGy TBI (n = 8/group). (G) At left, representative flow cytometric analysis of SLAM⁺KSL cells in Grb10 ^+/+ mice and Grb10 ^m/+ mice at day +10 following 550 cGy TBI and treatment with and without the mTORC1 inhibitor, CCI-779. At right, scatter plot shows the numbers of BM SLAM⁺KSL cells in Grb10 ^+/+ and Grb10 ^m/+ mice at day + 10 following 550 cGy TBI and treatment with and without CCI-779 or MK2206, an Akt inhibitor (n = 6/group). (H) Mean ratios of CFC numbers from BM KSL cells in response to SCF treatment or no SCF treatment for 3 days, showing enhanced response in BM KSL cells from Grb10 ^m/+ mice (n = 6/group). (I) At left, representative fluorescence microscopic images (63x, scale bar=10μm) show the binding of Grb10 and c-kit proteins via proximity ligation assay in BM KSL cells from Grb10 ^+/+ mice and Grb10 ^m/+ mice following 15 min of SCF treatment. Blue = DAPI; red = Grb10 - c-kit complex; at right, scatter plot shows the mean fluorescence signal intensity of BM KSL cells in each group (n = 21-30, t test)(all panels, means ± SEM). See also Figure S2 and Figure S3.