HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 2006-0727v1 25/6/1348    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kimura, T. Articles by Sonoda, Y. Search for Related Content PubMed PubMed Citation Articles by Kimura, T. Articles by Sonoda, Y.

TISSUE-SPECIFIC STEM CELLS

Identification of Long-Term Repopulating Potential of Human Cord Blood-Derived CD34^–flt3^– Severe Combined Immunodeficiency-Repopulating Cells by Intra-Bone Marrow Injection

^aDepartment of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Moriguchi, Osaka, Japan;
^bDepartment of Gynecology and Obstetrics, Fukuda Hospital, Kumamoto, Japan;
^cDepartment of Obstetrics and Gynecology, Aizenbashi Hospital, Osaka, Japan;
^dDepartment of Microbiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto, Japan;
^eDepartment of Industrial Science and Technology, Tokyo University of Science, Noda, Chiba, Japan;
^fFirst Department of Pathology, Transplantation Center,
^gFirst Department of Internal Medicine, Kansai Medical University, Moriguchi, Osaka, Japan

Key Words. Flt3 • Severe combined immunodeficiency-repopulating cell • Intra-bone marrow injection • Cord blood • Hematopoiesis

Correspondence: Yoshiaki Sonoda, M.D., Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan. Telephone: +81-6-6993-9435; Fax: +81-6-6992-3522; e-mail: sonoda{at}takii.kmu.ac.jp

Received November 8, 2006; accepted for publication February 6, 2007.
First published online in STEM CELLS EXPRESS   February 15, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Recently, we have identified human cord blood (CB)-derived CD34-negative (CD34^–) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003;101:2924). In contrast to murine CD34^– Kit⁺Sca-1⁺Lineage^– (KSL) cells, human CB-derived Lin^–CD34^– cells did not express detectable levels of c-kit by flow cytometry. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34^– SRCs. Both CD34⁺flt3^+/– cells showed SRC activity. In the CD34^– cell fraction, only CD34^–flt3^– cells showed distinct SRC activity by IBMI. Although CD34⁺flt3⁺ cells showed a rather weak secondary repopulating activity, CD34⁺flt3^– cells repopulated many more secondary recipient mice. However, CD34^–flt3^– cells repopulated all of the secondary recipients, and the repopulating rate was much higher. Next, we cocultured CD34^–flt3^– cells with the murine stromal cell line HESS-5. After 1 week, significant numbers of CD34⁺flt3^+/– cells were generated, and they showed distinct SRC activity. These results indicated that CB-derived CD34^–flt3^– cells produced CD34⁺flt3^– as well as CD34⁺flt3⁺ SRCs in vitro. The present study has demonstrated for the first time that CB-derived CD34^– SRCs, like murine CD34^– KSL cells, do not express flt3. On the basis of these data, we propose that the immunophenotype of very primitive long-term repopulating human hematopoietic stem cells is Lin^–CD34^–c-kit^–flt3^–.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
It is well documented that the tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse [1–10] and human hematopoiesis [1, 11–21]. Moreover, their respective ligands, stem cell factor (SCF) and flt3 ligand (FL), synergistically act with each other and play an important role in the regulation (generation, maintenance, proliferation, differentiation, and expansion) of early stages of murine and human candidate hematopoietic stem cells (HSCs) [1–21]. The most primitive HSCs in mammals, including mice and humans, have long been believed to be CD34 antigen-positive (CD34⁺) [22]. However, Osawa et al. [23] revealed that murine long-term lymphohematopoietic reconstituting HSCs are lineage marker-negative (Lin^–) c-kit⁺Sca-1⁺CD34-low/negative (CD34^lo/– KSL). In a murine model, it was recently reported that flt3^– KSL cells supported long-term multilineage hematopoietic reconstitution [24]. In contrast, flt3⁺ KSL cells are progenitors for the common lymphoid stage [24]. These flt3⁺ KSL cells have also been shown to lack erythro-megakaryocytic potential [25]. This notion was supported by the other reports that mice deficient in the expression of flt3 or FL showed deficient lymphopoiesis [26–28].

Recently, using the intra-bone marrow injection (IBMI) method, we have successfully identified human cord blood (CB)-derived CD34-negative (CD34^–) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability [29]. These CD34^– SRCs seemed to be more primitive HSCs than CD34⁺ SRCs [29, 30]. They could home into the BM niche only by IBMI, because they expressed lower levels of homing receptors, including CXCR4, and had poor SDF-1/CXCR4-mediated migration ability [29]. In contrast to the murine candidate HSCs (CD34^– KSL cells) [23], our identified CD34^– SRCs did not express detectable levels of c-kit tyrosine kinase receptor by flow cytometry. However, the degree to which flt3 is expressed on human HSCs, including CD34⁺ and CD34^– SRCs, which are capable of in vivo lymphomyeloid reconstitution, has not been fully elucidated.

Until now, a number of studies have reported that flt3 is expressed and functioned in the human CD34⁺ hematopoietic progenitor cells [11–17, 19, 20], including long-term culture-initiating cells (LTC-ICs) [15, 20, 31]. However, only two reports have demonstrated, using the conventional intravenous injection method, that human CB- and bone marrow (BM)-derived CD34⁺ HSCs capable of multilineage reconstitution in nonobese diabetic (NOD)/SCID mice express flt3 tyrosine kinase receptor [31, 32].

In this study, we have investigated, using the IBMI method, the function of flt3, which is expressed in early mouse [1–10] and human [11–21] hematopoiesis like c-kit, in our identified very primitive human CB-derived CD34^– SRCs [29, 30], as well as more committed CD34⁺ SRCs. Our data clearly demonstrate that part of human CB-derived CD34⁺ SRCs express flt3, as reported previously [31, 32]. However, only CD34⁺flt3^– cells showed significant secondary repopulating ability, even when a comparable number of CD34⁺flt3^– cells as CD34⁺flt3⁺ cells was transplanted. Moreover, CD34^–flt3^– cells showed distinct and potent SRC activity by IBMI, and they showed high and efficient secondary repopulating ability compared with CD34⁺flt3^– cells. The CD34^–flt3^– cells also produced CD34⁺flt3^– and CD34⁺flt3⁺ SRCs after the coculture with the murine stromal cell line HESS-5 in vitro, suggesting that these CD34^–flt3^– cells contained very primitive human CB-derived HSCs. The results of the present study are consistent with recent studies of the significance of flt3 expression in murine primitive hematopoiesis [24–28] and provide a new concept of hierarchy in the human primitive HSC compartment.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Collection of CB Samples and Processing
CB samples were obtained from normal full-term deliveries with signed informed consent and approved by the institutional review boards of Kansai Medical University and Kyoto Prefectural University of Medicine. CB-derived mononuclear cells (MNCs) were isolated using Ficoll-Paque (Amersham Biosciences AB, Uppsala, Sweden; http://www.amersham.com) density gradient centrifugation. The MNCs were further enriched by negative depletion of eight lineage-positive cells, including CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and Glycophorin A using a StemSep device (StemCell Technologies, Vancouver, BC, Canada; http://www.stemcell.com), as reported previously[29, 30].

Purification of Lin^–CD34⁺flt3^+/– and Lin^–CD34^–flt3^+/– Cells
The above-mentioned lineage-negative (Lin^–) cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD45 monoclonal antibody (mAb) (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com), PC5-conjugated anti-CD34 mAb (Beckman Coulter), and biotinylated anti-flt3 mAb (M22, Immunex, Seattle, http://immunex.com) followed by incubation with streptavidin-phycoerythrin (SA-PE; Becton Dickinson, Franklin Lakes, NJ, http://www.bd.com), as reported previously [20, 29, 30]. These stained cells were then sorted into four fractions, including CD34⁺flt3^+/– and CD34^–flt3^+/– cells, as shown in Figure 1C using a FACSVantage (Becton Dickinson) as reported [29, 30]. The viability of these sorted cells was consistently more than 99%. Approximately 80% of the CD34⁺ cell fraction in these Lin^– cells expressed flt3 receptor. On the other hand, only 30% of the CD34^– cell fraction in the same Lin^– cells expressed this receptor. In separate experiments, we stained these immunomagnetically separated Lin^– cells with 13 FITC-conjugated lineage-specific mAbs as reported previously [29], PC5-conjugated anti-CD34 mAb (Beckman Coulter), and PE-conjugated anti-c-kit mAb (Beckman Coulter) and examined the expression pattern of c-kit receptor (Fig. 1D).

Clonal Cell Culture
Human colony-forming cells (CFCs) were assayed using our standard methylcellulose cultures as reported previously [18–21, 29, 30, 33, 34]. Briefly, 200 or 500 sorted Lin^–CD34^+/–flt3^+/– cells were plated in 1 ml of culture containing 1.2% methylcellulose (Shinetsu Chemical, Tokyo, http://www.shinetsu.co.jp/e), 30% fetal calf serum (FCS; Hyclone, Laboratories, Logan, UT, http://www.hyclone.com), 1% bovine serum albumin (Sigma-Aldrich, St Louis, http://www.sigmaaldrich.com), 5 x 10^–5 mol/L 2-mercaptoethanol (Sigma-Aldrich), and various recombinant human (rh) cytokines, including SCF, interleukin (IL)-3, granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and erythropoietin (Epo) in 35-mm Lux suspension culture dishes (Nunc, Rochester, NY, http://www.nuncbrand.com). For culture of megakaryocyte colony-forming cell (CFU-Meg), 10% platelet-poor plasma (PPP) [33] instead of 30% FCS, and rh thrombopoietin (TPO) were used. Cytokines, including G-CSF, Epo, and TPO were provided by Kirin Brewery Company (Takasaki, Japan, http://www.kirin.com). SCF, IL-3, and GM-CSF were purchased from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com). Dishes were incubated at 37°C in a fully-humidified atmosphere flushed with a combination of 5% CO₂, 5% O₂, and 90% N₂. On days 12–14 of incubation, all colonies were scored under an inverted microscope according to their typical morphologic features, as reported elsewhere [18–21, 29, 30, 33]. CFU-Meg-derived pure megakaryocyte colonies were identified in situ as clusters of large cells, which were highly refractile and showed irregular contour and hyaline nongranulated cytoplasm. The types of colonies identified in situ were granulocyte (CFU-G), macrophage (CFU-M), granulocyte/macrophage (CFU-GM), erythroid burst (BFU-E), erythrocyte-containing mixed (CFU-Mix), and the above-mentioned CFU-Meg. The numbers of all types of hematopoietic colonies were determined as the mean of three independent experiments.

IBMI of Purified Cells
IBMI was carried out as reported previously [29, 30, 35]. Briefly, after sterilization of the skin around the left knee joint, the knee was flexed to 90 degrees and the proximal side of the tibia was drawn to the anterior. A 27-gauge needle was inserted into the joint surface of the tibia through the patellar tendon and then inserted into the BM cavity. Using a Hamilton's microsyringe, the number-specified donor cells per under 10 µl of -medium were carefully and slowly injected from the bone hole into the BM cavity.

SCID-Repopulating Cell Assay
An SRC assay was performed using the methods reported previously [36, 37] with modifications [29, 30, 38]. Five-week-old NOD/Shi-scid/scid (NOD/SCID) mice were purchased from Clea Japan (Tokyo, Japan, http://www.clea-japan.com). The animal experiments were approved by the Animal Care Committees of Kansai Medical University and Kyoto Prefectural University of Medicine. All mice were handled in sterile conditions and maintained in germ-free isolators located in the Central Laboratory Animal Facilities of Kansai Medical University and Kyoto Prefectural University of Medicine. In this study, purified 3 x 10⁴ to 5 x 10⁴ CB-derived Lin^–CD34⁺flt3^+/–, or 2 x 10⁴ to 7 x 10⁴ CB-derived Lin^–CD34^–flt3^+/– cells were transplanted by IBMI into sublethally irradiated (250 cGy using a ¹³⁷Cs- irradiator) 8–12-week-old mice. As we reported previously [29, 30], CB-derived CD34^– SRCs were detected only by the IBMI technique. Moreover, the repopulation rate of CD34⁺ SRCs by IBMI was significantly higher than that by the conventional tail-vein injection method [29]. Therefore, we used the IBMI technique to analyze SRC activities of Lin^–CD34^+/–Flt3^+/– cells in this study. The mice were killed 8–12 weeks after transplantation, and the BMs from the pairs of femurs, tibiae, and humeri of each mouse were flushed into -medium. The rates of human CD45⁺ cells in the murine BMs were analyzed by flow cytometry (FACS Calibur; Becton Dickinson) as described in the next section. Mice were scored as positive if over 0.1% of total murine BM cells were human CD45⁺.

Analysis of Human Cell Engraftment in NOD/SCID Mice by Flow Cytometry
The repopulation of human hematopoietic cells in murine BMs was determined by detecting the number of cells positively stained with PC5-conjugated anti-human CD45 mAb (Beckman Coulter) by flow cytometry. The cells were also stained with PE-conjugated anti-human CD34 mAb (Becton Dickinson), and FITC-conjugated mAbs for human lineage-specific Ags, including CD19 (eBioscience, San Diego, http://www.ebioscience.com), and CD33 (Beckman Coulter) for the detection of human lymphoid and myeloid hematopoietic cells, respectively.

Secondary Transplantation
For secondary transplantations, murine BM cells were obtained from the pairs of femurs, tibiae, and humeri of moderately engrafted primary recipient mice 8–12 weeks after transplantation with 3 x 10³ to 5 x 10³ Lin^–CD34⁺flt3⁺, 4 x 10³ to 5 x 10³ Lin^–CD34⁺flt3^–, or 2 x 10⁴ to 3 x 10⁴ Lin^–CD34^–flt3^– cells, respectively. The human cell repopulation rates in the primary recipients' BMs were comparable and approximately 4%–8%. Whole BM cells were transplanted by IBMI into sublethally (250 cGy) irradiated secondary recipient mice. Eight to 10 weeks after transplantation, the presence of human CD45⁺ cells in the secondary recipients' BMs was analyzed by flow cytometry, as described for primary transplantation.

Coculture with HESS-5 Cells and SRC Activity of Culture-Generated CD34⁺flt3^+/– Cells
A total of 5 x 10⁴ purified Lin^–CD34^–flt3^– cells per 12.5-cm² culture flask (BD Falcon; Becton Dickinson) onto preestablished irradiated HESS-5 [39] layers in StemPro-34 medium (Gibco Laboratories, Grand Island, NY, http://www.invitrogen.com) and a cocktail of cytokines, including 300 ng/ml SCF (R&D), 300 ng/ml TPO (Kirin), 10 ng/ml IL-3 (R&D), 10 units/ml IL-6 (provided by Dr. Akira Okano, Ajinomoto Co. Inc., Yokohama, Japan, http://www.ajinomoto.com), 10 ng/ml G-CSF (Kirin), and 5% FCS (Hyclone). After 1 week, all cells were collected by vigorous pipetting, and stained with PC5-conjugated anti-CD34 mAb (Beckman Coulter) and biotinylated anti-flt3 mAb (Immunex) as mentioned herein. Cells were then stained with SA-PE (Becton Dickinson). The rates of CD34⁺flt3^+/– cells were analyzed by flow cytometry. Simultaneously, these CD34⁺flt3^+/– cells were separately obtained by cell sorting (FACSVantage) for the detection of SRC activity. One to 2 x 10⁴ CD34⁺flt3⁺ or 2 x 10⁴ to 4 x 10⁴ CD34⁺flt3^– cells were transplanted by IBMI into sublethally (250 cGy) irradiated recipient mice. Eight weeks after transplantation, the presence of human CD45⁺ cells in the recipients' BMs was analyzed by flow cytometry, as described for primary transplantation.

Statistical Analysis
The significance of differences in the SRC assays and the numbers of hematopoietic colonies was determined using the Mann-Whitney U test and the two-tailed Student's t test, respectively.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Expression of flt3 and c-kit Receptors on Lin^–CD45⁺CD34^+/– Cells
First, we depleted the eight lineage-positive cells from CB-derived MNCs using the immunomagnetic beads system [29, 30]. Then, Lin^–CD45⁺CD34^+/– cells were gated as R2 as shown in Figure 1B. These Lin^–CD45⁺ cells were subdivided into four distinct populations on the basis of their surface CD34 and flt3 expression (Fig. 1C). We sorted these four fractions for further stem cell characterization. The phenotypic purity of the sorted cells consistently exceeded 98% when checked using postsorting flow cytometric analysis (data not shown). Importantly, these Lin^–CD34^– cells did not express detectable levels of c-kit receptors by flow cytometry, as shown in Figure 1D.

View larger version (41K): [in this window] [in a new window]   Figure 1. Expression of flt3 or c-kit receptor on cord blood-derived Lin– cells. (A): The forward scatter/SSC profile of immunomagnetically separated Lin– cells. The R1 gate was set on the lymphocyte window. (B): Lin–CD45+CD34+/– cells present in R1 gate were gated as R2. (C): The expression pattern of CD34 and flt3 on R2 gated cells is shown. Cells residing in the four cell fractions were classified as Lin–CD34+flt3+, Lin–CD34+flt3–, Lin–CD34–flt3+, and Lin–CD34–flt3– cells, respectively. Each sorting window is shown as a solid square. Figures in upper right corner show percentages of cells in each quadrant. (D): The expression pattern of c-kit on Lin–CD34+/– cells in a separate experiment. Abbreviations: FITC, fluorescein isothiocyanate; FSC, forward scatter; PE, phycoerythrin; SSC, side scatter.

View larger version (16K): [in this window] [in a new window]   Figure 2. Colony-forming capacities of Lin–CD34+flt3+/– cells. (A): The colony-forming capacities of 200 Lin–CD34+flt3+/– cells in the presence of stem cell factor, interleukin-3, granulocyte macrophage (GM) colony-stimulating factor (CSF), granulocyte (G) CSF, and erythropoietin. Open, shaded, closed, and gray bars represent the number of granulocyte/macrophage colony-forming units (CFUs; including CFU-G, CFU-macrophage, and CFU-GM), erythroid burst, CFU-Mix, and total colony, respectively. (B): The colony-forming capacities of 500 Lin–CD34+flt3+/– cells in the presence of thrombopoietin. Closed bars represent the number of megakaryocyte CFUs. The numbers of all types of colonies were determined as the mean of three independent experiments. Vertical bars represent standard deviation, and asterisks show statistical significance (p < .01) between the numbers of designated colonies formed by flt3+ and flt3– cells, respectively.

SRC Activity and Lymphomyeloid-Reconstituting Capacity of CB-Derived Lin^–CD34^+/–flt3^+/– Cells by IBMI
In this study, we have investigated the function of flt3 in our identified human CB-derived CD34 ^– SRCs. First, we studied the SRC activity of CB-derived Lin^–CD34⁺flt3^+/– or CD34^–flt3^+/– cells using IBMI, as shown in Figure 3. Both CD34⁺flt3^+/– cells repopulated all 20 recipient mice (10 mice each). The level of human CD45⁺ cells in the murine BMs that received transplants of CD34⁺flt3⁺ cells (n = 10; 29.3% to 90.8%; median, 65.4%) was higher than those that received transplants of CD34⁺flt3^– cells (n = 10; 0.3% to 45.1%; median, 16.9%).

View larger version (41K): [in this window] [in a new window]   Figure 3. Severe combined immunodeficiency-repopulating cell activities of Lin–CD34+/–flt3+/– cells by intra-BM injection (IBMI). (A): Each mouse transplanted with designated numbers of cord blood-derived Lin–CD34+flt3+, Lin–CD34+flt3–, Lin–CD34–flt3+, and Lin–CD34–flt3– cells was sacrificed 8–12 weeks after transplantation. Closed circles represent the repopulation rates in total murine BMs by the IBMI, respectively. Horizontal bars represent each median of the repopulation rates. (B–D): The human CD45+ cell reconstitution in the representative mouse presented in (A) received transplants of CD34+flt3+ (B), CD34+flt3– (C), and CD34–flt3– (D) cells, respectively. Percentages of cells in each quadrant are presented in the upper left corner. Abbreviations: BM, bone marrow; PE, phycoerythrin.

To further evaluate the function of flt3 expression in CD34⁺ and CD34^– SRCs, we studied their lymphomyeloid reconstitution abilities using IBMI. In our SRC assay system, all NOD/SCID mice transplanted either with 3 x 10⁴ to 5 x 10⁴ Lin^–CD34⁺flt3^+/– cells or 5 x 10⁴ to 7 x 10⁴ Lin^–CD34^–flt3^– cells by IBMI showed signs of human cell engraftment. The analyses of the three representative mice transplanted either with Lin^–CD34⁺flt3^+/– cells or Lin^– CD34^–flt3^– cells clearly indicate that these three classes of SRCs have an extensive differentiation capacity to B-lymphoid (CD19) and myeloid (CD33) lineages in vivo (Table 1).

Secondary Repopulating Ability of Lin^–CD34⁺ flt3^+/– or Lin^–CD34^–flt3^– Cells by IBMI
To further evaluate the long-term repopulating potential of these three populations (CD34⁺flt3⁺, CD34⁺flt3^–, and CD34^–flt3^– cells), BM cells obtained from each engrafted primary recipient mouse were assessed for their SRC activity by secondary transplantation by IBMI. Only one of six mice that received whole BM cells obtained from primary recipient mice that received transplants of CD34⁺flt3⁺ cells showed secondary repopulating activity (Fig. 4). On the other hand, 83% (five of six) of the secondary recipients that received whole BM cells from primary recipients that received CD34⁺flt3^– cells could be repopulated. Moreover, all five secondary recipient mice that received whole BM cells from primary recipients that received CD34^–flt3^– cells could be repopulated with a higher secondary repopulating rate (Fig. 4). These results demonstrated that CD34^–flt3^– SRCs have more potent secondary reconstituting abilities in comparison with the other two types of SRCs, and could sustain long-term human hematopoiesis in NOD/SCID mice.

View larger version (18K): [in this window] [in a new window]   Figure 4. Secondary repopulating capacities of Lin–CD34+flt3+/– or Lin–CD34–flt3– cells. Cells transplanted to primary recipients (PRs) by intra-BM injection (IBMI) numbered 3 x 103 to 5 x 103 CD34+flt3+cells, 4 x 103 to 5 x 103 CD34+flt3– cells, or 2 x 104 to 3 x 104 CD34–flt3– cells. Human cell repopulations of BMs in PRs (open circles) analyzed 8–12 weeks after transplantation were comparable and 4%–8%. Whole BM cells obtained from PRs were transplanted to secondary recipients (SRs) by IBMI. Human cell repopulation in SRs (closed circles) was analyzed 8–10 weeks after secondary transplantation. Horizontal bars represent each median of the repopulation rates in PRs and SRs, respectively. Abbreviation: BM, bone marrow.

View larger version (26K): [in this window] [in a new window]   Figure 5. Expression pattern of CD34 and flt3 on sorted Lin–CD34–flt3– cells after the 7-day coculture with HESS-5 cells. (A): Flow cytometry pattern of immunomagnetically separated cord blood-derived Lin– cells stained with anti-flt3 (PE) and anti-CD34 (PC5) monoclonal antibodies. Lin–CD34–flt3– cells were sorted for the coculture with HESS-5 cells. The sorting gate is indicated by the solid square. (B): Postsorting analysis of the sorted Lin–CD34–flt3– cells. (C): The expression pattern of flt3 on CD34+ cells derived from the 7-day cocultures of sorted Lin–CD34–flt3– cells with the murine stromal cell line, HESS-5, in the presence of a cocktail of cytokines. The sorting gates for culture-generated CD34+flt3+/– cells are indicated by two solid squares. Abbreviation: PE, phycoerythrin.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

On the other hand, the flt3 receptor has also been shown to be expressed and to function in murine candidate HSCs [1–10, 24–28], including Lin^–Sca-1⁺AA4⁺ fetal liver cells [5], CD34^+/– KSL BM cells [10, 24–26], and Thy-1.1^loKLS cells [9]. In particular, Jacobsen et al. have extensively studied the expression and functional significance of flt3 receptor on murine LTR-HSCs [7, 8, 10, 24–26]. Adolfsson et al. [24] first reported that the upregulation of flt3 on BM-derived KSL cells is accompanied by loss of self-renewal capacity. In other words, flt3⁺ KSL cells rapidly and efficiently reconstituted B and T lymphopoiesis, and only flt3^– KSL cells supported sustained multilineage reconstitution [24–26]. On the basis of these data, they proposed that flt3⁺ KSL cells are progenitors for the common lymphoid progenitor (CLP) [24–26]. Earlier, Kondo et al. [46] identified other CLPs that have the Lin^–Thy-1^–Sca-1^lowc-kit^lowIL-7R⁺ immunophenotype. In Adolfsson's study [25], it was shown that the flt3⁺ KSL cells produced this IL-7R⁺ CLP in vitro as well as in vivo. These results suggested that the flt3⁺ KSL population is distinct and most likely an intermediate between flt3^– KSL (LTR-HSC) and this IL-7R⁺ CLP. Interestingly, flt3⁺ KSL cells were found to be almost exclusively CD34⁺, whereas flt3^– KSL cells contained a small but significant (5%) fraction of CD34^– cells [24]. This notion was further supported by the recent study reported by Sitnicka et al. [26]. In this study, FL-deficient mice had severely (10-fold) reduced levels of CLP, although the numbers of common myeloid progenitors (CMPs) and CD34^– KSL were unaffected [26]. Very recently, Adolfsson et al. [25] have clearly demonstrated that the herein-mentioned flt3⁺ KSL cells sustain granulocyte, monocyte, and B- and T-cell potential, but fail to produce significant erythroid and megakaryocytic progeny. On the basis of these observations, they proposed an alternative road map for adult mouse blood lineage commitment [25].

In contrast to murine LTR-HSC, the expression and functional significance of flt3 and c-kit on human LTR-HSC has yet to be fully elucidated. Earlier studies have shown that most, if not all, long-term culture-initiating cell (LTC-IC) are c-kit⁺ [47, 48]. However, we observed that extended LTC-IC (ELTC-IC; assayed after 7–9 weeks of coculture with allogeneic BM stromal layer) are apparently enriched in a CB-derived CD34⁺c-kit^low/– cell population [21]. Of note was that ELTC-ICs assayed after 9 weeks of coculture were detected only in the CD34⁺c-kit^– cell population [21]. Our data are consistent with several other in vitro studies [49–51]. Sogo et al. [49] have clearly demonstrated that CB-derived CD34⁺c-kit^<low cells mature into CD34⁺c-kit^low and CD34⁺c-kit⁺ cells in vitro, suggesting that the upregulation of c-kit protein on c-kit^<low cells is the first maturational step of human HSCs [49]. Enrichment of human BM-derived primitive HSCs in the CD34⁺c-kit^low fraction was also confirmed using long-term engraftment studies in preimmune fetal sheep [51]. The findings showed that BM-derived CD34⁺c-kit^low cells transplanted to fetal sheep sustained long-term donor-derived hematopoiesis (up to 16 months) [52]. There is, however, no direct evidence yet for a distinct population of c-kit^– or c-kit^<low human primitive HSCs with long-term repopulating potential. Such a stem cell population is likely to be present at very low frequency in human BM- or CB-derived hematopoietic cells. Recently, we identified very primitive CD34^– SRCs in human CB detected only by the IBMI method [29, 30]. As shown in Figure 1D, the CB-derived Lin^–CD34^– cell population did not express detectable levels of c-kit protein by flow cytometry. Therefore, our identified CD34^– SRCs may correspond to such c-kit^– or c-kit^<low LTR-HSCs [29, 30].

In contrast to c-kit, the information regarding flt3 expression on human LTR-HSCs is much more limited. Recently, Sitnicka et al. [31], using the conventional intravenous injection method, clearly demonstrated that human BM- or CB-derived CD34⁺ HSC capable of multilineage engrafting NOD/SCID mice do express flt3 receptors. Moreover, they also showed that CB-derived CD34⁺flt3^– cells could repopulate recipient mouse BMs. On the basis of these data, they proposed that most BM- and CB-derived CD34⁺ SRCs express flt3, and that the expression pattern of flt3 and c-kit receptors on primitive mouse and human HSCs is different and contrasting. However, they did not investigate the secondary repopulating capacity of CD34⁺flt3^+/– cells as well as the repopulation capacity of the CD34^– counterpart.

In this study, we have investigated the SRC activity of CB-derived Lin^–CD34⁺flt3^+/– cells as well as Lin^–CD34^–flt3^+/– cells using the IBMI method. First, we confirmed that CB-derived Lin^–CD34⁺flt3^+/– cells showed distinct SRC activity by IBMI (Fig. 3; Table 1). Interestingly, we demonstrated for the first time that Lin^–CD34^–flt3^– cells showed significant and potent SRC activity by IBMI. Moreover, our secondary transplantation study clearly indicated that the secondary repopulating capacity is most potently observed in CD34^–flt3^– cells in comparison with CD34⁺flt3^+/– cells (Fig. 4). Finally, we observed that these Lin^–CD34^–flt3^– cells could produce CD34⁺flt3^+/– SRCs after being cocultured with HESS-5 cells in the presence of a cocktail of cytokines (Fig. 5). These results suggest that CD34^–flt3^– SRCs are the precursor for CD34⁺flt3^+/– SRCs. On the basis of the results of our present study, we propose that the immunophenotype of very primitive human LTR-HSCs is Lin^–CD34^–c-kit^–flt3^–. Primitive human LTR-HSCs may express lower levels of c-kit and flt3 receptors on their surfaces when they commit to more mature short-term repopulating HSCs (STR-HSCs). It is still unclear whether such a distinct pattern of c-kit and flt3 expression might identify distinct subpopulations of LTR-HSC or STR-HSC within the human HSC hierarchy.

From another point of view, we and many other investigators have planned to expand candidate human HSCs ex vivo using several cytokines, including SCF, FL, TPO, and IL-6/soluble IL-6 receptor (or fusion protein) [53–56]. However, the present study and other reported studies [24–26, 41–43,49–52] have demonstrated/suggested that very primitive LTR-HSCs do not express their receptors, such as c-kit and flt3. Furthermore, Lin^–CD34^–c-kit^–flt3^– cells are still heterogeneous, and putative human LTR-HSC may express other potentially important stem cell molecules. Therefore, for clinical application in the near future, further studies will be required to elucidate the proposed model of the human HSC hierarchy as well as to identify hitherto unidentified molecules that are important (indispensable) for stem cell expansion.

In conclusion, the present study provides evidence that human CB-derived CD34^– SRCs do not express flt3 tyrosine kinase receptors, like murine candidate HSCs CD34^– KSL cells. According to our data, the immunophenotype of human LTR-HSC is Lin^–CD34^–c-kit^–flt3^–. Therefore, further studies will be required to identify positive markers, such as Sca-1 for murine CD34^– KSL cells, for these primitive human LTR-HSCs in the near future.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
We thank Dr. Takeshi Todo of Department of Mutagenesis, Radiation Biology Center, Kyoto University, for his advice on the irradiation of NOD/SCID mice; Kirin Brewery Co. Ltd. (Tokyo) and Ajinomoto Co. Inc. (Yokohama, Japan) for providing the various growth factors used in this study; and Yuko Masai for assistance in preparation of the manuscript. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (Grant number 15039227) and for Scientific Research C (Grant number 15591015) from the Ministry of Education, Science and Culture of Japan, a grant from Haiteku Research Center of the Ministry of Education, a grant from the Science Frontier Program of the Ministry of Education, a grant from the 21st Century Center of Excellence (COE) program of the Ministry of Education, a grant from the Promotion and Mutual Aid Corporation for Private Schools of Japan, a grant from Kansai Medical University (Research Grant B), a grant from the Japan Leukemia Research Foundation, and a grant from the Mitsubishi Pharma Research Foundation.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 

1. Lyman SD, Jacobsen SEW. c-kit ligand and flt3 ligand: Stem/progenitor cell factors with overlapping yet distinct activities. Blood 1998;91:1101–1134.[Free Full Text]

2. Matthews W, Jordan CT, Wiegand GW et al. A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 1991;65:1143–1152.[CrossRef][Medline]

3. Lyman SD, James L, Bos TV et al. Molecular cloning of a ligand for the flt3/flk2 tyrosine kinase receptor: A proliferative factor for primitive hematopoietic cells. Cell 1993;75:1157–1167.[CrossRef][Medline]

4. Hannum C, Culpepper J, Cambell D et al. Ligand for flt3/flk2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs. Nature 1994;368:643–648.[CrossRef][Medline]

5. Zeigler FC, Bennett B, Jordan CT et al. Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells. Blood 1994;84:2422–2430.[Abstract/Free Full Text]

6. Hirayama F, Lyman SD, Clark SC et al. The flt3 ligand supports proliferation of lymphohematopoietic progenitors and early B-lymphoid progenitors. Blood 1995;85:1762–1768.[Abstract/Free Full Text]

7. Jacobsen SEW, Okkenhaug C, Myklebust J et al. The flt3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: Synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factor. J Exp Med 1995;181:1357–1363.[Abstract/Free Full Text]

8. Veiby OP, Jacobsen FW, Cui L et al. The flt3 ligand promotes the survival of primitive hematopoietic progenitor cells with myeloid as well as B lymphoid potential. J Immunol 1996;157:2953–2960.[Abstract]

9. Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 2001;98:14541–14546.[Abstract/Free Full Text]

10. Yang L, Bryder D, Adolfsson J et al. Identification of Lin-Sca1⁺kit⁺CD34⁺Flt3^– short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood 2005;105:2717–2723.[Abstract/Free Full Text]

11. Lyman SD, James L, Johnson L et al. Cloning of the human homologue of the murine flt3 ligand: A growth factor for early hematopoietic progenitor cells. Blood 1994;83:2795–2801.[Abstract/Free Full Text]

12. Muench MO, Roncarolo MG, Menon S et al. Flk-2/flt-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver. Blood 1995;85:963–972.[Abstract/Free Full Text]

13. Shah AJ, Smogorzewska EM, Hannum C et al. Flt3 ligand induces proliferation of quiescent human bone marrow CD34⁺CD38^– cells and maintains progenitor cells in vitro. Blood 1996;87:3563–3570.[Abstract/Free Full Text]

14. Rusten LS, Lyman SD, Veiby OP et al. The flt3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34⁺ bone marrow progenitor cells in vitro. Blood 1996;87:1317–1325.[Abstract/Free Full Text]

15. Rappold I, Ziegler BL, Kohler I et al. Functional and phenotypic characterization of cord blood and bone marrow subsets expressing flt3 (CD135) receptor tyrosine kinase. Blood 1997;90:111–125.[Abstract/Free Full Text]

16. Gotze KS, Ramirez M, Tabor K et al. Flt3^high and flt3^low CD34⁺ progenitor cells isolated from human bone marrow are functionally distinct. Blood 1998;91:1947–1958.[Abstract/Free Full Text]

17. Xiao M, Oppenlander BK, Plunkett JM et al. Expression of flt3 and c-kit during growth and maturation of human CD34⁺CD38^– cells. Exp Hematol 1999;27:916–927.[CrossRef][Medline]

18. Sonoda Y, Sakabe H, Ohmizono Y et al. Synergistic actions of stem cell factor and other burst-promoting activities on proliferation of CD34⁺ highly purified blood progenitors expressing HLA-DR or different levels of c-kit protein. Blood 1994;84:4099–4106.[Abstract/Free Full Text]

19. Sonoda Y, Kimura T, Sakabe H et al. Human flt3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34⁺ blood progenitors expressing different levels of c-kit protein. Eur J Haematol 1997;58:257–264.[Medline]

20. Sakabe H, Kimura T, Zeng ZZ et al. Haematopoietic action of flt3 ligand on cord blood-derived CD34-positive cells expressing different levels of flt3 or c-kit tyrosine kinase receptor: Comparison with stem cell factor. Eur J Haematol 1998;60:297–306.[Medline]

21. Sakabe H, Yahata N, Kimura T et al. Human cord blood-derived primitive progenitors are enriched in CD34⁺c-kit^– cells: Correlation between long-term culture-initiating cells and telomerase expression. Leukemia 1998;12:728–734.[CrossRef][Medline]

22. Krause DS, Fackler MJ, Civin CI et al. CD34: Structure, biology, and clinical utility. Blood 1996;87:1–13.[Free Full Text]

23. Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic cell. Science 1996;273:242–245.[Abstract]

24. Adolfsson J, Borge OJ, Bryder D et al. Upregulation of flt3 expression within the bone marrow Lin^–Sca1⁺c-kit⁺ stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 2001;15:659–669.[CrossRef][Medline]

25. Adolfsson J, Mansson R, Buza-Vidas N et al. Identification of flt3⁺ lympho-myeloid stem cells lacking erythro-megakaryocytic potential: A revised road map for adult blood lineage commitment. Cell 2005;121:295–306.[CrossRef][Medline]

26. Sitnicka E, Bryder D, Theilgaard-Monch K et al. Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool. Immunity 2002;17:463–472.[CrossRef][Medline]

27. Mackarehtschian K, Hardin JD, Moore KA et al. Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity 1995;3:147–161.[CrossRef][Medline]

28. McKenna HJ, Socking KL, Miller RE et al. Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood 2000;95:3489–3497.[Abstract/Free Full Text]

29. Wang J, Kimura T, Asada R et al. SCID-repopulating cell activity of human cord blood-derived CD34^– cells assured by intra-bone marrow injection. Blood 2003;101:2924–2931.[Abstract/Free Full Text]

30. Kimura T, Wang J, Matsui K et al. Proliferative and migratory potentials of human cord blood-derived CD34^– severe combined immunodeficiency repopulating cells that retain secondary reconstituting capacity. Int J Hematol 2004;79:328–333.[Medline]

31. Sitnicka E, Buza-Vidas N, Larsson S et al. Human CD34⁺ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: Distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cell. Blood 2003;102:881–886.[Abstract/Free Full Text]

32. Ebihara Y, Wada M, Ueda T et al. Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice by clonal cells expanded from single CD34⁺CD38^– cells expressing flk2/flt3. Br J Haematol 2002;119:525–534.[CrossRef][Medline]

33. Kimura T, Sakabe H, Tanimukai S et al. Simultaneous activation of signals through gp130, c-kit, and interleukin-3 receptor promotes a trilineage blood cell production in the absence of terminally acting lineage-specific factors. Blood 1997;90:4767–4778.[Abstract/Free Full Text]

34. Kimura T, Wang J, Minamiguchi H et al. Signal through gp130 activated by soluble interleukin (IL)-6 receptor(R) and IL-6 or IL-6R/IL-6 fusion protein enhances ex vivo expansion of human peripheral blood-derived hematopoietic progenitors. STEM CELLS 2000;18:444–452.[Abstract/Free Full Text]

35. Kushida T, Inaba M, Hisha H et al. Intra-bone marrow injection of allogeneic bone marrow cells: A powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood 2001;97:3292–3299.[Abstract/Free Full Text]

36. Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy. Nat Med 1996;2:1329–1337.[CrossRef][Medline]

37. Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4:1038–1045.[CrossRef][Medline]

38. Ogata K, Satoh C, Tachibana M et al. Identification and hematopoietic potential of CD45^– clonal cells with very immature phenotype (CD45^–CD34^–CD38^–Lin^–) in patients with myelodysplastic syndromes. STEM CELLS 2005;23:619–630.[Abstract/Free Full Text]

39. Tsuji T, Ogasawara H, Aoki Y et al. Characterization of murine stromal cell clones established from bone marrow and spleen. Leukemia 1996;10:803–812.[Medline]

40. Osawa M, Nakamura K, Nishi N et al. In vivo self-renewal of c-kit⁺Sca-1⁺Lin^low/– hemopoietic stem cells. J Immunol 1996;156:3207–3214.[Abstract]

41. Ogawa M, Matsuzaki Y, Nishikawa S et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991;174:63–71.[Abstract/Free Full Text]

42. Orlic D, Fischer R, Nishikawa S et al. Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor. Blood 1993;82:762–770.[Abstract/Free Full Text]

43. Doi H, Inaba M, Yamamoto Y et al. Pluripotent hemopoietic stem cells are c-kit^<low. Proc Natl Acad Sci U S A 1997;94:2513–2517.[Abstract/Free Full Text]

44. Ortiz M, Wine JW, Lohrey N et al. Functional characterization of a novel hematopoietic stem cell and its place in the c-kit maturation pathway in bone marrow cell development. Immunity 1999;10:173–182.[CrossRef][Medline]

45. Randall TD, Weissman IL. Characterization of a population of cells in the bone marrow that phenotypically mimics hematopoietic stem cells: Resting stem cells or mystery population? STEM CELLS 1998;16:38–48.[Abstract/Free Full Text]

46. Kondo M, Weissman IL, Akashi K. Ientification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 1997;91:661–672.[CrossRef][Medline]

47. Simmons PJ, Aylett GW, Niutta S et al. C-kit is expressed by primitive human hematopoietic cells that give rise to colony-forming cells on stroma-dependent or cytokine-supplemented culture. Exp Hematol 1994;22:157–165.[Medline]

48. Briddell RA, Broudy VC, Bruno E et al. Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a monoclonal antibody to the c-kit receptor. Blood 1992;79:3159–3167.[Abstract/Free Full Text]

49. Sogo S, Inaba M, Ogata H et al. Induction of c-kit molecules on human CD34⁺/c-kit^<low cells: Evidence for CD34⁺/c-kit^<low cells as primitive hematopoietic stem cells. STEM CELLS 1997;15:420–429.[Abstract/Free Full Text]

50. Gunji Y, Nakamura M, Osawa H et al. Human primitive hematopoietic progenitor cells are more enriched in KIT^low cells than in KIT^high cells. Blood 1993;82:3283–3289.[Abstract/Free Full Text]

51. Laver JH, Abboud MR, Kawashima I et al. Characterization of c-kit expression by primitive hematopoietic progenitors in umbilical cord blood. Exp Hematol 1995;23:1515–1519.[Medline]

52. Kawashima I, Zanjani ED, Almaida-Porada G et al. CD34⁺ human marrow cells that express low levels of kit protein are enriched for long-term marrow-engrafting cells. Blood 1996;87:4136–4142.[Abstract/Free Full Text]

53. Ohmizono Y, Sakabe H, Kimura T et al. Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand. Leukemia 1997;11:524–530.[CrossRef][Medline]

54. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997;186:619–624.[Abstract/Free Full Text]

55. Conneally E, Cashman J, Petzer A et al. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 1997;94:9836–9841.[Abstract/Free Full Text]

56. Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, flk2/flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest 2000;105:1013–1021.[Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: 2006-0727v1 25/6/1348    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions