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c-Kit Expression and Stem Cell Factor-Induced Hematopoietic Cell Proliferation Are Up-Regulated in Aged B6D2F1 Mice

¹ Hematology Branch
² Flow Cytometry Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.

Address correspondence to Dr. Jichun Chen, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Clinical Research Center Room 3-5132, 10 Center Drive, Bethesda, MD 20892-1202. E-mail: chenji{at}nhlbi.nih.gov

	Abstract

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Expression of c-Kit (CD117) and stem cell factor/c-Kit-mediated cell proliferation were tested in vitro in young and old B6D2F1 mice to study the role of c-Kit signaling in hematopoietic stem cell (HSC) senescence. Increasing age is associated with a significant increase in bone marrow (BM) cells without affecting mature blood cells. The number of c-Kit-expressing BM cells increased significantly in old mice when compared to young controls, to 201% in total BM cells, 261% in Lin^– cells, 517% in Lin^–CD34⁺Sca1⁺ progenitor cells, and 1272% in Lin^–CD34^–Sca1⁺ HSCs. Sorted Lin^–Sca1⁺CD117⁺ BM cells from an old mouse expanded 5-fold when cultured in vitro for 72 hours with stem cell factor at 25 ng/ml, which was significantly higher than a 2.5-fold expansion of the same cells from a young donor. HSCs and progenitor cells from B6D2F1 mice maintain extremely high proliferative potentials and do not reach proliferative arrest at old age during a normal life span.

An important pair of cytokine-receptor signaling molecules in the regulation of HSC function and senescence are stem cell factor (SCF) and its receptor, c-Kit (CD117), a transmembrane receptor tyrosine kinase expressed on primitive HSCs and progenitor cells (14–18). Expression of CD117 has been used as a marker to characterize HSCs in mice and in humans (15,16,19–21). SCF and c-Kit interaction triggers a chain of signaling events which activate downstream pathways leading to cell proliferation and/or differentiation (14,22). Spontaneous mutations in mice of the c-Kit gene result in the characteristic white spotting with serious consequences, including functional failure in hematopoiesis (17,18,23–25). Thus, c-Kit signaling plays a significant role in maintaining normal hematopoiesis.

In the current study, we specifically tested the effects of aging on c-Kit expression and c-Kit signaling in bone marrow (BM) hematopoietic cells. First, we found that the proportion and total number of c-Kit⁺ cells are significantly increased in aged B6D2F1 mice in various BM cell fractions. We then sorted BM cells into Lin^–Sca1⁺CD117⁺ and Lin^–Sca1⁺CD117^– fractions and cultured them in vitro to test cell expansion with or without SCF stimulation. Our data support the view that HSCs from B6D2F1 mice have very high proliferative potentials and do not reach proliferative arrest even at an old age during a normal life span. Increasing age is associated with an enlarged HSC pool and an enhanced HSC response to SCF stimulation in B6D2F1 mice.

	METHODS

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Mice and Cell Preparation
Hybrid B6D2F1 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were housed in the National Institutes of Health animal facility under standard care and nutrition conditions. We used young (2–6 months) and old (20–30 months) females in the current study.

Peripheral blood samples were obtained through retro-orbital sinus bleeding. Complete blood counts were performed using a Hemavet 1500 analyzer (Drew Scientific, Oxford, CT). BM cells were flushed from two femurs and two tibias of each mouse into 2 mL of flow buffer (2.68 mM KCl, 1.62 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 137 mM NaCl, 7.69 mM NaN₃, and 1% bovine serum albumin) using a 25-gauge needle, and filtered through a 90-µm nylon mesh (Small Parts, Miami Lakes, FL) to prepare for single cell suspensions. BM cells were counted using a model Z2 Coulter Counter (Coulter, Hialeah, FL) after erythrocytes were lysed with ZAP-OGLOBIN II lytic reagent (Coulter). Total BM cells were calculated based on the assumption that two tibiae and two femurs contain 25% of all BM cells in the body.

Flow Cytometry
BM cell composition was analyzed by flow cytometry. Cells were incubated in Gey's solution (130.68 mM NH₄Cl, 4.96 mM KCl, 0.82 mM Na₂HPO₄, 0.16 mM KH₂PO₄, 5.55 mM dextrose, 1.03 mM MgCl₂, 0.28 mM MgSO₄, 1.53 mM CaCl₂, and 13.39 mM NaHCO₃) for 10 minutes on ice to lyse red blood cells, stained first with a premixed antibody cocktail and then with diluted streptavidin-conjugated Quantum Red (SAQR), and analyzed using a BD LSR II flow cytometer (BD Biosciences,<--1-->San Jose, CA). Monoclonal antibodies for murine CD3 (clone 145-2C11), CD4 (clone GK 1.5), CD8 (clone 53-6.72), CD11b (clone M1/70), CD19 (clone ID3), CD34 (clone RAM34), CD45R (B220, clone RA3-6B2), CD117 (c-Kit, clone 2B8), erythroid cells (clone Ter119), granulocytes (Gr1/Ly6-G, clone RB6-8C5), and stem cell antigen 1 (Sca1, clone E13-161) were all purchased from BD Biosciences (San Diego, CA). Each antibody was conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), biotin, or allophycocyanin (APC). SAQR was purchased from Sigma (St. Louis, MO). Each acquisition was stopped when 20,000 or 1,000,000 cells were collected, depending on the type of analysis and the availability of cells.

Cell Sorting
BM cells from two tibias and two femurs of each young or old B6D2F1 mouse were flushed into 2 mL of Iscove's modified Dulbecco's medium (IMDM; American Type Culture Collection, Manassas, VA). Residual erythrocytes were lysed using Gey's solution, and cells were stained for 30 minutes on ice with a premixed antibody cocktail containing Sca1- FITC, CD117-PE, and Lineage-Biotin (CD3, CD4, CD8, CD11b, CD19, Gr1, and Ter119). After a second staining with SAQR for 30 minutes, cells were sorted into Lin^–Sca1⁺CD117⁺ and Lin^–Sca1⁺CD117^– fractions using a MoFlo cell sorter (DAKO Cytomation, Ft. Collins, CO). For each sample, 30–40 x 10⁶ original cells were stained for sorting.

SCF-Stimulated Cell Proliferation
Unfractionated BM cells, sorted Lin^–Sca1⁺CD117⁺ cells, and sorted Lin^–Sca1⁺CD117^– cells were cultured in 12-well polystyrene tissue culture plates (Corning, Corning, NY) at an initial concentration of 5000 cells per well in 2 mL of Dulbecco's modified essential medium (Cellgro Mediatech, Herndon, VA) supplemented with 15% heat-inactivated fetal bovine serum, 1% penicillin, 1% streptomycin, 1% L-glutamine, interleukin-3 at 5 µg/mL, and interleukin-6 at 5 µg/mL. Cells were cultured with or without the presence of SCF at 25 ng/mL (R & D Systems, Minneapolis, MN) in duplicate or triplicate (depending on the number of cells available) at 37°C with 5% CO₂. After 2 days in culture, cells were examined under a phase-contrast microscope and were photographed. After 3 days in culture, cells were collected and counted under a light microscope to calculate cell expansion (defined as the ratio between the number of cells recovered and the number of cells used to initiate the culture). Cultured cells were also stained with antibodies and were analyzed by flow cytometry using similar antibody combinations as described earlier to define the phenotype of the expanded cells.

Data Analysis
Blood, spleen, and BM cellular composition data as well as cell expansion data were analyzed using one-way or two-way analysis of variance platforms provided by the JMP Statistical Discovery Software (SAS Institute, Cary, NC) (26). Results are shown as mean and standard error, and statistical significance was declared at p <.05 and p <.01 levels.

	RESULTS

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Higher Proportion of CD117⁺ Cells in the BM of Old B6D2F1 Mice
The primary focus of this study was to test the effects of increasing age on c-Kit (CD117) expression and hematopoietic cell response to SCF stimulation, as the SCF/c-Kit ligand/receptor pair is vital to the normal function of HSCs and progenitor cells. First, we extracted BM from young and old B6D2F1 mice and stained the cells with various antibodies to test CD117 expression. Of the 1,000,000 cells collected for each sample, the proportion of CD117⁺ cells was slightly higher (not significant, p >.05) in old than in young B6D2F1 mice within total BM cells (4.4 ± 0.5% vs 3.4 ± 0.4%; Figure 1, A and B), and was significantly higher (p <.05) in old than in young mice in Lin^– BM cells (0.72 ± 0.09% vs 0.43 ± 0.08%; Figure 1, C and D). When BM cells were further divided into Lin^–CD34^– and Lin^–CD34⁺ cell fractions (Figure 2, A and B), the proportions of CD117⁺ cells were significantly higher (p <.05) in old versus young B6D2F1 mice in both the Lin^–CD34⁺ (0.58 ± 0.08% vs 0.36 ± 0.06%; Figure 2, C and D) and the Lin^–CD34^– cell fractions (0.14 ± 0.02% vs 0.08 ± 0.02%; Figure 2, E and F). Furthermore, the percentage of Lin^–CD34⁺Sca1⁺CD117⁺ progenitor cells was three times higher (0.0363 ± 0.0070% vs 0.0113 ± 0.0031%, p <.05; Figure 2, C and D), whereas the percentage of Lin^–CD34^–Sca1⁺CD117⁺ HSCs was more than eight times higher (0.0375 ± 0.0110% vs 0.0045 ± 0.0013%, p <.05; Figure 2, E and F), in old than in young B6D2F1 mice. Thus, in old B6D2F1 mice, CD117 expression was up-regulated in all fractions of BM cells, especially in the fraction that contains the most primitive HSCs.

View larger version (52K): [in this window] [in a new window]   Figure 1. Bone marrow (BM) cells extracted from two tibiae and two femurs of each young and old B6D2F1 mice were first counted, and then stained with an antibody cocktail containing CD34-fluorescein isothiocyanate (FITC) + Sca1- phycoerythrin (PE) + Lineage-Biotin/streptavidin-conjugated Quantum Red (CD3, CD4, CD8, CD11b, CD19, Gr1, and Ter119) + CD117- allophycocyanin (APC). Expression of CD117 on total BM cells (A and B) and on Lin– BM cells (C and D) were shown as flow cytometry dot plots. Data presented were representatives of five pairs of young (A and C) and old (B and D) mice used for the analysis

View larger version (37K): [in this window] [in a new window]   Figure 2. After flow cytometry analysis, bone marrow cells were first gated into Lin–CD34– and Lin–CD34+ cell fractions (A and B). Expression of Sca1 and CD117 on Lin–CD34+ (C and D) and Lin–CD34– (E and F) cells were shown as representatives of five pairs of young (A, C, and E) and old (B, D, and F) B6D2F1 mice used for the analysis

View larger version (19K): [in this window] [in a new window]   Figure 3. Calculated total bone marrow cells (A), CD117+ cells (B), Lin–CD117+ cells (C), Lin–Sca1+CD117+ cells (D), Lin–Sca1+CD117+CD34+ cells (E), and Lin–Sca1+CD117+CD34– cells (F) were shown as means with standard error bars from five young and five old B6D2F1 female mice. Young and old differences were all at a p <.01 significance level except for Lin–Sca1+CD117+CD34+ cells (E) which is at a p <.05 significance level

SCF-Stimulated Lin^–Sca1⁺CD117⁺ Cell Proliferation Is Elevated in Old B6D2F1 Mice
The fact that total numbers of Lin^–CD34^–Sca1⁺CD117⁺ and Lin^–CD34⁺Sca1⁺CD117⁺ cells were both significantly increased in aged B6D2F1 mice provided evidence indicating that immature hematopoietic cell proliferation was up-regulated in the BM of old B6D2F1 mice. One possibility is that cells from old mice are more sensitive to a normal cytokine environment. The other possibility is that the old environment provides higher concentrations of cytokines that stimulate cell proliferation. We cultured total BM cells and sorted Lin^–Sca1⁺CD117⁺ cells and Lin^–Sca1⁺CD117^– cells in vitro for 3 days, with or without the presence of SCF, to test the hypothesis that BM cell response to SCF stimulation was up-regulated in old B6D2F1 mice.

When unfractionated total BM cells were cultured, we observed no age or SCF effect on cell proliferation (Figure 4A). Young and old cells expanded 1.57 ± 0.09-fold and 1.90 ± 0.15-fold, respectively, showing no significant age effect. Cells expanded 1.77 ± 0.12-fold and 1.70 ± 0.12-fold, respectively, with or without SCF (Figure 4A), indicating that total BM cell proliferation was not stimulated by the concentration of SCF used in the study. Similarly, for sorted Lin^–Sca1⁺CD117^– cells, there was no SCF effect on cell proliferation, as cells expanded 1.74 ± 0.13-fold and 1.68 ± 0.13-fold, respectively, with or without SCF simulation (Figure 4B). However, Lin^–Sca1⁺CD117^– cells from old B6D2F1 mice proliferated slightly better (p <.01) than did those from young B6D2F1 mice. On average, old cells expanded 2.07 ± 0.14-fold, whereas young cells expanded 1.36 ± 0.12-fold (Figure 4B).

View larger version (14K): [in this window] [in a new window]   Figure 4. Total bone marrow cells (A), sorted Lin–Sca1+CD117+ cells (B), and sorted Lin–Sca1+CD117– cells (C) from young and old B6D2F1 mice were each cultured in Dulbecco's modified essential medium at 37°C with 5% CO2 with or without the presence of stem cell factor at 25 ng/mL. Three days later, cells were harvested and counted under a regular light microscope. Fold of cell expansion was calculated based on the number of cells harvested and the number of cells used to initiate the culture. Data are presented as means with standard error bars from triplicate measurements

We further analyzed cultured cells by flow cytometry for the expression of CD117 and Sca1 on Lin^– cells (Figure 5). Essentially, there was no CD117 or Sca1 expression on Lin^– cells from cultured young (Figure 5A) and old (Figure 5B) total BM cells. This was also the case in Lin^– cells from cultured young (Figure 5C) and old (Figure 5D) Lin^–Sca1⁺CD117^– cells. Only cultured Lin^–Sca1⁺CD117⁺ cells from young (Figure 5E) and old (Figure 5F) mice contained larger cell fractions expressing both CD117 and Sca1. However, the proportions of CD117⁺ and CD117⁺Sca1⁺ cells were much larger in cultured cells from old B6D2F1 mice (Figure 5F).

View larger version (21K): [in this window] [in a new window]   Figure 5. Cultured cells were harvested and stained with the antibody cocktail Sca1-fluorescein isothiocyanate (FITC)+ CD117-phycoerythrin (PE) + Lineage-Biotin/streptavidin-conjugated Quantum Red (CD3, CD4, CD8, CD11b, CD19, Gr1, and Ter119). Expression of Sca1 and CD117 on Lin– cells from cultured total bone marrow cells (A and B), sorted Lin–Sca1+CD117– cells (C and D), and sorted Lin–Sca1+CD117+ cells (E and F) from young (A, C, and E) and old (B, D, and F) B6D2F1 mice were shown as dot plots after 3 days culture in Dulbecco's modified essential medium at 37°C with 5% CO2 and stem cell factor at 25 ng/mL. Cells from triplicate culture wells of each sample were pooled for the staining

	DISCUSSION

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Defining HSCs and progenitor cells by marker phenotypes has been a widely accepted practice in recent years. Spangrude and colleagues (11,12) were the first to use cell surface markers to define HSCs. More recently, Osawa and colleagues (21) sorted out single cells, tested them in a competitive engraftment assay, and found that cells with long-term engraftment ability were found in the CD34^– fraction of Lin^–Sca1⁺CD117⁺ cells, whereas the CD34⁺ fraction of Lin^–Sca1⁺CD117⁺ cells contained less primitive progenitor cells. In a separate experiment, Zhao and colleagues (20) confirmed that long-term functional HSCs are in the CD38⁺CD34^– fraction of Lin^–Sca1⁺kit⁺ cells. In addition, Orlic and colleagues (27) indicated that the Lin^–CD117⁺ cell fraction contains primitive HSCs, whereas the Lin^–CD117^– cell fraction does not. In the case of HSC senescence, Morrison and colleagues (28) found that old B6 mice had a 5- to 7-fold increase in cells with the HSC marker phenotype and a 2-fold increase in HSC engraftment in vivo. Results from our current study are consistent with these findings from earlier studies that the HSC-containing Lin^–Sca1⁺CD117⁺ cells are highly responsive to SCF stimulation (Figure 4B).

As a key receptor, c-Kit plays a vital role in the regulation of HSC proliferation and differentiation. This proto-oncogene lies on mouse chromosome 5 at the White-spotting locus. Mutations at the White-spotting locus affect proliferation and/or migration of cells during early embryogenesis and cause intrinsic defects in the HSC hierarchy (17,29). Mutations at different portions of the c-Kit gene result in various levels of functional loss, affecting embryonic survival and hematopoiesis (14,18,23,24). In our current study, increasing age caused significant increases in the proportion and total number of CD117⁺ cells in various cell fractions, especially the Lin^–CD34^–Sca1⁺CD117⁺ primitive HSCs (Figures 1 and 3). Our result is consistent with observations in B6 mice, where increasing age is associated with an increased number and function of HSCs (4,9,28,30).

Two elements are to be considered in studying HSC senescence: HSC number and functional ability per HSC. In BALB mice, HSC frequency was unchanged and total HSCs per mouse was slightly higher in old mice as a result of the increased total BM cellularity. However, functional ability per HSC was significantly reduced in old BALB mice and accounted for the overall decline in HSC functionality (3,4). In the current study, sorted Lin^–Sca1⁺c-Kit⁺ cells from old B6D2F1 mice had a 5-fold expansion when cultured in vitro for 72 hours in the presence of SCF, twice that of the 2.5-fold expansion seen in sorted Lin^–Sca1⁺c-Kit⁺ cells from young mice cultured under the same conditions (Figures 4 and 5). There are two possible explanations for this result: 1) each old Lin^–Sca1⁺c-Kit⁺ cell proliferated twice as fast as each young Lin^–Sca1⁺c-Kit⁺ cell; 2) the old Lin^–Sca1⁺c-Kit⁺ cell population contains twice as many cells in the proliferative state as young Lin^–Sca1⁺c-Kit⁺ cells.

That HSC function does not decline with increasing age is not an uncommon phenomenon. In the mouse model, inbred B6 and D2 mice, the parental strains of B6D2F1 mice, have distinctive phenotypes of HSC senescence (30). D2 mice are short-lived but have higher cobblestone area forming cell frequencies in fetal liver cells in comparison to B6 mice (30,31). B6 mice have twice as many Lin^–Sca1⁺c-Kit⁺ and Lin^–Sca1⁺c-Kit^– cells in their BM than do D2 mice (32,33), but D2 Lin^–Sca1⁺c-Kit⁺ cells contain twice as many cells with cobblestone area forming cell activity (9,30). Mice transplanted with 1000 D2 Lin^–Sca1⁺c-Kit⁺ cells recover much faster than do animals transplanted with the same number of B6 cells. Data from early studies using B6-based strains showed that marrow or spleen grafts from aged mice produced antibody-forming cells as effectively as did grafts from younger donors in recipients. In addition, grafts from aged and younger donors gave similar responses when stimulated with various doses of antigens (6,7). Marrow cell lines from old donors also functioned as well as those from young donors after transplantation into either W/W^v anemic or lethally-irradiated normal recipients (7). In the B6-D2 allophenic mice, D2 HSCs showed high functionality early in life, but were then eclipsed by HSCs of the B6 genotype (34). When B6-D2 allophenic marrow cells were engrafted into lethally irradiated B6D2F1 recipients, D2 hematopoiesis was dominant early after transplantation but B6 hematopoiesis ascended over the subsequent months to become dominant later in life (35,36). Our data, which show a higher response of old Lin^–Sca1⁺c-Kit⁺ cells to SCF-stimulated cell proliferation (Figure 4), is consistent with previously published results and may represent the dominant fashion by which the B6 phenotype is inherited.

In a recent report, Chen and colleagues (37) found that freshly isolated CD4⁺ and CD8⁺ T cells from aged B6 mice expressed increased levels of chemokines such as interferon--inducible protein 10 and macrophage inflammatory protein (MIP)-1 and -1ß, and that the age-related difference in T-cell chemokine expression has an important functional consequence. In studying in vitro proliferative potential of human skin biopsies from members of the Baltimore Longitudinal Study of Aging, Smith and colleagues (38) found a significant decline in proliferative potential with donor age in females, but not in males. In addition, the proliferative potential was significantly greater for donors under the age of 30 years when compared with all donors over the age of 30 years (38). We speculate that HSCs from B6D2F1 and other B6-based strains may have higher proliferative potentials or lower proliferative rates. This might also be the case for the skin biopsies from some of the older men in the study by Smith and colleagues (38). The larger HSC pool in old B6D2F1 mice is associated with insignificantly lower mature cell counts in the peripheral blood. It is possible that the enlarged HSC pool in old B6D2F1 mice is a response to the aged environment, in which up-regulation in HSC proliferation is necessary to maintain hematopoiesis at a normal level.

We studied the effect of aging on HSC number and functional response to SCF stimulation in vitro in B6D2F1 mice. Increasing age is associated with a significant increase in the total number of HSCs and progenitor cells. Sorted HSCs from old B6D2F1 mice also showed significantly higher levels of response to SCF stimulation in vitro. Thus, not only the HSC pool size, but also the functional ability per HSC, are increased in aged B6D2F1 mice during a normal life span.

	Acknowledgments

 
We thank Dr. Neal S. Young for his critical review and constructive comments. We also thank Ms. Leigh Samsel (Flow Cytometry Core Facility) and Mr. David Caden (Laboratory Animal Medicine & Surgery) of the National Heart, Lung, and Blood Institute for technical assistance in cell sorting and complete blood counts.

	Footnotes

 
Decision Editor: James R. Smith, PhD

Received October 1, 2004

Accepted December 8, 2004

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