Regulation of the hematopoietic stem cell pool by C-Kit-associated trogocytosis

Abstract

Hematopoietic stem cells (HSCs) are routinely mobilized from the bone marrow (BM) to the blood circulation for clinical transplantation. However, the precise mechanisms by which individual stem cells exit the marrow are not understood. This study identified cell-extrinsic and molecular determinants of a mobilizable pool of blood-forming stem cells. We found that a subset of HSCs displays macrophage-associated markers on their cell surface. Although fully functional, these HSCs are selectively niche-retained as opposed to stem cells lacking macrophage markers, which exit the BM upon forced mobilization. Macrophage markers on HSCs could be acquired through direct transfer by trogocytosis, regulated by receptor tyrosine-protein kinase C-Kit (CD117), from BM-resident macrophages in mouse and human settings. Our study provides proof of concept that adult stem cells utilize trogocytosis to rapidly establish and activate function-modulating molecular mechanisms.

Fig. 1.. F4/80 is expressed on hematopoietic stem cells.

(A) Representative flow cytometry plots showing the percentage of F4/80⁺ HSCs in total BM HSCs. (B) Immunofluorescence images of sorted HSCs stained for F4/80. Scale bar, 10 μm. (C) Cell-cycle analyses of HSCs by flow cytometry using anti-K_i-67 and Hoechst 33342. Representative plots (left) and quantification (right; each symbol represents a different mouse) are shown; n = 8 mice per group. (D) Experimental design for competitive bone marrow transplantation (BMT) of 200 sorted CD45.2 F4/80⁻ or F4/80⁺ HSCs along with 250,000 CD45.1 donor cells into lethally irradiated [12 grays (Gy)] CD45.1 mice. For secondary transplantations, bone marrow cells were flushed from femurs of primary recipients and cells from a half femur were transplanted into lethally irradiated CD45.1 mice. (E) Donor-derived chimerism (CD45.2⁺) of total leukocytes from blood of primary (white background) and secondary (gray background) transplanted recipients as shown in (D). n = 7 to 22 mice per group. Data are represented as mean ± SEM and are representative of two (C) or three (E) independent experiments. Statistical significance was assessed by two-way ANOVA with Sidak multiple comparison test [(C) and (E)]. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 2.. F4/80+ HSC defines a non-mobilizable HSC pool.

(A) Blood HSPC (LSKF⁻: Lin⁻ Sca-1⁺ c-Kit⁺ Flt3⁻) numbers after PBS or G-CSF treatment; n = 11 to 12 mice. (B) Blood LSKF⁻ numbers after PBS or AMD3100 (5 mg/kg) treatment; n = 10 to 11 mice. (C) Blood LSKF⁻ numbers after PBS or clodronate liposomes treatment; n = 10 to 11 mice. (D) (Left) Representative flow cytometry plots and (right) percentage of F4/80⁺ HSCs in the bone marrow in young and old mice; n = 4 mice (E) Blood HSPC (LSKF⁻: Lin⁻ Sca-1⁺ c-Kit⁺ Flt3⁻) numbers in young and old mice; n = 4 mice. (F) Blood F4/80⁺ and F4/80⁻ HSPC numbers in young and old mice; n = 4 mice. (G) (Left) Representative flow cytometry plots showing F4/80 and TdTomato expression in bone marrow HSCs from iTdTomato and CD169-Cre/iTdTomato mice. (Right) Percentage of TdTomato⁺ HSCs in F4/80⁻ and F4/80⁺ HSCs from CD169-Cre/iTdTomato mice. n = 7 mice. (H to J) Blood LSKF⁻ numbers after (E) PBS or G-CSF treatment in CD169-Cre/iTdTomato mice; n = 4 to 5 mice; (F) PBS or AMD3100 (5 mg/kg) treatment in CD169-Cre/iTdTomato mice, n = 6 mice; and (G) clodronate liposomes treatment in CD169-Cre/iTdTomato mice; n = 6 to 7 mice. Data in (A) to (J) are represented as mean ± SEM and are representative of two independent experiments; each symbol represents a different mouse. Statistical significance was assessed using two-way ANOVA with Sidak multiple comparisons test [(A) to (C) and (H) to (J)] or two-tailed unpaired t test [(D) to (G)]. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 3.. HSCs acquire membrane signaling components from BM macrophages, thereby enhancing CXCR4-mediated anchoring in bone marrow.

(A) Experimental design for competitive transplantation of 200 sorted TdTomato⁻ or TdTomato⁺ from CD169-Cre/iTdTomato mice (CD45.2) with 250,000 CD45.1 cells into lethally irradiated (12 Gy) CD45.1 recipients. (B) TdTomato expression in total BM (left) and BM HSCs (right) in mice transplanted with TdTomato⁻ or TdTomato⁺ HSCs; n = 6 to 7 mice. (C) Experimental design for noncompetitive BM transplantation of 3 × 10⁶ cells from CD45.1 C57BL/6 mice into lethally irradiated iTdTomato control mice or CD169-Cre/iTdTomato mice (CD45.2). (D) Representative plots of TdTomato expression in BM donor HSCs from control iTdTomato or CD169-Cre/iTdTomato mice transplanted as shown in (C). (E) Representative confocal images showing localization of the TdTomato signal on the cell membrane. Scale bar, 10 μm. (F) Representative flow cytometry plots and quantification of mean fluorescence intensities (MFI) in CXCR4 expression on BM macrophages (Gr-1⁻ F4/80⁺ CD115^int SSC^int/lo) and HSCs (Lin⁻ Sca-1⁺ c-Kit⁺ CD150⁺ CD34⁻); n = 3 mice. (G) Quantification of MFI in CXCR4 expression on F4/80⁻ and F4/80⁺ HSCs; n = 5 mice. (H) Quantification of Cxcr4 mRNA levels in sorted F4/80⁺ and F4/80⁻ HSCs from the bone marrow by quantitative reverse transcription polymerase chain reaction; n = 5 biological replicates. (I) Expression of F4/80 and CXCR4 within BM donor TdTomato⁻ and TdTomato⁺ HSCs from CD169-Cre/iTdTomato recipients; n = 7 mice. (J) The number of macrophages, HSCs, and F4/80⁺ HSCs per femur and the percentage of CXCR4⁺ HSCs in the bone marrow from WT and CD169-DTR mice following DT treatment; n = 5 to 10 mice per group. (K) Experimental design to test F4/80 transfer from mouse macrophages to human HSCs in vivo in immunodeficient huNOG mice transplanted with human CD34⁺ HSPCs. (L) MFI of mouse F4/80 signal on human HSCs; n = 5 mice. Data are represented as mean ± SEM and are representative of two [(B), (F) to (I), and (L)] or three (J) independent experiments; each data point represents a different mouse. Statistical significance was assessed using two-tailed paired t test [(F) to (I); paired dots are connected by lines in (G) to (I)] or two-tailed unpaired t test [(B), (J), and (L)]. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 4.. HSCs acquire membrane signaling components from BM macrophages through c-Kit–mediated trogocytosis.

(A) Transwell (no direct cell contact) and coculture (direct-contact) experiments in which TdTomato⁺ BM macrophages (from Rosa^mT/mG mice) were incubated in the presence of GFP⁺ lineage-depleted BM cells (from UBC-GFP mice). Flow plots show the percentage of TdTomato⁺ GFP⁺ HSCs in total HSCs (defined by Lin⁻ Sca-1⁺ c-Kit⁺ CD150⁺ CD34⁻) in transwell or direct-contact setting. (B) Representative confocal images showing plasma membrane transfer from TdTomato⁺ BM-derived macrophages to GFP⁺ lineage-depleted BM cells. GFP⁺ lineage-depleted BM cells were cocultured with or without TdTomato⁺ macrophages for 1 hour and then taken out for cytospin before confocal microscopy analysis. Scale bar, 5 μm. (C) Time course of percentage of TdTomato⁺ GFP⁺ HSCs after coculture for 5, 15, 30, 60, 120, and 240 min; n = 4 biological replicates (D) Percentage of TdTomato⁺ GFP⁺ double-positive HSCs after coculturing of GFP⁺ lineage-depleted cells with TdTomato⁺ macrophages in the presence of actin depolymerizing agent cytochalasin D; n = 6 biological replicates. (E) MFI of CXCR4 on TdTomato⁻ and TdTomato⁺ HSCs and percentage of CXCR4⁺ HSCs within TdTomato⁻ and TdTomato⁺ HSCs; n = 7 biological replicates. (F) Representative flow cytometry plots and percentage of TdTomato⁺ GFP⁺ double-positive HSCs after coculturing of GFP⁺ lineage-depleted cells with TdTomato⁺ macrophages with PBS or imatinib; n = 5 biological replicates. (G and H) MFI of c-Kit signal on BM HSCs (G) and percentage of TdTomato⁺ GFP⁺ double-positive cells after coculturing of TdTomato⁺ BM macrophages with GFP⁺ lineage-depleted BM cells in the presence of 0, 10, 50, 100 ng/ml SCF (with or without PP2 treatment) (H); n = 3 biological replicates. (I) Percentage of F4/80⁺ HSCs in the bone marrow with vehicle or SCF treatment; n = 9 to 10 mice per group. (J to L) Percentage of F4/80⁺ HSCs in the c-Kit^high, c-Kit^med, and c-Kit^low HSC populations; n = 8 mice. Data in (C) to (L) are represented as mean ± SEM and are representative of two independent experiments; each symbol represents a different biological sample [(D) to (H)] or mouse [(I), (K), and (L)]. SSC, skeletal stem cell. Statistical significance was assessed using two-tailed paired t test [(D) to (F); paired dots are connected by lines] or two-tailed unpaired t test (I) or one-way ANOVA with Tukey’s multiple comparisons test [(G), (H), and (K)] or Ozone correlations (L). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 5.. Macrophage markers are present on human HSCs.

(A) Representative flow plot showing that human BM HSCs (CD34⁺ CD38⁻ CD90⁺ CD45RA⁻ CD49f⁺) stained positive for macrophage markers, CD11b, CD14, and CD163. (B and C) Quantification of macrophage markers on human hematopoietic stem and progenitor populations in the BM; n = 9, 9, 9, and 5 biological replicates for isotype, CD11b, CD14, and CD163, respectively. (D) Quantification of macrophage markers on human BM HSCs and PB HSCs; n = 9, 9, 9, and 5 biological replicates for isotype, CD11b, CD14, and CD163, respectively. (E) Quantification of macrophage markers on HSCs from paired BM and PB samples; n = 5, 5, 5, and 3 biological replicates for isotype, CD11b, CD14, and CD163, respectively. (F to H) Percentage of CD11b⁺ (F), CD14⁺(G), and CD163⁺ (H) HSCs within the C-Kit^low and C-Kit^high HSC populations in human bone marrow samples; n = 9, 9, and 5 biological replicates for (F), (G), and (H), respectively. Data are represented as mean ± SEM and are representative of five (E) or nine [(B) to (D) and (F) to (H)] independent experiments; each symbol represents a different biological sample. Statistical significance was assessed using two-tailed paired t test [(E) to (H); paired dots are connected by lines] or two-tailed unpaired t test (D). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.