High c-Kit expression identifies hematopoietic stem cells with impaired self-renewal and megakaryocytic bias

Abstract

Hematopoietic stem cells (HSCs) are heterogeneous with respect to their self-renewal, lineage, and reconstitution potentials. Although c-Kit is required for HSC function, gain and loss-of-function c-Kit mutants suggest that even small changes in c-Kit signaling profoundly affect HSC function. Herein, we demonstrate that even the most rigorously defined HSCs can be separated into functionally distinct subsets based on c-Kit activity. Functional and transcriptome studies show HSCs with low levels of surface c-Kit expression (c-Kit(lo)) and signaling exhibit enhanced self-renewal and long-term reconstitution potential compared with c-Kit(hi) HSCs. Furthermore, c-Kit(lo) and c-Kit(hi) HSCs are hierarchically organized, with c-Kit(hi) HSCs arising from c-Kit(lo) HSCs. In addition, whereas c-Kit(hi) HSCs give rise to long-term lymphomyeloid grafts, they exhibit an intrinsic megakaryocytic lineage bias. These functional differences between c-Kit(lo) and c-Kit(hi) HSCs persist even under conditions of stress hematopoiesis induced by 5-fluorouracil. Finally, our studies show that the transition from c-Kit(lo) to c-Kit(hi) HSC is negatively regulated by c-Cbl. Overall, these studies demonstrate that HSCs exhibiting enhanced self-renewal potential can be isolated based on c-Kit expression during both steady state and stress hematopoiesis. Moreover, they provide further evidence that the intrinsic functional heterogeneity previously described for HSCs extends to the megakaryocytic lineage.

Figure 1.

Differential expression of c-Kit subdivides HSCs into distinct populations. (A) c-Kit^hi and c-Kit^lo HSCs were defined as the top 30% or lowest 30% of c-Kit expressors, respectively, among immunophenotypically defined HSCs (Lin⁻ c-Kit⁺ Sca-1⁺ SLAM⁺ CD34⁻). (B) c-Kit^hi and c-Kit^lo HSCs were double FACS-sorted into liquid culture media supplemented with differentiation-inducing cytokines. After 5 d in culture, total cell output was quantified using a hemocytometer. (C) TetOP-H2B.GFP mice were given doxycycline-treated water for 6 wk before experimentation. 100 TetOP-H2B.GFP⁺ c-Kit^hi or c-Kit^lo HSCs were double FACS-sorted into cytokine supplemented liquid culture. After 5 d, label retention by c-Kit⁺ (green), c-Kit^lo (blue), and c-Kit^hi (red) HSCs was measured by flow cytometry. (D) The GFP mean fluorescence intensity (MFI) in c-Kit^lo and c-Kit^hi HSCs was quantified. (E) Differentiation kinetics were assessed by determining the cellular composition of progeny derived from HSCs using flow cytometry after 5 d in liquid culture. (F) Development of mature progenitors by C57BL/6 c-Kit^hi and c-Kit^lo HSCs was assessed by flow cytometry after 5 d in culture (preGM, L⁻S⁻K⁺ CD41⁻ CD16/32⁻ CD105⁻ CD150⁻; preCFUe, L⁻S⁻K⁺ CD41⁻ CD16/32⁻ CD105⁺ CD150⁺; preMegE, L⁻S⁻K⁺ CD41⁻ CD16/32⁻ CD105⁻ CD150⁺; MkP, L⁻S⁻K⁺ CD41⁺ CD150⁺). (G) 100 c-Kit^hi and c-Kit^lo HSCs were double FACS-sorted into cytokine supplemented methylcellulose media, and total colony output was scored at indicated time points. After 14 d of primary culture, 100,000 cells from each culture were replated into fresh methylcellulose media. (H) Colony types were scored in primary and secondary colony assays after 14 d in culture. (I) Cell cycle analysis of freshly isolated bone marrow was performed using Ki67 and Hoechst 33342, and positive gates were drawn based on isotype controls. Results are representative of three independent experiments, and shown as mean ± SEM. n = 4–5 mice. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

Figure 2.

c-Kit^hi HSCs exhibit reduced engraftment potential. 400 c-Kit^hi and c-Kit^lo HSCs were double FACS-sorted from 3-mo-old C57BL/6 (CD45.2⁺) mice and competitively transplanted into lethally irradiated C57BL/6.SJL (CD45.1⁺) recipients with 300,000 recipient-type bone marrow cells. Donor CD45⁺ (A), Gr1⁺Mac1⁺ granulocyte (B), B220⁺ B cell (C), and CD3⁺ T cell chimerism (D) levels were evaluated in the peripheral blood of primary recipients at the indicated time points. Each data point represents an individual recipient. (E) At week 18 after transplant, bone marrow cells from primary recipients were harvested, and donor HSC (F) and MPP (L⁻S⁺K⁺ CD34⁺; G) chimerism levels were compared with bone marrow aspirates drawn at week 7 after transplant. (H) 400 donor L⁻S⁺K⁺ cells were double FACS-sorted from recipients of c-Kit^hi or c-Kit^lo HSCs and competitively transplanted into secondary, lethally irradiated recipients with 300,000 recipient-type bone marrow cells. Donor CD45 chimerism levels in the peripheral blood of secondary recipients were measured. (I) 400 donor L⁻S⁺K⁺ Slam⁺ CD34⁻ HSCs were double-FACS sorted from an independent cohort of primary recipients that received c-Kit^hi or c-Kit^lo HSCs, and then competitively transplanted into secondary, lethally irradiated recipients with 300,000 recipient-type marrow. Donor HSC chimerism levels in the bone marrow of secondary recipients were measured. (J) 600 pCxeGFP⁺ c-Kit^lo and c-Kit^hi HSCs were double FACS-sorted and transplanted into lethally irradiated mouse recipients. The total number of bone marrow lodged donor HSCs was evaluated 24 h after transplant. (K) qPCR of mRNA transcripts isolated from 2,000 double-sorted c-Kit^hi or c-Kit^lo HSCs. Results are representative of at least two independent experiments, and shown as mean ± SEM. n = 7–9 mice. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

Figure 3.

Hierarchical organization of c-Kit^lo and c-Kit^hi HSCs. (A) 100 c-Kit^lo and c-Kit^hi HSCs were double FACS-sorted into cytokine supplemented liquid culture using the depicted sorting gate (far left). After 24 h of culture, cells were restained and surface c-Kit expression was reevaluated to measure surface levels of c-Kit on c-Kit^lo (middle) and c-Kit^hi (far right) HSCs. (B) The frequency of c-Kit^hi HSCs produced by c-Kit^lo or c-Kit^hi HSCs was calculated by dividing the number of total c-Kit^hi HSCs in the well by the total number of HSCs remaining in each well. (C) Surface c-Kit expression on donor HSCs in recipient bone marrow at week 18 after transplant was compared with surface c-Kit expression on donor c-Kit^hi and c-Kit^lo HSCs at the time of transplant by flow cytometry. Results are representative of four independent experiments. n = 7 mice.

Figure 4.

c-Kit^hi HSCs exhibit a megakaryocytic differentiation bias. (A) CD150 expression on c-Kit^hi and c-Kit^lo HSCs from freshly isolated bone marrow cells was measured by flow cytometry. (B) Donor leukocyte contributions to peripheral blood T cells (CD3⁺), B cells (B220⁺), and granulocytes (Gr1⁺Mac1⁺) was assessed by flow cytometry in mice competitively transplanted with 400 c-Kit^hi or c-Kit^lo HSCs. (C) Single c-Kit^hi or c-Kit^lo HSCs were sorted into cytokine supplemented media and the number of individual HSCs that produced megakaryocytes was evaluated 7 d later. Light microscope images of representative wells are presented with arrows indicating megakaryocytes (left). Bars, 100 µm. c-Kit^hi HSCs gave rise to megakaryocytes in a statistically significant manner compared with c-Kit^lo HSCs (P = 0.002). Graphic depiction of the frequency of individual c-Kit^hi and c-Kit^lo HSCs giving rise to megakaryocytes from two independent experiments (right). (D) Frequency of immunophenotypically defined myeloid progenitors arising from 400 c-Kit^hi and c-Kit^lo HSCs transplanted into lethally irradiated recipients 5 d after transplant. (E) Lethally irradiated mice were transplanted with 400 double-sorted GFP⁺ c-Kit^hi or GFP⁺ c-Kit^lo HSCs from pCxeGFP transgenic mice. Bone marrow of primary recipients was harvested at day 5 and 7 after transplant to evaluate early myeloid commitment and polyploid, CD41⁺ megakaryocyte maturation, respectively. (F) Donor platelet chimerism levels were evaluated in lethally irradiated mice competitively transplanted with 400 double FACS-sorted GFP⁺ c-Kit^hi or c-Kit^lo HSCs with 300,000 recipient-type bone marrow cells. Numbers shown in red represent percent donor platelet chimerism. (G) The ratio of platelet production by donor c-Kit^hi HSCs was normalized to that of donor c-Kit^lo HSC. (H) qPCR of mRNA transcripts isolated from 2,000 double-FACS sorted c-Kit^hi or c-Kit^lo HSCs. Results are representative of two to three independent experiments and are shown as mean ± SEM. n = 4–6 mice. *, P < 0.05; **, P < 0.01.

Figure 5.

5-Fluorouracil treatment preferentially eliminates c-Kit^hi HSCs and enriches for c-Kit^lo HSCs with preserved function. (A) 100 c-Kit^lo, c-Kit^hi, and total c-Kit⁺ HSCs were double FACS-sorted into cytokine-supplemented media with 5-FU or vehicle controls. The percent of c-Kit^hi or c-Kit^lo HSCs (out of total L⁻S⁺K⁺CD150⁺CD34⁻ HSCs remaining in the well) was assessed after 48 h by flow cytometry. (B) Total cell counts remaining after 5-FU treatment were measured manually using a hemocytometer. (C) The frequency of mature progenitors (L⁻S⁻K⁺ and L⁻S⁻K⁻) cells produced after 4 d of culture was measured by flow cytometry. (D) Representative plot of c-Kit expression changes on HSCs after in vivo 5-FU treatment (167 mg/kg) at 3 d after treatment. (E) Megakaryocyte production from 10 c-Kit^hi and c-Kit^lo HSCs isolated from vehicle or 5-FU–treated mice were assessed by flow cytometry after 9 d of culture. Results are representative of two independent experiments, and shown as mean ± SEM, n = 4. *, P < 0.05; **, P < 0.01.

Figure 6.

HSCs with differential c-Kit expression exhibit varying levels of c-Kit signaling. (A and B) Histograms of phosphorylated Stat5 and Stat3 levels in freshly isolated c-Kit^lo HSCs (blue), c-Kit^hi HSCs (red), and isotype control (orange). Frequencies of phosphorylated Stat5⁺ or Stat3⁺ cells among c-Kit^hi and c-Kit^lo HSCs were evaluated by flow cytometry. (C and D) 100 c-Kit^hi and c-Kit^lo HSCs were double FACS-sorted into cytokine-supplemented liquid culture and treated for 24 h with imatinib (1.0 µM) or vehicle controls, after which time cells were placed into fresh media. After 7 d of culture, total cells produced by imatinib or vehicle-treated c-Kit^hi and c-Kit^lo HSCs were measured. Bar, 400 µm. (E) Total number of L⁻S⁻K⁺ progenitors produced by vehicle or imatinib-treated c-Kit^hi or c-Kit^lo HSCs was measured by flow cytometry. (F) After 7 d of daily imatinib treatment in vivo (25 mg/kg), the absolute numbers of myeloid progenitors in the bone marrow of vehicle or imatinib-treated mice were assessed using flow cytometry (CMP: Lin⁻ c-Kit⁺ Sca-1⁻ CD16/32⁻ CD34⁺, GMP: Lin⁻ c-Kit⁺ Sca-1⁻ CD16/32⁺ CD34⁺; MEP: Lin⁻ c-Kit⁺ Sca-1⁻ CD16/32⁻ CD34⁻). (G) 100 c-Kit^hi or c-Kit^lo HSCs were double FACS-sorted into wells containing varying amounts of SCF ranging from 0 to 1 ng/ml. Megakaryocyte number in each well was assessed (after 5 d of megakaryocyte production) by c-Kit^hi HSCs normalized to that of c-Kit^lo HSCs (left). Light microscope images of representative wells are shown. Arrows indicate megakaryocytes (right). Bar, 100 µm. (H) 100 c-Kit^hi or c-Kit^lo HSCs were double FACs-sorted into cytokine-supplemented liquid culture and incubated with anti–c-Kit antibody (ACK2) or isotype controls. Total number of cells produced in each well was assessed manually using a hemocytometer. (I) 600 c-Kit^hi and c-Kit^lo HSCs were double FACS-sorted into cytokine-supplemented liquid culture and treated with ACK2 or isotype control for 24 h, after which they were competitively transplanted into lethally irradiated recipients (CD45.1⁺) with 300,000 recipient-type bone marrow cells. Donor CD45 chimerism levels in the peripheral blood of mice transplanted with c-Kit^lo HSCs (top) or c-Kit^hi HSCs (bottom) were assessed at indicated time points. (J) Donor HSC chimerism levels were evaluated in bone marrow aspirates from primary recipients of isotype or ACK2-treated c-Kit^hi HSCs at week 12 after transplant. Results are representative of at least two independent experiments, and shown as mean ± SEM. n = 3 mice. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

Figure 7.

Loss of c-Cbl activity promotes the transition from c-Kit^lo to c-Kit^hi HSCs. (A) The level of phosphorylated c-Cbl in wild-type c-Kit^lo and c-Kit^hi HSCs was assessed by intracellular staining, followed by flow cytometry. (B) 400 c-Kit^hi or c-Kit^lo HSCs were double FACS-sorted into cytokine-supplemented liquid culture containing PP2 (0.01 M) or vehicle controls. Surface c-Kit levels were assessed by flow cytometry after 24 h of treatment. (C and D) Mice were treated daily with PP2 (5 mg/kg) or vehicle control for 9 d, and frequencies of c-Kit^lo HSCs, c-Kit^hi HSCs, and myeloid progenitors were determined per two femurs and tibias. (E and F) Frequencies of myeloid progenitors were evaluated in two femurs and tibias of c-Cbl^−/− and WT mice. (G and H) Circulating platelet numbers were assessed in WT mice treated with PP2 or vehicle control, as well as in c-Cbl^−/− mice using a Hemavet counter. (I) 100 WT or c-Cbl^−/− c-Kit^hi or c-Kit^lo HSCs were sorted into media, and the production of progenitors by each cell type was assessed after 5 d. The frequency of progenitors produced by c-Kit^hi HSCs was normalized to the frequency produced by c-Kit^lo HSCs. (J) Production of donor c-Kit^hi HSCs was measured in mice competitively transplanted with WT or c-Cbl^−/− c-Kit^lo HSCs. Results are representative of at least two independent experiments, and shown as mean ± SEM, n = 3 to 4 mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001.