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Functional Differences between Subpopulations of Mobilized Peripheral Blood-Derived CD34⁺ Cells Expressing Different Levels of HLA-DR, CD33, CD38 and c-kit Antigens

^a Second Department of Internal Medicine, Shiga University of Medical Science,
^b Department of Pediatrics,
^c Second Department of Surgery,
^d Department of Hygiene, Kyoto Prefectural University of Medicine, Kyoto, Japan; Department of Biology, Faculty of Science, Niigata University, Niigata, Japan

Key Words. PBSCT • G-CSF • CD34⁺ • CD33 • CD38 • c-kit • LTC-IC

Dr. Yoshiaki Sonoda, Department of Hygiene, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602, Japan.

	Abstract

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We have investigated the functional characteristics of peripheral blood-derived CD34⁺ cells mobilized by a combination of chemotherapy and G-CSF (mobilized peripheral blood-derived [MPB] CD34⁺ cells). In this study, subpopulations of MPB CD34⁺ cells have been directly compared in clonal cultures, long-term cultures with bone marrow (BM) stromal cells, and single-cell cultures. MPB CD34⁺ cells could be subdivided by expression levels of HLA-DR (DR), CD38, CD33 and c-kit antigens. The majority of MPB CD34⁺ cells expressed DR and CD38 antigens. In contrast, approximately 60% and 20% of the MPB CD34⁺ cells expressed CD33 and c-kit antigens, respectively. Interestingly, MPB CD34⁺ cells can be subdivided into three fractions which express high, low or negative levels of c-kit receptor. All types of committed progenitors were observed in populations of CD34⁺DR⁺, CD34⁺DR^–, CD34⁺CD33^–, CD34⁺CD38⁺ and CD34⁺ c-kit^low cells. Colony forming unit-granulocyte/macrophage was highly enriched in the population of CD34⁺CD33⁺ cells, whereas BFU-E was highly enriched in the population of CD34⁺ c-kit^high cells. In the population of CD34⁺CD38^– cells, however, a few myeloid progenitors were detected. In addition, limiting dilution analyses clearly showed that the long-term culture-initiating cell (LTC-IC) is enriched in the populations of CD34⁺DR^–, CD34⁺CD33^– and CD34⁺c-kit^{– or low} cells, but very few in CD34⁺ c-kit^high cells, and that CD38 antigen is not a useful marker for the enrichment of LTC-IC derived from MPB CD34⁺ cells. Moreover, single cell clone sorting experiments clearly demonstrated the functional differences between CD34⁺CD38⁺ and CD34⁺CD38^– cells as well as CD34⁺ cells expressing different levels of c-kit receptor. Our results suggest that an immunophenotype of LTC-IC is different between BM-, cord blood- and MPB-derived CD34⁺ cells and that primitive and committed progenitors existing in these sources may be functionally different.

	Introduction

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It is well-established that a combination of chemotherapy and G-CSF effectively mobilizes hematopoietic progenitor cells into peripheral blood (PB) [1-4]. Therefore, PB-derived CD34⁺ cells are alternatives to bone marrow (BM) cells as a source for autologous and allogeneic stem cell transplantation. Recently, PB stem cell transplantation (PBSCT) has been widely used to reconstitute the hematopoietic system in cancer patients who were treated with high-dose chemotherapy [3]. This is because PBSCT can achieve restoration of sustained hematopoietic functions more rapidly than bone marrow transplantation (BMT) [4]. Siena et al. demonstrated that PB-derived CD34⁺ cells mobilized by high-dose chemotherapy with or without GM-CSF possess qualitatively normal hematopoietic colony-forming ability and high cloning efficiency [5]. PB-derived CD34⁺ cells were reported to express CD33 or HLA-DR (DR) antigen on their surfaces which are comparable to BM-derived CD34⁺ cells [5]. On the other hand, the proto-oncogene c-kit, which encodes a transmembrane tyrosine kinase receptor [6], plays an important role in murine and human hematopoiesis [7-9]. Several investigators have demonstrated that murine and human primitive hematopoietic progenitors express different levels of c-kit protein [10-12]. However, an expression level of c-kit protein on the surfaces of PB-derived CD34⁺ cells has not been clearly established.

Although previous studies have provided unique characteristics of committed progenitors in CD34⁺ cells mobilized by a combination of chemotherapy and G-CSF (mobilized peripheral blood-derived [MPB] CD34⁺ cells) [2, 13], the characteristics of more immature progenitor cells, long-term culture-initiating cells (LTC-IC), have not been fully established.

In this study, we divided MPB CD34⁺ cells depending on the expression levels of DR, CD38, CD33 and c-kit antigens using a fluorescence-activated cell sorter (FACS), and compared the colony-forming ability of each subpopulation in clonal cell cultures. Moreover, we evaluated LTC-IC in each subpopulation simultaneously. Interestingly, MPB CD34⁺ cells which express different levels of DR, CD38, CD33 and c-kit antigens can be subdivided into two or three fractions such as positive or negative cells, or high, low or negative cells. Each of nine subpopulations of MPB CD34⁺ cells showed different stem cell characteristics. In addition, LTC-IC was highly enriched in the populations of CD34⁺DR^– and CD34⁺CD33^– cells. In contrast, CD38 antigen is not applicable for the enrichment of LTC-IC derived from MPB CD34⁺ cells. These unique characteristics of subpopulations of MPB CD34⁺ cells were further elucidated by single cell clone sorting experiments. These results suggest that MPB-derived committed progenitors could be immunophenotypically separated, and that MPB-derived LTC-IC may represent a functionally different class of primitive progenitor cell compared to that in BM- or cord blood (CB)-derived CD34⁺ cells.

	Materials and Methods

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Recombinant Factors
Purified bacterially derived recombinant human (rHu) interleukin 3 (IL-3), GM-CSF, G-CSF, stem cell factor (SCF) and purified CHO-cell-derived rHu erythropoietin (EPO) were generous gifts from Kirin Brewery Co., Ltd. (Tokyo, Japan). Purified E. coli-derived rHu IL-6 was kindly provided by Ajinomoto Co., Inc. (Yokohama, Japan).

Cell Preparation
After informed consent was obtained, PB mononuclear (MN) cells were collected by leukapheresis using Fenwall CS-3000 (Fenwall Laboratories, Inc.; Deerfield, IL) from 10 patients with testicular tumors. The details of the methods used for cell collection have been reported elsewhere [1, 14]. Briefly, hematopoietic progenitor cells were mobilized into PB by a combination of myeloablative chemotherapy and G-CSF. Cells in the collection bag were centrifuged at 150 g for 15 min and buffy-coat cells were transferred to the second bag. These cells were diluted with an equal volume of RPMI 1640 medium (Bio Whittaker; Walkersville, MD) and were subsequently frozen using a rate-controlled programmed freezer for autotransplantation as reported elsewhere [15]. Prior to freezing, a portion of the cells were removed and used for the present studies. These PB MN cells contained an average 5.9 ± 3.4% (n = 11) CD34⁺ cells by flow cytometric analysis. These cells were washed twice with medium (Dainippon Pharmaceutical Co., Ltd.; Australia) containing 10% fetal calf serum (FCS; Dainippon) and nonadherent cells were recovered by overnight adherence to plastic dishes. The MN nonadherent (MNNA) cell fractions were further enriched by negative selection using soybean agglutinin (SBA) MicroCELLector flask (Applied Immune Science Inc.; Menlo Park, CA) as reported [16]. The PB MNNA cells were applied at 0.8-2.0 (median, 1.2) x 10⁷ cells/flask (n = 10). After one-hour incubation, 2.5-9.3 (median, 5.0) x 10⁶ SBA^– cells were recovered. These SBA^– cells contained an average 28.7 ± 11.7% CD34⁺ cells according to FACS analysis.

Purification of Hematopoietic Progenitor Cells by FACS
Highly-purified progenitor cells were separated from PB-derived SBA^– cells using a FACStar Plus (Becton Dickinson Immunocytometry System; San Jose, CA) equipped with an argon laser tuned at 488 nm as reported elsewhere [16, 17]. First, PB-derived SBA^– cells were washed twice with Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS^–) containing 2% FCS (staining medium) and pelleted before staining with monoclonal antibodies (mAbs). The mAbs used were as follows: fluorescein isothiocyanate (FITC)-conjugated CD34 (mouse IgG₁, HPCA-2; Becton Dickinson); phycoerythrin (PE)-conjugated DR (mouse IgG_2a; Becton Dickinson); PE-conjugated CD38 (mouse IgG₁, Leu17; Becton Dickinson); PE-conjugated CD33 (mouse IgG_2b, My9, Coulter Immunology; Hialeah, FL); purified antihuman c-kit mAb (mouse IgM, Immunotech S.A.; Marseilles Cedex, France); and goat antimouse IgM with PE (Immunotech S.A.). For double staining, cells were incubated with 20 µl of CD34 (FITC)/10⁶ cells, and 20 µl of one of these mAbs: HLA-DR (PE) and CD38 (PE)/10⁶ cells, or 5 µl of CD33 (PE)/10⁶ cells for 30 min on ice, and then washed twice with staining medium. Cells were also stained with 20 µl of unconjugated c-kit mAb/5 x 10⁵ cells for 30 min at room temperature. After washing with staining medium, cells were incubated with 20 µl of goat antimouse IgM with PE/5 x 10⁵ cells for 30 min at room temperature. These cells were washed twice with staining medium and then were incubated with 10 µl of normal mouse serum for 15 min on ice to block the free-binding sites of the secondary PE-conjugated antibody. These cells were then stained with CD34 (HPCA-2) as described. All of the stained cells were passed through a stainless steel mesh and were kept on ice for cell sorting. For negative controls, unstained cells and cells stained only with second antibody or isotype control IgG with FITC or PE were included.

Stained cells were sorted using a FACStar Plus single-laser flow cytometer system. Sorting gates were first established for both intermediate forward scattering and low side scattering. A dual-parameter dot diagram displaying FITC (CD34) and PE (DR, CD38, CD33 and c-kit) fluorescence was then generated from the gated events. Using these gated dot diagrams, each subpopulation of CD34⁺ cells was sorted for clonal cultures. Data acquisition was performed using FACStar Plus Research software. A total of at least 20,000 events was analyzed for each sample. We isolated nine subpopulations of CD34⁺ cells as specified later. The phenotypic purity of sorted cells determined by post-sort flow cytometric reanalysis exceeded 90%. After sorting, the recovered cells were washed twice with medium and cultured as described below.

Clonal Cell Culture
Cultures were carried out in 35 mm Lux suspension culture dishes (No. 171099, Nunc Inc.; Naperville, IL) as we reported elsewhere [16-18]. One milliliter of culture contained 200 sorted cells, 1.2% of 1,500 centipoise methylcellulose (Shinetsu Chemical; Tokyo, Japan), 30% FCS, 1% deionized fraction V bovine serum albumin ([BSA], Sigma Chemical Co.; St. Louis, MO), 5 x 10^–5 mol/L 2-mercaptoethanol (Sigma), and various combinations of colony stimulating factors (CSFs). Final concentrations of each CSF were as follows: IL-3, 10 ng/ml; GM-CSF, 10 ng/ml; G-CSF, 20 ng/ml; EPO, 2 U/ml; SCF, 20 ng/ml. These concentrations supported maximal total colony formation in preliminary titration experiments (data not shown). In some experiments, we added 100 U/ml (20 ng/ml) IL-6, which produced maximal effects on maturation of megakaryocyte progenitors in the culture [19].

Dishes were incubated at 37°C in a fully humidified atmosphere flushed with a combination of 5% CO₂, 5% O₂ and 90% N₂. On day 14 of incubation, all colonies were scored on an inverted microscope according to their typical morphologic appearance as reported [16-20]. Colony types identified in situ were granulocyte colonies (colony forming units-granulocyte [CFU-G]), macrophage colonies (colony forming units-macrophage [CFU-M]), granulocyte/macrophage colonies (CFU-granulocyte/macrophage [-GM]), erythroid-bursts (BFU-E), eosinophil colonies (CFU-Eo), and mixed colonies containing erythromyeloid cells and megakaryocytes (CFU-Mix).

Clone Sorting and Single Cell Culture
Clone sorting was performed on a FACStar Plus using Automated Cell Deposition Unit (ACDU). Sorting windows were established as described above. In these experiments, single cells were deposited in each well of 96-well flat-bottomed microtiter plates (Falcon 3072, Becton Dickinson and Company; Lincoln Park, NJ). Each well contained 0.2 ml of serum-free culture medium consisting of ASF 104 medium (Ajinomoto Co., Ltd) [21], 1% BSA, 300 µg/ml transferrin (Sigma), 10 µg/ml lecithin (Sigma), 6 µg/ml cholesterol (Sigma), and six cytokines including SCF, interleukin 6 (IL-6), IL-3, GM-CSF, G-CSF and EPO. After single cell sorting of specified subpopulations of MPB CD34⁺ cells, cultures were incubated for two weeks. Then numbers of cells in positive wells were counted directly under an inverted microscope or using a counting chamber as reported [16]. Clones generated from single cells were then replated into secondary methylcellulose cultures containing SCF, IL-6, IL-3, GM-CSF, G-CSF and EPO to test their colony forming cell (CFC) producing abilities.

Limiting Dilution Analysis of LTC-IC
Cocultures were established by incubating cells originated from nine sorted CD34⁺ subfractions including DR^+/–, CD33^+/–, CD38^+/– and c-kit^high/low/– cells in 96-well plates precoated with murine stromal cell line, MS-5 [22], instead of allogeneic BM stromal cells as reported [23]. In brief, the MS-5 stromal cell line was maintained in medium supplemented with 10% FCS. For cocultures with enriched human progenitors, unirradiated MS-5 feeders were pre-established in 96-well plates (3 x 10³ cells/well). To avoid early detachment of stromal layer, we used gelatin-coated plates (MS-0096G, Sumitomo Bakelite Co., Ltd.; Tokyo, Japan). In these limiting dilution experiments, each CD34⁺ subfraction was seeded at four different initial cell concentrations (2 to 100 cells/well) with a mean of 78 ± 29 replicate wells for 2 to 10 cells, and 22 ± 5 for 20 to 100 cells, respectively.

Cocultures were initiated in stromal medium ( medium, 10% FCS, 10% horse serum (Bio Whittaker), 5 x 10^–5 mol/L 2-mercaptoethanol) and fed every week by half-medium change. Cultures were sacrificed after five weeks, and the progenitor content of each well was assayed in above-mentioned methylcellulose cultures containing optimal concentrations of IL-3, GM-CSF, G-CSF, EPO, SCF and IL-6 as reported [24].

Statistical Analysis
The significance of differences in means was determined using the two-tailed Student's t-test. The significance of differences in replating efficiencies was determined using x-square test. The frequency of LTC-IC in the initial population was calculated by Poisson statistics as reported [24, 25].

	Results

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Comparison of Colony Formation by PB-Derived MN Cells and Purified CD34⁺ Cells
There is a possibility that certain populations of CD34⁺ cells may have been lost during our purification process. To rule out such a possibility, colony formation by PB MN cells and FACS-sorted CD34⁺ cells were compared (Table 1). The number of colonies varied between different samples; however, the percentage of each colony derived from different progenitors is almost identical. These results suggest that purified CD34⁺ cells contained the same population of progenitors which existed in the original apheresis products.

Colony Formations by MPB CD34⁺DR^+/–, CD34⁺CD38^+/– and CD34⁺CD33^+/– Cells
The representative scattergrams of MPB SBA^– cells taken by the expression of CD34 versus each of the three antigens are shown in Figure 1. Each sorting window was established as described in Materials and Methods. The representative colony-forming patterns of five independent experiments are shown in Figure 2. In the populations of CD34⁺DR⁺, CD34⁺DR^–, CD34⁺CD38⁺ and CD34⁺CD33^– cells, all of the committed progenitors including BFU-E, CFU-G, CFU-M, CFU-GM, CFU-Eo and CFU-Mix were detected in the presence of a combination of SCF, IL-3, GM-CSF, G-CSF and EPO (CSFs). Plating efficiency (PE) of the population of CD34⁺DR⁺ cells in the presence of CSFs was approximately 50% to 70% (Fig. 2A). In contrast, PE was much lower in the population of CD34⁺DR^– cells than in that of CD34⁺DR⁺ cells. The PE of the population of CD34⁺CD38⁺ cells was almost comparable to that of CD34⁺DR⁺ cells (Fig. 2B). In contrast, the PE of CD34⁺CD38^– cells was significantly lower than that of CD34⁺CD38⁺ cells even in the presence of CSFs. The PE of CD34⁺CD33^– cells was approximately 70% to 80%. The PE of CD34⁺CD33⁺ cells was slightly lower than that of CD34⁺CD33^– cells; however, CFU-GM was highly enriched in this population of progenitors (Fig. 2C).

View larger version (22K): [in this window] [in a new window]   Figure 1. Representative scattergrams of HLA-DR (A), CD38 (B) and CD33 (C) antigens on MPB CD34+ cells. First, sorting gates were established for both forward scattering and side scattering. Dual-parameter dot diagrams displaying FITC (CD34) and PE (HLA-DR or CD38 or CD33) fluorescence were then generated from the gated events. The vast majority of CD34+ cells expressed HLA-DR and CD38 antigens. Approximately 60% of CD34+ cells expressed CD33 antigen.

View larger version (23K): [in this window] [in a new window]   Figure 2. Colony formations by 200 MPB (A) CD34+DR+/–, (B) CD34+CD38+/– and (C) CD34+CD33+/– cells in the presence of 30% FCS and SCF, IL-3, GM-CSF, G-CSF and EPO are shown. CFU-GM (including CFU-G, CFU-M, CFU-Eo and CFU-GM) were highly enriched in the population of CD34+CD33+ cells. The CD34+DR+ or –, CD34+CD38+ and CD34+CD33– cell populations contained all types of committed progenitors including CFU-GM, BFU-E and CFU-Mix. In contrast, the CD34+CD38– cell population depleted committed progenitors, and contained a few CFU-GM.

View larger version (29K): [in this window] [in a new window]   Figure 3. Expressions of c-kit antigen on MPB CD34+ cells. MPB SBA– cells were stained with CD34 (FITC) and c-kit (PE) mAbs (B, D) and only with control second antibody (PE) or isotype control IgG with FITC (A, C). MPB CD34+ cells were subdivided into three fractions, namely CD34+ c-kithigh, CD34+ c-kitlow and CD34+ c-kit– cells (B, D). CD34+ c-kithigh cells consistently expressed a lower level of CD34 antigen compared to MPB CD34+ cells which expressed a low or negative level of c-kit antigen (D).

View larger version (11K): [in this window] [in a new window]   Figure 4. Colony formations by 200 MPB CD34+ c-kithigh, CD34+ c-kitlow and CD34+ c-kit– cells in the presence of 30% FCS and SCF, IL-3, GM-CSF, G-CSF and EPO are shown. BFU-E and CFU-GM were highly enriched in the populations of CD34+ c-kithigh and CD34+ c-kit– cells, respectively. CD34+ c-kitlow cell population contained all types of committed progenitors.

View larger version (32K): [in this window] [in a new window]   Figure 5. Representative data of limiting dilution analyses are shown. Decreasing numbers of each of nine subpopulations of MPB CD34+ cells were seeded onto MS-5 feeders and the numbers of clonogenic cells detectable after five weeks of cocultures were determined. In these experiments, the frequencies of LTC-IC in the initial cell populations were as follows: 1 per 57 cells for CD34+DR+; 1 per 12 cells for CD34+DR–; 1 per 58 cells for CD34+CD38+; 1 per 84 cells for CD34+CD38–; 1 per 66 cells for CD34+CD33+; 1 per 32 cells for CD34+CD33–; 1 per 663 cells for CD34+ c-kithigh; 1 per 110 cells for CD34+ c-kitlow; and 1 per 90 cells for CD34+ c-kit–.

Proliferative and Replating Potential of Clone-Sorted Subpopulations of MPB CD34⁺ Cells
In order to further examine the functional differences between MPB CD34⁺CD38^{+ or –} and CD34⁺ c-kit^high/low/- cells, we carried out single cell clone sorting experiments. Data pooled from three independent experiments are shown in Tables 2 and 3. Our data demonstrated that approximately 80% of single cells derived from CD34⁺CD38⁺, CD34⁺ c-kit^high, or CD34⁺ c-kit^low cells proliferated in the presence of SCF, IL-6, IL-3, GM-CSF, G-CSF and EPO. In contrast, only 40% to 50% of single cells derived from CD34⁺CD38^– or CD34⁺ c-kit^– cells proliferated in the same culture condition. Interestingly, single MPB CD34⁺CD38⁺ cells generated significantly larger (p < 0.01) clones than those from CD34⁺CD38^– cells (Table 2). In addition, clones from CD34⁺ c-kit^low cells generated significantly larger clones than those from CD34⁺c-kit^– or CD34⁺ c-kit^high cells (p < 0.01). Moreover, clones from MPB CD34⁺CD38⁺ cells produced significantly more CFCs (p < 0.05) than those from MPB CD34⁺CD38^– cells (Table 3). Approximately, 40% of clones derived from CD34⁺c-kit^low or c-kit^– cells produced secondary CFCs. However, only 7% of clones derived from CD34⁺c-kit^high cells yielded secondary CFCs. This difference is statistically significant (p < 0.01). Numbers of secondary CFCs derived from these three subpopulations are almost comparable.

	Discussion

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We then subdivided MPB CD34⁺ cells into two fractions according to the expression level of DR, CD33 or CD38 antigen, respectively. Our data clearly showed that MPB CD34⁺ cells which do not express CD33 antigen and do express DR or CD38 antigen contained all types of committed progenitors including CFU-Mix. The PEs of these subpopulations were almost comparable. In contrast, the CD34⁺CD33⁺ population contained a large number of CFU-GM as previously reported [5, 33]. In contrast, CD34⁺CD38^– cell population contained a few CFU-GM. The expression of c-kit receptor on MPB CD34⁺ cells was approximately 20% and this was significantly lower than that of BM- or CB-derived CD34⁺ cells which express high levels of c-kit receptor [7, 11]. In addition, MPB CD34⁺ cells were subdivided into three fractions (c-kit^high, c-kit^low and c-kit^– cells) which showed different stem cell characteristics as described. Recently, Gunji et al. [11] demonstrated that myeloid progenitors are enumerated in CD34⁺c-kit^high cells and erythroid progenitors are more enriched in CD34⁺c-kit^low cells when BM-derived progenitors were used as the target cells. However, our results clearly indicate that erythroid progenitors are highly enriched in MPB CD34⁺c-kit^high cells, and that CFU-GM is enriched in MPB CD34⁺c-kit^– cells. Our data are consistent with an earlier report which demonstrated the characteristics of CB-derived CD34⁺ cells expressing different levels of c-kit receptor [12]. Collectively, CB- or MPB-derived CD34⁺ cells expressing different levels of c-kit protein might differ from BM-derived CD34⁺ cells which express comparable levels of c-kit protein.

Next, we investigated the capacity of nine sorted subpopulations of MPB CD34⁺ cells to yield CFC in the long-term BM culture system. It was reported that LTC-IC can be detected in MPB CD34⁺ cells [34, 35]; however, little is known about the immunophenotype of LTC-IC in these cells. Our limiting dilution analyses clearly demonstrated that LTC-IC was highly enriched in MPB CD34⁺HLA-DR^– and CD34⁺CD33^– cell populations. These data are consistent with several previous reports [27, 28, 36, 37]. In contrast, the frequency of LTC-IC in MPB CD34⁺CD38⁺ cells was slightly larger than that in CD34⁺CD38^– cells. This is a striking contrast to previous reports in which the CD34⁺CD38^– immunophenotype defines a primitive subpopulation of progenitor cells [23, 26, 28, 29]. In our LTC-IC assay system, however, CB-derived LTC-IC was highly enriched in the population of CD34⁺CD38^– cells [Sakabe et al., unpublished data]. Moreover, CB-derived CD34⁺CD38^– cells were able to generate CFCs nine weeks after the initiation of cocultures with BM stromal cells [Sakabe et al., unpublished data]. These results are consistent with the earlier reports [26, 29]. In addition, our single cell clone sorting experiments demonstrated that single MPB CD34⁺CD38⁺ cells generated significantly larger clones than those from CD34⁺CD38^– cells, and that clones from MPB CD34⁺CD38⁺ cells produced significantly more CFCs than those from MPB CD34⁺CD38^– cells. These data suggest that MPB CD34⁺CD38⁺ cells have higher proliferative and replating potential than their CD38^– counterpart. On the contrary, it was reported that clones produced from single CB or BM CD34⁺CD38^– cells contained more cells than those from CD34⁺CD38⁺ cells, showing the higher proliferative potential of the more primitive CD34⁺CD38^– cells [26]. These discrepancies may be explained by the functional differences between CB, BM and MPB CD34⁺ cells.

According to the c-kit expression on MPB CD34⁺ cells, LTC-IC was detected in CD34⁺ c-kit^{– or low} cell population, but very few in CD34⁺ c-kit^high cell population. In addition, single cell clone sorting experiments demonstrated that CD34⁺ c-kit^low cells show significantly higher proliferative potential than that of CD34⁺ c-kit^{– or high} cells, and that clones derived from CD34⁺ c-kit^{– or low} cells showed higher replating efficiencies than those from CD34⁺ c-kit^high cells. Our data suggest that primitive progenitors with self-renewal potential may present in the MPB CD34⁺ c-kit^{– or low} cell population. However, it was reported that LTC-IC is highly enriched in BM-derived CD34⁺ c-kit^low cells and that the number of LTC-IC in CD34⁺ c-kit^– cells is very low compared to that in the CD34⁺ c-kit^low or c-kit^high cell population [11]. Very recently, Laver et al. reported that the CB-derived CD34⁺ c-kit^low cell population contains the majority of cell cycle dormant progenitors and blast cell colony forming cells [12]. These discrepancies may also suggest that MPB CD34⁺ cells differ from BM- or CB-derived CD34⁺ cells even if they express the same levels of c-kit protein.

Our data are partly consistent with earlier reports which showed that LTC-IC is enriched in BM- and PB-derived CD34⁺DR^{low or –} cell populations [36, 37], and BM-derived CD34⁺CD33^– cell population [37]. There is, however, a conflicting result. Traycoff et al. reported that the CB-derived CD34⁺DR⁺ cell population is more primitive than the CD34⁺DR^– cell population [27] in contrast to their previous findings in adult BM where the LTC-IC was shown to be CD34⁺DR^– [38]. These results lead us to propose that an immunophenotype of LTC-IC may be different between BM-, CB- and MPB-derived CD34⁺ cells.

In conclusion, these data, including ours, suggest that the CD34⁺CD38^– or CD34⁺CD33^– or CD34⁺c-kit^{– or low} or CD34⁺DR^– immunophenotype defines primitive nature of progenitor cells in fetal liver, fetal BM, adult BM, MPB and CB [1-12,16,27-32,37-40]; there may be significant functional differences between these sources. Further precise study using multicolor flow cytometry and cell sorting will be required to fully characterize the immunophenotype of LTC-IC derived from MPB CD34⁺ cells.

	Acknowledgments

 
The authors are particularly indebted to Professor Tadao Banba, Second Department of Internal Medicine, Shiga University of Medical Science. The authors are grateful to Dr. Hiroaki Asano of Kyoto Prefectural University of Medicine for his advice on statistical analysis, and to Drs. Yoshihide Fujiyama and Keiko Hodohara, Second Department of Internal Medicine, Shiga University of Medical Science for their kind support. The authors also wish to thank Yoichi Miyamoto of the Department of Radiation Therapy, Kyoto Prefectural University Medicine for his excellent technical assistance.

Supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 08261213) from the Ministry of Education, Science and Culture of Japan.

	References

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