Serial transplantation reveals a critical role for endoglin in hematopoietic stem cell quiescence

Abstract

Transforming growth factor β (TGF-β) is well known for its important function in hematopoietic stem cell (HSC) quiescence. However, the molecular mechanism underlining this function remains obscure. Endoglin (Eng), a type III receptor for the TGF-β superfamily, has been shown to selectively mark long-term HSCs; however, its necessity in adult HSCs is unknown due to embryonic lethality. Using conditional deletion of Eng combined with serial transplantation, we show that this TGF-β receptor is critical to maintain the HSC pool. Transplantation of Eng-deleted whole bone marrow or purified HSCs into lethally irradiated mice results in a profound engraftment defect in tertiary and quaternary recipients. Cell cycle analysis of primary grafts revealed decreased frequency of HSCs in G₀, suggesting that lack of Eng impairs reentry of HSCs to quiescence. Using cytometry by time of flight (CyTOF) to evaluate the activity of signaling pathways in individual HSCs, we find that Eng is required within the Lin^-Sca⁺Kit⁺-CD48^- CD150⁺ fraction for canonical and noncanonical TGF-β signaling, as indicated by decreased phosphorylation of SMAD2/3 and the p38 MAPK-activated protein kinase 2, respectively. These findings support an essential role for Eng in positively modulating TGF-β signaling to ensure maintenance of HSC quiescence.

Graphical abstract

Figure 1.

Characterization of Eng cKO mice. (A) Scheme for pIpC treatment. To induce Eng deletion, Eng^fl/Δ;Mx1-Cre⁺ mice (blue) and control mice (red), Eng^fl/wt;Mx1-Cre⁺ or Eng^fl/Δ;Mx1-Cre⁻, were injected intraperitoneally with 5 doses of pIpC (250 µg) every other day for 10 days. (B-D) Eng deletion in HSCs. BM from pIpC-treated control mice (left panels) and Eng^fl/Δ;Mx1-Cre⁺ mice (right panels) were analyzed by fluorescence-activated cell sorting (FACS) 2 weeks after the last pIpC injection. (B) Representative gating strategy for the LSKCD48⁻CD150⁺ HSC fraction are showed in the top 3 rows. Control LSKCD48⁻CD150⁺ HSCs are homogenously positive for Eng (bottom left panel), whereas HSCs from pIpC-treated Eng^fl/Δ;Mx1-Cre⁺ mice have significantly reduced levels of this receptor (bottom right panel). Representative histogram plots (C) and respective quantification (D) confirm Eng deletion in HSCs from pIpC-treated Eng^fl/Δ;Mx1-Cre⁺ mice, whereas HSCs from controls express Eng at very high levels. (D) Box plot whiskers represent minimum and maximum values for each cohort (n = 5 per group). (E-G) Blood counts. Time course analyses of white blood cells (WBC) (E), red blood cells (RBC) (F), and platelets (PLT) (G) in pIpC-treated Eng^fl/Δ;Mx1-Cre⁺ mice and control mice. Error bars represent standard error of the mean for each cohort (n = 8 per group). (H) Survival curve shows that Eng deletion does not affect the survival rate of pIpC-treated Eng^fl/Δ;Mx1-Cre⁺ mice compared with controls. A total of 14 mice was analyzed (7 for each group). Mice were followed for a total of 30 weeks. ***P < .001.

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 Eng^fl/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 Eng^fl/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.

Figure 3.

Hematopoietic defect is cell autonomous. (A) Outline of transplantation experiments. A total of 100 purified HSCs from pIpC-treated Eng^fl/Δ;Mx1-Cre⁺ mice (blue) or control mice (red) was injected into lethally irradiated CD45.1 recipient mice, along with 2 × 10⁵ CD45.1 total BM competitor cells. For subsequent serial transplantation, 1 × 10⁶ total pooled BM cells were injected into lethally irradiated CD45.1 recipients. (B) Percentage of CD45.2 chimerism in primary to tertiary recipients (left to right) at 6 and 16 weeks following HSC transplantation confirm defective hematopoietic reconstitution in tertiary recipient mice. Box plot whiskers represent minimum and maximum values for each cohort. (C) Contribution of CD45.2⁺ donor cells to macrophages, granulocytes, B lymphocytes, and T lymphocytes. Error bars indicate standard error of the mean for each cohort. *P < .05.

Figure 4.

CyTOF analysis reveals HSC defect is due to reduced canonical and noncanonical TGF-β signaling. (A-C) CyTOF analysis. (A) Representative SPADE tree of c-Kit⁺ cells (gated on nonapoptotic live Lin⁻c-Kit⁺ cells) from BM cells from 1 representative control mouse. Each node in the tree represents 1 cluster of phenotypically similar cells, whereas node size corresponds to the number of cells. Nodes are colored according to the mean expression of the indicated marker in that particular node. A core of 9 markers was used to generate the SPADE trees, as well as to assign clusters to specific cell fractions. c-Kit expression in all nodes is shown. (B) Expression of the 9 markers used for cluster classification. (C) Mean expression of Eng in cKO Eng mice (right tree) and control mice (left tree) shows that Eng is highly expressed in HSC and MEP fractions from control mice (arrows) but not in the respective cell fractions from mice transplanted with cells lacking Eng (arrows) (n = 4 per group). (D-E) Expression levels of pSMAD2/3 and pMAPKAPK2 (or pMK2), the canonical and noncanonical downstream effectors of TGF-β, respectively, in cKO Eng mice and control mice reveal reduced activation of the TGF-β signaling pathway in the absence of Eng. (D) Heat map shows Eng, pSMAD2/3, and pMK2 arcsinh ratios for HSCs and MEPs from control mice and cKO Eng mice. Each row represents an individual animal (n = 4 per group). Raw expression was arcsinh transformed and fold change was calculated to the column’s maximum value (arcsinh ratio). (E) Quantification of raw expression for pSMAD2/3 and pMK2 in HSCs and MEPs from control mice (red) and cKO Eng mice (blue). Box plot whiskers represent minimum and maximum values for each cohort. *P < .05, **P < .005.

Figure 5.

Proposed model for the function of Eng in HSC quiescence. During transplantation-induced stress (left panel), Eng enhances TGF-β signaling in HSCs, resulting in activation of canonical (SMADs) and noncanonical (TAK1/p38/MK2) pathways, which ultimately leads to regulation of genes that control the cell cycle, and inhibition of the proliferation of HSCs, which ultimately promotes the HSC return to quiescence. However, lack of Eng (right panel) results in decreased signaling of canonical and noncanonical TGF-β pathways, resulting in enhanced proliferation of HSCs and impaired HSC quiescence.