Figure 2.
Figure 2. Serial transplantation reveals Eng as an important regulator of HSC quiescence. (A) Outline of transplantation experiments. Total BM cells (5 × 105 cells) from pIpC-treated Engfl/Δ;Mx1-Cre+ or control mice (all CD45.2 background) were injected into lethally irradiated CD45.1 recipient mice. Competitor cells consisted of 2 × 105 total CD45.1 BM cells. For subsequent serial transplantation, 1 × 106 total BM cells were injected into lethally irradiated CD45.1 recipients. (B) Percentage of CD45.2 chimerism in primary recipients at 6 and 16 weeks posttransplantation (short term and long term, respectively) shows no differences in mice injected with Eng-deleted BM or control BM. Box plot whiskers represent minimum and maximum values for each cohort (n = 8-10 per group). (C) Contribution of CD45.2+ donor cells to several blood lineages: macrophages (Mac-1), granulocytes (Gr-1), B lymphocytes (CD19), and T lymphocytes (CD3). Error bars indicate standard error of the mean for each cohort. (D-E) BM analysis of primary recipients. (D) Frequency of total HSCs in the BM of transplanted mice. Box plot whiskers represent minimum and maximum values for each cohort (n = 5 per group). (E) Percentage of donor contribution to several cell fractions within the BM, including LSK cells, HSCs (LSKCD150+CD48−), and multipotent progenitor cells (MPP; LSKCD150−CD48−). Total BM is shown as reference. Box plot whiskers represent minimum and maximum values for each cohort (n = 5 per group). (F) Serial transplantation data. Percentage of CD45.2 chimerism in secondary to quaternary recipients (left to right) at 6 and 16 weeks posttransplantation reveals defective hematopoietic reconstitution in tertiary and quaternary recipient mice. Box plot whiskers represent minimum and maximum values for each cohort. (G-H) Cell cycle analysis. (G) Representative fluorescence-activated cell sorting plots of BM LSK CD48− CD150+ HSCs isolated from primary recipient animals that had been transplanted with pIpC-treated Engfl/Δ;Mx1-Cre+ mice or control mice (in blue and red, respectively), analyzed in combination with Ki67 (proliferation) and propidium iodide (PI; for DNA content) 6 months posttransplantation. (H) Quantification of cell cycle analysis. Error bars represent standard error of the mean. *P < .05 **P < .01, ***P < .001.

Serial transplantation reveals Eng as an important regulator of HSC quiescence. (A) Outline of transplantation experiments. Total BM cells (5 × 105 cells) from pIpC-treated Engfl/Δ;Mx1-Cre+ or control mice (all CD45.2 background) were injected into lethally irradiated CD45.1 recipient mice. Competitor cells consisted of 2 × 105 total CD45.1 BM cells. For subsequent serial transplantation, 1 × 106 total BM cells were injected into lethally irradiated CD45.1 recipients. (B) Percentage of CD45.2 chimerism in primary recipients at 6 and 16 weeks posttransplantation (short term and long term, respectively) shows no differences in mice injected with Eng-deleted BM or control BM. Box plot whiskers represent minimum and maximum values for each cohort (n = 8-10 per group). (C) Contribution of CD45.2+ donor cells to several blood lineages: macrophages (Mac-1), granulocytes (Gr-1), B lymphocytes (CD19), and T lymphocytes (CD3). Error bars indicate standard error of the mean for each cohort. (D-E) BM analysis of primary recipients. (D) Frequency of total HSCs in the BM of transplanted mice. Box plot whiskers represent minimum and maximum values for each cohort (n = 5 per group). (E) Percentage of donor contribution to several cell fractions within the BM, including LSK cells, HSCs (LSKCD150+CD48−), and multipotent progenitor cells (MPP; LSKCD150−CD48−). Total BM is shown as reference. Box plot whiskers represent minimum and maximum values for each cohort (n = 5 per group). (F) Serial transplantation data. Percentage of CD45.2 chimerism in secondary to quaternary recipients (left to right) at 6 and 16 weeks posttransplantation reveals defective hematopoietic reconstitution in tertiary and quaternary recipient mice. Box plot whiskers represent minimum and maximum values for each cohort. (G-H) Cell cycle analysis. (G) Representative fluorescence-activated cell sorting plots of BM LSK CD48− CD150+ HSCs isolated from primary recipient animals that had been transplanted with pIpC-treated Engfl/Δ;Mx1-Cre+ mice or control mice (in blue and red, respectively), analyzed in combination with Ki67 (proliferation) and propidium iodide (PI; for DNA content) 6 months posttransplantation. (H) Quantification of cell cycle analysis. Error bars represent standard error of the mean. *P < .05 **P < .01, ***P < .001.

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