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Induction of c-kit Molecules on Human CD34⁺/c-kit^<low Cells: Evidence for CD34⁺/c-kit^<low Cells as Primitive Hematopoietic Stem Cells

^a Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan;
^b First Department of Pathology,
^c Department of Pediatrics,
^d Department of Obstetrics and Gynecology, Kansai Medical University, Moriguchi City, Osaka, Japan

Key Words. c-kit • CD34 • Human cord blood • Hematopoietic stem cells

Dr. Susumu Ikehara, First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570, Japan.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
c-kit, a receptor for stem cell factor, has been widely accepted as a distinctive marker for hematopoietic stem cells. However, the level of c-kit expression on pluripotent hematopoietic stem cells is still controversial in mice and humans. We purified CD34⁺/c-kit^<low cells (phenotypically c-kit-negative but only detectable at the message level) from human cord blood and examined their maturational steps in relation to the expression of c-kit molecules. When the CD34⁺/c-kit^<low cells were cultured with cytokines (flt 3 ligand, interleukin 6 and interleukin 7) plus immobilized anti-CD34 monoclonal antibody (to crosslink CD34 molecules), c-kit molecules were clearly induced within 24 h. The c-kit expression gradually increased until day 8. When CD34⁺/c-kit^low or CD34⁺/c-kit⁺ cells that had been induced from CD34⁺/c-kit^<low cells were resorted and recultured using a methylcellulose culture system, they showed the same colony-forming ability as the freshly isolated CD34⁺/c-kit^low or CD34⁺/c-kit⁺ cells, respectively. Furthermore, CD34⁺/c-kit^<low cells have a similar hematopoietic potential to CD34⁺/c-kit^low cells in assays for long-term culture initiating cell and colony-forming unit culture generated from long-term cultures. These findings suggest that CD34⁺/c-kit^<low cells mature into CD34⁺/c-kit^low and CD34⁺/c-kit⁺ cells, and acquire the reactivity to various humoral hematopoietic stimuli. Moreover, CD34⁺/c-kit^<low cells showed a low level of rhodamine 123 retention, suggesting that CD34⁺/c-kit^<low cells have multidrug resistance. Therefore, the CD34⁺/c-kit^<low cells without colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte activity are also a pluripotent hematopoietic stem cell population, and the expression of c-kit on c-kit^<low cells is the first maturational step of hematopoiesis.

	Introduction

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Pluripotent hematopoietic stem cells (P-HSCs) are defined as cells with the capacity to eternally self-renew and to differentiate into all hematopoietic lineage cells. In humans, HSCs have been characterized as CD34⁺ cells; they possess colony-forming activity when bone marrow (BM), cord blood (CB), and mobilized peripheral blood are cultured [1-4]. Other immunophenotypes defining primitive HSCs have been reported to be CD34⁺/CD38^–, CD34⁺/Thy-1⁺, CD34⁺/Lineage(Lin)^–/CD41a^–, CD34⁺/HLA-DR^– or CD34⁺/HLA-DR⁺/Rhodamine 123 (Rh123)^dull, based on the assays for high proliferative potential colony-forming cells (HPP-CFC) or long-term culture-initiating cells (LTC-IC). CD34⁺/CD38^– cells can generate colony-forming unit-culture (CFU-C) in LTC-IC assay [5]. Most LTC-IC, cobblestone area-forming cells, and cells reconstituting the thymus implant in severe combined immunodeficiency human mice are contained in the CD34⁺/Thy-1^low population [6-8]. The CD34⁺/Lin^–/CD41a^– population in the fetal BM contains a significantly high frequency of cobblestone area-forming cells [9]. The cells possessing the capacity for self-renewal and commitment to multipotential hematopoietic differentiation were found in the CD34⁺/HLA-DR^– population [10], though there has been a report that LTC-IC are present in the CD34⁺/HLA-DR⁺ population, and that HPP-CFC are in the CD34⁺/HLA-DR⁺/Rh123^dull population [11].

Another functionally and phenotypically important molecule is c-kit. c-kit is known as a receptor for stem cell factor (SCF) which plays an important role in the early stage of hematopoiesis [12-15]. In previous studies, it was found that the CD34⁺/c-kit^low population contained the majority of cycle-dormant progenitors, multilineage colony-forming cells (colony-forming unit granulocyte-erythroid-macrophage-megakaryocyte [CFU-GEMM]) and LTC-IC [16-18]. In addition, long-term reconstituting activity (LTRA) was found to be enriched in the CD34⁺/c-kit^low population using an in utero transplantation system [19]. In contrast, CD34⁺/c-kit^– cells were rejected as P-HSCs because of their low proliferative responses or the absence of such responses to several stimuli [14, 16-20], in spite of the existence of these cells in the human BM and CB. However, it has also been shown that the expression of c-kit molecules on HSCs is dependent on the differentiation steps [14, 20, 21]. Therefore, there is a possibility that the induction of c-kit molecules is a first step in the differentiation of P-HSCs, being accompanied by the acquirement of reactivity to SCF. Furthermore, taking the dormancy of HSCs into consideration, it would be logical for HSCs not to express c-kit on their surface and so protect them from stimuli that would induce them to enter the cell cycle. In this sense, CD34⁺/c-kit^<low cells could be more primitive HSCs. Actually, in mice, we have very recently found that Lin^–/CD71^–/MHC class I^high/c-kit^<low cells (phenotypically negative, but c-kit mRNA was detected by reverse transcriptase polymerase chain reaction [RT-PCR] assay) have LTRA in comparison with Lin^–/CD71^–/MHC class I^high/c-kit^low cells [22]. This finding prompted us to examine whether human P-HSCs are c-kit^<low, c-kit^low, or c-kit⁺. In this report, we show that c-kit molecules can be induced on the purified CD34⁺/c-kit^<low cells as a first maturational step of hematopoiesis, and we also compare the colony-forming activity of CD34⁺/c-kit^<low cells with that of induced and freshly isolated CD34⁺/c-kit^low or c-kit⁺ cells. In addition, we show that CD34⁺/c-kit^<low cells have a potential as P-HSCs in the long-term culture with the BM stromal cell line, MS-5.

	Materials and Methods

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Cell Preparation and Purification of CD34⁺/c-kit^– (CD34⁺/c-kit^<low) Cells
The CB cells were collected in acid-citrate-dextrose according to the guidelines of the Cord Blood Bank, Kansai Medical University (Osaka, Japan; fully informed consent was obtained from the mothers). BM cells were obtained from normal healthy volunteers. RBCs were sedimented out by the addition of a half volume of 2% Dextran, and mononuclear cells were isolated by using Lymphoprep^TM (Nycomed; Oslo, Norway) density gradient centrifugation. CD34-bearing cells were positively isolated using anti-CD34 class II monoclonal antibodies (mAb) (QBend-10) and CD34 multisort kit (Miltenyi Biotec; Bergisch Gladbach, Germany) followed by the Mini MACS system (Miltenyi Biotec). The purified CD34⁺ cells were then stained with fluorescein isothiocyanate (FITC)-conjugated mAb against CD34 class III (HPCA2; Becton Dickinson Immunocytometry Systems; San Jose, CA), which did not share the epitope recognized by anti-CD34 class II specific mAb (QBend-10) [23], and phycoerythrin (PE)-conjugated anti-c-kit (anti-CD117) mAb (Immunotech; Marseille, France). The cells with phenotype of CD34⁺/c-kit⁺, CD34⁺/c-kit^low, and CD34⁺/c-kit^– were isolated using a FACStar^TM (Becton Dickinson Immunocytometry Systems), as shown in Figure 1.

View larger version (13K): [in this window] [in a new window]   Figure 1. Preparation of c-kit–, c-kitlow, and c-kit+ populations from CD34+ cells. CD34+ cells isolated from the CB were stained with FITC-conjugated anti-CD34 mAb and PE-conjugated anti-c-kit mAb. A) Forward scatter versus side scatter pattern of CD34+ cells: Live CD34+ cells were gated as shown in R1. B) CD34 versus c-kit pattern of the cells: R2, R3 and R4 are the gates for sorting CD34+/c-kit–, CD34+/c-kitlow and CD34+/c-kit+ populations, respectively. C) FACS profile of the cells stained with isotype-matched control mAbs.

Flow Cytometric Analyses and Colony-Forming Assays
Cells were harvested and double-stained with FITC-conjugated anti-CD34 (HPCA2) and PE-conjugated anti-c-kit mAbs. Expression of cell-surface antigens was evaluated using a FACScan^TM (Becton Dickinson Immunocytometry Systems). Sorted cells, or cells harvested from the liquid culture of CD34⁺/c-kit^– cells, were assayed for their ability to produce burst forming unit-erythroid (BFU-E), colony-forming unit-granulocyte-macrophage (CFU-GM), colony-forming unit-macrophage (CFU-M) and CFU-GEMM in a methylcellulose assay system. Briefly, cells (50 cells/well) were added to 0.8 ml of culture mixture (Methocult GF H4434; Stem Cell Technologies, Inc.) consisting of 0.9% methylcellulose, 30% FBS, 1% bovine serum albumin, 10^–4M 2-mercaptoethanol, 2 mM L-glutamate, rHu erythropoietin (EPO), rHuSCF, rHuGM-CSF, rHuG-CSF and rHuIL-3 (concentrations of CSFs were optimally conditioned to form BFU-E, CFU-GM, CFU-M and CFU-GEMM) in 12-well culture plates (Flow Laboratories, Inc.) The plates were incubated for 14 days, and the numbers of colonies were counted under an inverted microscope. The average number of colonies and standard errors were calculated from quadruplicated wells.

LTC-IC Assay
The assay for LTC-IC was carried out according to the modified method described by Young et al. [24]. Briefly, sorted CD34⁺/c-kit^<low cells or CD34⁺/c-kit^low cells (10³ cells/flask) were plated on murine BM stromal cell line (MS-5) in the presence of rHuIL-3 (1 ng/ml), rHuIL-6 (1 ng/ml), rHuG-CSF (1 ng/ml), rHuSCF (1 ng/ml), rHu leukemia inhibitory factor (1 ng/ml), and rHu FL (20 ng/ml) in Iscove's modified Dulbecco's medium with 10% FBS. At weekly intervals, the supernatant was removed and replaced by fresh medium containing cytokines. The nonadherent cells in the supernatant were counted. Nonadherent cells harvested from LTC-IC were further cultured for the colony-forming assay (CFU-C) under the same conditions described above.

Detection of c-kit Transcript by RT-PCR
The sorted CD34⁺/c-kit^– and CD34⁺/c-kit^low cells (3.6 x 10³ cells, each) were lyzed with RNA STAT-60 (TEL-TEST"B"; Friendswood, TX) in the presence of 2 mg of yeast ribosomal RNA (TOYOBO; Tokyo, Japan) as a carrier, and total RNA was extracted according to the manufacturer's protocol. The RT-PCR was performed using oligo-dT primers for RT; human c-kit 5' sense (ATGGCACGGTTGAATGTAAGGC) and 3' antisense (TCTCCTCAACAACCTTCCACTG) were used as primers for PCR.

Rh123 Staining and Efflux
The staining and efflux procedure for Rh123 was described previously [25] and was modified as follows. Briefly, CD34⁺ cells were incubated with 100 ng/ml of Rh123 (Molecular Probes; Eugene, OR) for 20 min at 37°C. The cells were washed and incubated at 4°C or 37°C for 40 min to determine Rh123 dye efflux. The cells were then washed and stained with PE-conjugated anti-c-kit mAb and PE-Cyanin 5-conjugated anti-CD34 mAb. The fluorescence intensity of Rh123 was detected in FL1 (535 nm) using a FACScan^TM (Becton Dickinson Immunocytometry Systems).

	Results

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Purification of CD34⁺/c-kit^– Cells
Positively isolated CD34⁺ cells from the CB cells were stained with FITC-conjugated anti-CD34 class III and PE-conjugated anti-c-kit mAbs. The forward scatter versus side scatter pattern and the expression of CD34/c-kit are represented in Figure 1A and 1B, respectively. The regions (R) in Figure 1B show the sorting gates for purifying CD34⁺/c-kit^– cells (R2), CD34⁺/c-kit^low (R3), and CD34⁺/c-kit⁺ (R4). Figure 1C represents a FACS profile stained with FITC- or PE-conjugated isotype-matched control mAbs to determine the sorting regions for each cell population. The populations (R2-R4) sorted using a FACStar^TM were reanalyzed using a FACScan^TM to confirm the intensity of c-kit expression. Peak intensities of c-kit expression in the sorted populations corresponded to the sorting gates (R2-R4). In particular, the intensity of c-kit^<low cells was lower than that of the negative controls stained by isotype-matched control mAb (mouse IgG1) or unstained.

c-kit mRNA in CD34⁺/c-kit^– Cells
The c-kit transcripts in CD34⁺/c-kit^low cells and CD34⁺/c-kit^– cells were next compared by RT-PCR analyses. Substantial expression of c-kit mRNA was detected even in the c-kit^– cells (data not shown). Based on this finding, we defined the cells as "c-kit^<low" instead of "c-kit^–", although the cells appeared to be "c-kit^–" in FACS analyses, as shown in Figure 1B.

Colony-Forming Ability of CD34⁺/c-kit^<low Cells
The colony-forming ability of the sorted CD34⁺/c-kit^<low, CD34⁺/c-kit^low, and CD34⁺/c-kit⁺ cells was next determined using a methylcellulose culture system containing SCF, EPO, GM-CSF, G-CSF and IL-3. The ability of CD34⁺/c-kit^<low cells to form BFU-E, CFU-GM, CFU-M or CFU-GEMM was less than that of the other two populations ( Table 1). It should be noted that no CFU-GEMM colony was detected in the culture of CD34⁺/c-kit^<low cells. Thus, CD34⁺/c-kit^<low cells have less reactivity to these stimuli.

View larger version (24K): [in this window] [in a new window]   Figure 2. CD34+/c-kit<low cells stimulated with FL, IL-6, IL-7 and immobilized anti-CD34 mAb. A) Expression patterns of c-kit molecules in precultured (day 0), one-day cultured and three-day cultured cells. B) Kinetics of c-kit expression in cultured CD34+/c-kit<low cells in vitro.

View larger version (23K): [in this window] [in a new window]   Figure 3. Induction of c-kit molecules on CD34+/c-kit<low cells cultured with various stimuli. Sorted CD34+/c-kit<low cells (7,000 cells) were cultured with: A) FL + IL-6 + IL-7 + immobilized anti-CD34 mAb; B) FL + IL-6 + IL-7; C) FL + IL-6; D) immobilized anti-CD34 mAb; E) IL-6; F) FL, and G) medium alone for three days. Each cell was harvested, counted and assessed for its expression of c-kit molecules. Recovered cell counts were 5,500 (A), 6,000 (B), 3,000 (C), 1,500 (D), 2,500 (E), 3,000 (F) and 500 (G), respectively.

View larger version (10K): [in this window] [in a new window]   Figure 4. Essential stimuli in induction of c-kit molecules. CD34+/c-kit<low cells were suspended with Iscove's modified Dulbecco's medium containing 10% FBS (A, C) or serum-free medium based on Iscove's modified Dulbecco's medium (B). Cells were cultured in culture-coated plates for cell adhesion (A, B) or in polypropylene tubes (C) for 24 h. The cells were harvested, stained with FITC-anti-CD34 class III mAb plus PE-anti-c-kit mAb, and analyzed using a FACScanTM.

Capacity of the Sorted CD34⁺/c-kit^<low Cells to Maintain and Produce CFU-C in LTC
The LTC is considered to be the most informative in vitro assay for stem cell activity. As shown in Figure 5A, the numbers of nonadherent cells generating from CD34⁺/c-kit^<low cells were comparable to those in the culture of CD34⁺/ c-kit^low cells over the culture period (until 90 days). The ability to form CFU-C in nonadherent cells from the LTC was further investigated. Cells harvested from the LTC of CD34⁺/c-kit^<low cells have a similar CFU-C activity to that of CD34⁺/c-kit^low cells ( Fig. 5B). These findings clearly indicate that CD34⁺/c-kit^<low cells have the potential of P-HSCs.

View larger version (16K): [in this window] [in a new window]   Figure 5. LTC-IC assay. A) Sorted CD34+/c-kit<low cells (closed circle) or CD34+/c-kitlow cells (open circle) (103 cells/flask) were plated on murine stromal cell line (MS-5) in the presence of hematopoietic cytokines. At weekly intervals, the number of nonadherent cells was counted. B) Nonadherent cells harvested from the LTC were further cultured (300 cells/well) for colony-forming assay under the same conditions described in Table 1. Values in A and B represent mean of four cultures.

View larger version (14K): [in this window] [in a new window]   Figure 6. Rh123 efflux from CD34+/c-kit<low cells. CD34+ cells from the CB were stained with Rh123, washed, and incubated for 40 min at 4°C or 37°C (A). Rh123 retention was evaluated using CD34+/c-kit<low cells, CD34+/c-kitlow cells or CD34+/c-kit+ cells (B).

	Discussion

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To prove this hypothesis, highly purified CD34⁺/c-kit^<low cells were cultured with various stimuli that regulate hematopoiesis: FL [31], IL-6 [32-34], IL-7 [35] and immobilized anti-CD34 mAb to crosslink CD34 molecules [36, 37]. In this report, we have shown that the colony-forming ability of sorted CD34⁺/c-kit^<low cells is less than that of the other two populations (CD34⁺/c-kit⁺, CD34⁺/c-kit^low), and that the stimulation with FL, IL-6, IL-7 and CD34 crosslinking induces the expression of c-kit molecules on CD34⁺/c-kit^<low cells within 24 h of in vitro culture, then increases and reaches a plateau on day 8 of culture. It should be noted that the CD34⁺/c-kit⁺ and CD34⁺/c-kit^low cells generated from CD34⁺/c-kit^<low cells acquired the ability to form multilineage colonies, as did the freshly isolated CD34⁺/c-kit⁺ or CD34⁺/c-kit^low populations, respectively. These data indicate that the changes in the expression of c-kit molecules reflect the changes not only in their immunophenotype but also in functional maturation of CD34⁺/c-kit^<low cells. This is in accordance with the report by Gunji et al. [18] where: A) CD34⁺/c-kit^high cells are induced from CD34⁺/c-kit^low cells after four-week culture with stromal cells and B) LTC-ICs are enriched in the CD34⁺/c-kit^low population, but not in differentiated CD34⁺/c-kit^high cells with the ability to form CFU-GM. However, they only reported the induction of differentiation from c-kit^low to c-kit^high. Our purified CD34⁺/c-kit^<low or ^low and CD34⁺/c-kit⁺ cells may correspond to CD34⁺/c-kit^– and CD34⁺/c-kit^low cells, respectively, in the previous reports, based on FCM pattern and colony-forming ability [16, 19]. Therefore, our results clearly show that the changes in the intensity of c-kit molecules are closely related to the earliest maturational phase of hematopoiesis. In our murine system, the substantial expression of c-kit mRNA was also detected using RT-PCR analyses even in the c-kit^– cells [22]. To explain this discrepancy, there are two possibilities below: A) this CD34⁺/c-kit^– cell expresses a very low level of c-kit molecules in spite of the "c-kit^–" immunophenotype in FACS analyses or B) c-kit protein synthesis from c-kit transcript is blocked by some system. Taking these findings into consideration, both in humans and mice, CD34⁺/c-kit^<low cells appear to be an earlier population than CD34⁺/c-kit^low cells, and the changes in the immunophenotype (c-kit^– to c-kit^low) are the first maturational step of P-HSCs. Furthermore, both the CD34⁺/c-kit^<low and CD34⁺/c-kit^low populations had a repopulating activity not only in long-term (>90 days) culture in the presence of hematopoietic cytokines but also in CFU-C assays from LTC. This may be due to the transition of CD34⁺/c-kit^low cells from the initial CD34⁺/c-kit^<low cells during the culture period, since the induction of CD34⁺/c-kit^low cells from CD34⁺/c-kit^<low cells occurs within three days ( Fig. 2).

As shown in Figure 2, the expression of c-kit molecules was immediately inducible in CD34⁺/c-kit^<low cells, and this transition was unidirectional and irreversible in our in vitro culture system. However, only a small but steady number of CD34⁺/c-kit^<low cells were detected in the CB, suggesting that a niche constructed by stromal cells or some cytokine(s) negatively regulate the induction of c-kit molecules and the reactivity to SCF to maintain dormant P-HSCs. The report that TNF- and TGF-ß downregulate not only the c-kit m-RNA but also c-kit protein in CD34⁺/c-kit^high cells and CD34⁺ leukemia cells might support this possibility [38-40].

In our system, the transition of c-kit^<low to c-kit^low occurred even in the absence of exogenous stimuli (hematopoietic cytokines, FBS and HS) ( Figs. 3 and 4). The addition of TGF-ß, macrophage inflammatory protein-1 and TNF- inhibits the transition from c-kit⁺ cells to c-kit^high cells in our system. However, these cytokines did not affect the transition from c-kit^<low cells to c-kit⁺ cells (data not shown). Though factor(s) regulating the c-kit induction in the CD34⁺/c-kit^<low population were still unclear, the induction of c-kit molecules was completely inhibited when the cells were cultured in polypropylene tubes ( Fig. 4C). It is well known that cell adhesion is weaker in polypropylene tubes than in usual culture plates (polystyrene). Therefore, it is feasible that some adhesive signal(s) essentially regulates the induction of c-kit molecules on CD34⁺/c-kit^<low cells, and the exogenous cytokines used in our system may support the survival of the cells and act synergistically with the adhesive signals. If this is the case, our findings may explain why the CD34⁺/c-kit^<low population showed no colony formation in the methylcellulose culture system or in utero transplantation assay, where it is possible that no essential adhesion occurs [18, 19].

In the BM microenvironment, stromal cells appear to support quiescent P-HSCs and induce them to proliferate by the production of various soluble factors and cell-to-cell adhesion [41-46]. Primitive hematopoiesis could therefore be regulated by these mechanisms [47]. When CD34⁺/c-kit^<low cells isolated from the BM were cultured, CD34⁺/c-kit^low or CD34⁺/c-kit⁺ cells developed from the CD34⁺/c-kit^<low cells within 24 h, although a significant population of CD34⁺/c-kit^<low cells still remained (data not shown), while only a minute population was present in the CB. This may indicate that the CD34⁺/ c-kit^<low cells in the CB are more mature and, therefore, more sensitive to hematopoietic stimuli than those in the BM. These findings raise the possibility that real pluripotent HSCs are indeed c-kit^– cells that do not express even c-kit mRNA, and can develop into c-kit^<low, c-kit^low and c-kit⁺ cells after the cognate interaction with some regulatory cells (i.e., stromal cells).

Rh123 is another useful reagent for defining the subset of P-HSCs [25], and the efflux of Rh123 is dependent on an efflux pump such as P-glycoprotein (the product of multidrug resistance gene) on hematopoietic cells [48]. In our experiment, there was less retention of Rh123 by the CD34⁺/c-kit^<low cells than the CD34⁺/c-kit^low or CD34⁺/c-kit⁺ cells, indicating that the cells with a high level of Rh123 efflux are enriched in the CD34⁺/c-kit^<low cells ( Fig. 6). Therefore, the CD34⁺/c-kit^<low cells have multidrug resistance and can be protected from various cytotoxic substances. This is in accordance with the recent reports that P-HSCs in the BM are highly enriched in the CD34⁺/Thy-1⁺/Rh123^low cells in humans [25] and that c-kit^<low or c-kit^– cells remained after the injection of 5-fluorouracil in mice [22, 49, 50].

In conclusion, our findings clearly indicate that the immunophenotypical change from CD34⁺/c-kit^<low to CD34⁺/c-kit^low or ⁺ is the first step in functional maturation in hematopoiesis. Even though the CD34⁺/c-kit^<low cells have no ability to form CFU-GEMM, unlike CD34⁺/c-kit^low cells, both populations have similar repopulating activity in long-term culture, suggesting that CD34⁺/c-kit^<low cells without CFU-GEMM activity are another P-HSC population and the functional maturation of the cells occurs immediately in this condition in association with immunophenotypical changes. Therefore, the presence of a CD34⁺/c-kit^<low population in situ is physiologically important as a reservoir of dormant P-HSCs with multidrug resistance. Studies to clarify the mechanism(s) behind the regulation of the transition from c-kit^<low to c-kit^low are now under way.

	Acknowledgments

 
We thank Mr. F. Ishida (Research Center of Kansai Medical University; Osaka, Japan) for flow cytometry studies, and Ms. K. Ando for preparing the manuscript.

	References

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