Identification of Long-Term Repopulating Potential of Human Cord Blood-Derived C - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:54 |只看该作者 |倒序浏览 |打印

作者：Takafumi Kimuraa, Rumiko Asadaa, Jianfeng Wanga, Takashi Kimuraa,g, Miho Moriokaa, Kazuo Matsuib, Katsuya Kobayashic, Kae Henmic, Shiro Imaic, Masakazu Kitad, Takashi Tsujie, Yutaka Sasakia, Susumu Ikeharaf, Yoshiaki Sonodaa作者单位：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;dD 7 X( @$ P: w! s- e3 h

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      【摘要】& Q: s8 Z9 }! a. b9 r: U' }+ W
   Recently, we have identified human cord blood (CB)-derived CD34-negative (CD34¨C) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003;101:2924). In contrast to murine CD34¨C Kit Sca-1 Lineage¨C (KSL) cells, human CB-derived Lin¨CCD34¨C 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¨C SRCs. Both CD34 flt3 /¨C cells showed SRC activity. In the CD34¨C cell fraction, only CD34¨Cflt3¨C cells showed distinct SRC activity by IBMI. Although CD34 flt3  cells showed a rather weak secondary repopulating activity, CD34 flt3¨C cells repopulated many more secondary recipient mice. However, CD34¨Cflt3¨C cells repopulated all of the secondary recipients, and the repopulating rate was much higher. Next, we cocultured CD34¨Cflt3¨C cells with the murine stromal cell line HESS-5. After 1 week, significant numbers of CD34 flt3 /¨C cells were generated, and they showed distinct SRC activity. These results indicated that CB-derived CD34¨Cflt3¨C cells produced CD34 flt3¨C as well as CD34 flt3  SRCs in vitro. The present study has demonstrated for the first time that CB-derived CD34¨C SRCs, like murine CD34¨C 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¨CCD34¨Cc-kit¨Cflt3¨C.* q4 }# O9 S) \3 F% h/ C* k
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Disclosure of potential conflicts of interest is found at the end of this article. * _7 [: H6 H  e, M" \3 Z
      【关键词】 Flt Severe combined immunodeficiency-repopulating cell Intra-bone marrow injection Cord blood Hematopoiesis
   INTRODUCTION' {- L# \$ B* V) t1 G1 L) {
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It is well documented that the tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse ., w. l6 W. T% H  C* m5 n
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Recently, using the intra-bone marrow injection (IBMI) method, we have successfully identified human cord blood (CB)-derived CD34-negative (CD34¨C) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability , our identified CD34¨C 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¨C 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 .
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In this study, we have investigated, using the IBMI method, the function of flt3, which is expressed in early mouse  and provide a new concept of hierarchy in the human primitive HSC compartment.& {/ Y( G0 e% X$ k  _

MATERIALS AND METHODS8 K" ]" R% u7 h% S

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.  y  _+ ^" y4 t; f! l
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Purification of Lin¨CCD34 flt3 /¨C and Lin¨CCD34¨Cflt3 /¨C Cells
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The above-mentioned lineage-negative (Lin¨C) 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 , 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).
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Clonal Cell Culture

Human colony-forming cells (CFCs) were assayed using our standard methylcellulose cultures as reported previously . 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.
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IBMI of Purified Cells
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IBMI was carried out as reported previously . 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.7 g! p" l% R. n& `3 o; c9 N3 g5 q
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SCID-Repopulating Cell Assay/ R' k0 f2 ?0 @1 \5 j; l* A) P

An SRC assay was performed using the methods reported previously . Therefore, we used the IBMI technique to analyze SRC activities of Lin¨CCD34 /¨CFlt3 /¨C cells in this study. The mice were killed 8¨C12 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 .1 J) N% G0 ?1 I! D
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Analysis of Human Cell Engraftment in NOD/SCID Mice by Flow Cytometry
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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.- t- t1 ]0 E2 q  m3 k( @$ X; v1 ?5 D
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Secondary Transplantation3 @" j' ^& }2 I7 ~; U2 C" g1 D" q

For secondary transplantations, murine BM cells were obtained from the pairs of femurs, tibiae, and humeri of moderately engrafted primary recipient mice 8¨C12 weeks after transplantation with 3 x 103 to 5 x 103 Lin¨CCD34 flt3 , 4 x 103 to 5 x 103 Lin¨CCD34 flt3¨C, or 2 x 104 to 3 x 104 Lin¨CCD34¨Cflt3¨C cells, respectively. The human cell repopulation rates in the primary recipients' BMs were comparable and approximately 4%¨C8%. 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.! d6 w6 T  ^  Z9 o& l

Coculture with HESS-5 Cells and SRC Activity of Culture-Generated CD34 flt3 /¨C Cells
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A total of 5 x 104 purified Lin¨CCD34¨Cflt3¨C cells per 12.5-cm2 culture flask (BD Falcon; Becton Dickinson) onto preestablished irradiated HESS-5  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 /¨C cells were analyzed by flow cytometry. Simultaneously, these CD34 flt3 /¨C 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¨C 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.
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Statistical Analysis
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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.! b+ l2 X7 Y2 `$ L+ [+ e3 |: C
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RESULTS& \+ S9 W% d# T3 i

Expression of flt3 and c-kit Receptors on Lin¨CCD45 CD34 /¨C Cells

First, we depleted the eight lineage-positive cells from CB-derived MNCs using the immunomagnetic beads system . Then, Lin¨CCD45 CD34 /¨C cells were gated as R2 as shown in Figure 1B. These Lin¨CCD45  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¨CCD34¨C cells did not express detectable levels of c-kit receptors by flow cytometry, as shown in Figure 1D.; k# [4 `, w. O8 U
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Figure 1. Expression of flt3 or c-kit receptor on cord blood-derived Lin¨C cells. (A): The forward scatter/SSC profile of immunomagnetically separated Lin¨C cells. The R1 gate was set on the lymphocyte window. (B): Lin¨CCD45 CD34 /¨C 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¨CCD34 flt3 , Lin¨CCD34 flt3¨C, Lin¨CCD34¨Cflt3 , and Lin¨CCD34¨Cflt3¨C 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¨CCD34 /¨C cells in a separate experiment. Abbreviations: FITC, fluorescein isothiocyanate; FSC, forward scatter; PE, phycoerythrin; SSC, side scatter.

Characteristics of Colony-Forming Capacity by CB-Derived Lin¨CCD34 /¨Cflt3 /¨C Cells

The colony-forming capacities of these four fractions were quite different. The plating efficiency of each Lin ¨CCD34 flt3  or Lin¨CCD34 flt3¨C cell fraction was approximately 50% and comparable (Fig. 2A). Lin¨CCD34 flt3  cells contained approximately 81% CFU-GM, 17% BFU-E, and 2% CFU-Mix. In contrast, Lin¨CCD34 flt3¨C cell fraction contained 21% CFU-GM, 66% BFU-E, and 12% CFU-Mix. The Lin¨CCD34¨Cflt3 /¨C cell fractions showed almost no colony formation (data not shown). On the other hand, the vast majority of CFU-Megs (more than 90%) were detected in the Lin¨CCD34 flt3¨C cell fraction (Fig. 2B).
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Figure 2. Colony-forming capacities of Lin¨CCD34 flt3 /¨C cells. (A): The colony-forming capacities of 200 Lin¨CCD34 flt3 /¨C 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¨CCD34 flt3 /¨C 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
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These results clearly demonstrate that CB-derived Lin¨CCD34 flt3  cells display weak erythroid and megakaryocytic potentials. These findings were consistent with a recent study in which murine flt3  KSL cells failed to produce significant erythroid and megakaryocytic progeny .
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SRC Activity and Lymphomyeloid-Reconstituting Capacity of CB-Derived Lin¨CCD34 /¨Cflt3 /¨C Cells by IBMI0 v1 F1 @( Z# H7 w( G

In this study, we have investigated the function of flt3 in our identified human CB-derived CD34 ¨C SRCs. First, we studied the SRC activity of CB-derived Lin¨CCD34 flt3 /¨C or CD34¨Cflt3 /¨C cells using IBMI, as shown in Figure 3. Both CD34 flt3 /¨C 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¨C cells (n = 10; 0.3% to 45.1%; median, 16.9%).

Figure 3. Severe combined immunodeficiency-repopulating cell activities of Lin¨CCD34 /¨Cflt3 /¨C cells by intra-BM injection (IBMI). (A): Each mouse transplanted with designated numbers of cord blood-derived Lin¨CCD34 flt3 , Lin¨CCD34 flt3¨C, Lin¨CCD34¨Cflt3 , and Lin¨CCD34¨Cflt3¨C cells was sacrificed 8¨C12 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¨CD): The human CD45  cell reconstitution in the representative mouse presented in (A) received transplants of CD34 flt3  (B), CD34 flt3¨C (C), and CD34¨Cflt3¨C (D) cells, respectively. Percentages of cells in each quadrant are presented in the upper left corner. Abbreviations: BM, bone marrow; PE, phycoerythrin.% n4 J0 ]8 [+ A

On the other hand, the seven mice that received transplants of CD34¨Cflt3  cells did not show human cell repopulation. Only CD34¨Cflt3¨C cells repopulated all seven recipient mice, and the level of human CD45  cells in the murine BMs was 4.1% to 63.3% (median, 37.9%). These results indicated for the first time that the CB-derived Lin¨CCD34¨Cflt3¨C cell population contained SRCs, as detected by IBMI.$ W' x  Z  |5 a# a2 X

To further evaluate the function of flt3 expression in CD34  and CD34¨C 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¨CCD34 flt3 /¨C cells or 5 x 104 to 7 x 104 Lin¨CCD34¨Cflt3¨C cells by IBMI showed signs of human cell engraftment. The analyses of the three representative mice transplanted either with Lin¨CCD34 flt3 /¨C cells or Lin¨C CD34¨Cflt3¨C 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).

Table 1. Lymphomyeloid reconstitution abilities of CD34 flt3 /¨C and CD34¨Cflt3¨C severe combined immunodeficiency repopulating cells by intra-bone marrow (BM) injection2 r% Q2 P: X+ k% ?$ n5 R* P
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Next, the percentages of lineage-positive cells expressing CD19 and CD33 were compared (Table 1). These results demonstrated that all three classes of SRCs could supply lymphoid as well as myeloid cells at 8¨C12 weeks after the transplantation. Interestingly, CD34 flt3  SRCs showed a lymphoid-dominant repopulation pattern compared with the other two classes of SRCs. These results are consistent with the notion that cells in the Lin¨CSca-1 c-kit  murine HSC compartment coexpressing flt3 tyrosine kinase receptor sustain lymphoid potential .8 b/ W4 T  y5 ^/ H

Secondary Repopulating Ability of Lin¨CCD34  flt3 /¨C or Lin¨CCD34¨Cflt3¨C Cells by IBMI3 [, l2 z& N) J* P% P3 Z9 k. m
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To further evaluate the long-term repopulating potential of these three populations (CD34 flt3 , CD34 flt3¨C, and CD34¨Cflt3¨C 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¨C cells could be repopulated. Moreover, all five secondary recipient mice that received whole BM cells from primary recipients that received CD34¨Cflt3¨C cells could be repopulated with a higher secondary repopulating rate (Fig. 4). These results demonstrated that CD34¨Cflt3¨C 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.+ `6 D, ?8 A8 W4 l

Figure 4. Secondary repopulating capacities of Lin¨CCD34 flt3 /¨C or Lin¨CCD34¨Cflt3¨C 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¨C cells, or 2 x 104 to 3 x 104 CD34¨Cflt3¨C cells. Human cell repopulations of BMs in PRs (open circles) analyzed 8¨C12 weeks after transplantation were comparable and 4%¨C8%. Whole BM cells obtained from PRs were transplanted to secondary recipients (SRs) by IBMI. Human cell repopulation in SRs (closed circles) was analyzed 8¨C10 weeks after secondary transplantation. Horizontal bars represent each median of the repopulation rates in PRs and SRs, respectively. Abbreviation: BM, bone marrow.
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SRC Activity of Culture-Generated CD34 flt3 /¨C Cells by IBMI6 T9 G( R9 A& F2 h& N' m

Recently, we reported that our identified CD34¨C SRCs could produce CD34  SRCs after being cocultured with the murine stromal cell line HESS-5 . After 1 week, significant numbers of CD34 flt3¨C and CD34 flt3  cells were generated, as shown in Figure 5C. We then sorted these two populations (CD34 flt3 /¨C cells) and tested their SRC activities by IBMI. Seven of 10 and 5 of 10 mice that received either CD34 flt3  or CD34 flt3¨C cells were repopulated with human cells. (Table 2). Human cell repopulation rates in mice that received transplants of either CD34 flt3  or CD34 flt3¨C cells were 1.2%¨C8.8% (median, 4.5%) and 1.4%¨C7.8% (median, 3.7%), respectively.( p$ t4 Y. X4 H, U' C* E. q
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Figure 5. Expression pattern of CD34 and flt3 on sorted Lin¨CCD34¨Cflt3¨C cells after the 7-day coculture with HESS-5 cells. (A): Flow cytometry pattern of immunomagnetically separated cord blood-derived Lin¨C cells stained with anti-flt3 (PE) and anti-CD34 (PC5) monoclonal antibodies. Lin¨CCD34¨Cflt3¨C 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¨CCD34¨Cflt3¨C cells. (C): The expression pattern of flt3 on CD34  cells derived from the 7-day cocultures of sorted Lin¨CCD34¨Cflt3¨C 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 /¨C cells are indicated by two solid squares. Abbreviation: PE, phycoerythrin.
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Table 2. Severe combined immunodeficiency (SCID) repopulating cell activity of culture-generated CD34 flt3 /¨C cells8 s1 {3 f( O7 F; J! }
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DISCUSSION
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A number of studies have demonstrated that flt3 tyrosine kinase receptor plays a pivotal role in the regulation of primitive murine  reported that c-kit¨C pluripotent stem cells can give rise to c-kit  cells with colony-forming unit in spleen (CFU-S) activity, suggesting that c-kit  HSCs are recruited from a more primitive quiescent c-kit¨C HSC population. Collectively, these reported studies suggest that most of the murine LTR-HSCs express a low to high level of c-kit on their surfaces, but also that there is a less frequent subpopulation expressing less than a low level of c-kit coexisting in murine BMs.
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On the other hand, the flt3 receptor has also been shown to be expressed and to function in murine candidate HSCs .

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  .
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In contrast to c-kit, the information regarding flt3 expression on human LTR-HSCs is much more limited. Recently, Sitnicka et al. , 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¨C 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 /¨C cells as well as the repopulation capacity of the CD34¨C counterpart.
      【参考文献】

Lyman SD, Jacobsen SEW. c-kit ligand and flt3 ligand: Stem/progenitor cell factors with overlapping yet distinct activities. Blood 1998;91:1101¨C1134.

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¨C1152.1 A/ r" q. V4 T2 i+ n
1 J5 F3 H& r4 ?1 g7 s* C/ u$ d
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¨C1167.

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¨C648.
) n# v* B- M. c
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¨C2430.

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¨C1768.

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¨C1363.  g( E4 t6 f9 D7 |+ Z
, P8 R1 w8 z) r% W, o# s
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¨C2960.

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¨C14546.: O: Q* h. o+ _
; ^; s" k/ Q. D
Yang L, Bryder D, Adolfsson J et al. Identification of Lin-Sca1 kit CD34 Flt3¨C short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood 2005;105:2717¨C2723.

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¨C2801.

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¨C972.
# @( ~1 l1 ~+ {0 Z" p' t
Shah AJ, Smogorzewska EM, Hannum C et al. Flt3 ligand induces proliferation of quiescent human bone marrow CD34 CD38¨C cells and maintains progenitor cells in vitro. Blood 1996;87:3563¨C3570.

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¨C1325.8 t0 ]/ e' }6 U3 o# m0 j' ?$ s
. H, P6 |0 ?  T1 g. ?. u
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¨C125.* |4 N& L; k+ Q

Gotze KS, Ramirez M, Tabor K et al. Flt3high and flt3low CD34  progenitor cells isolated from human bone marrow are functionally distinct. Blood 1998;91:1947¨C1958.) o3 R6 w9 x- ?
* u& c3 u7 ^0 b
Xiao M, Oppenlander BK, Plunkett JM et al. Expression of flt3 and c-kit during growth and maturation of human CD34 CD38¨C cells. Exp Hematol 1999;27:916¨C927.9 A3 y0 [* c$ _& a  t+ o& t, |
* _! a$ E! Y' t& A: {# r4 r
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¨C4106.

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¨C264.! m  b- _5 d  y& Y' U, s

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¨C306.7 {- {3 S! {% A2 b
7 k8 H: e8 {/ p3 [- j1 M9 N
Sakabe H, Yahata N, Kimura T et al. Human cord blood-derived primitive progenitors are enriched in CD34 c-kit¨C cells: Correlation between long-term culture-initiating cells and telomerase expression. Leukemia 1998;12:728¨C734.

Krause DS, Fackler MJ, Civin CI et al. CD34: Structure, biology, and clinical utility. Blood 1996;87:1¨C13.

Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic cell. Science 1996;273:242¨C245.8 b9 r( x  Z6 _! Y( t
1 u. X$ p1 K7 v5 z+ J5 _
Adolfsson J, Borge OJ, Bryder D et al. Upregulation of flt3 expression within the bone marrow Lin¨CSca1 c-kit  stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 2001;15:659¨C669.

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¨C306.' {- h' W4 t1 l( n0 |9 k, L

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¨C472.

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¨C161.
7 c1 G7 {' }- k8 n
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¨C3497.

Wang J, Kimura T, Asada R et al. SCID-repopulating cell activity of human cord blood-derived CD34¨C cells assured by intra-bone marrow injection. Blood 2003;101:2924¨C2931.
( H% E# K/ P" m+ G7 R3 O3 T/ ]
Kimura T, Wang J, Matsui K et al. Proliferative and migratory potentials of human cord blood-derived CD34¨C severe combined immunodeficiency repopulating cells that retain secondary reconstituting capacity. Int J Hematol 2004;79:328¨C333.& n1 ]4 M, m. t8 f+ P
* j) o+ J+ k% h9 J) U/ c8 A' ~
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¨C886.. C* m7 d- G& k+ i" X

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¨C cells expressing flk2/flt3. Br J Haematol 2002;119:525¨C534./ J: Q. l6 V  W. Z1 [8 t

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¨C4778.8 i7 \6 X& y* D" V! e

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¨C452.) a0 l) |6 E, |1 K' y) Y
# Z* Q' T4 b* S2 s
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¨C3299.
  C$ d4 J/ x2 k0 p# [
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¨C1337.

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¨C1045.
3 C3 F1 |7 ]" Z
Ogata K, Satoh C, Tachibana M et al. Identification and hematopoietic potential of CD45¨C clonal cells with very immature phenotype (CD45¨CCD34¨CCD38¨CLin¨C) in patients with myelodysplastic syndromes. STEM CELLS 2005;23:619¨C630.
; d, t) a9 k! i
Tsuji T, Ogasawara H, Aoki Y et al. Characterization of murine stromal cell clones established from bone marrow and spleen. Leukemia 1996;10:803¨C812.
' C% w+ V& f! D& o- p$ q4 u
Osawa M, Nakamura K, Nishi N et al. In vivo self-renewal of c-kit Sca-1 Linlow/¨C hemopoietic stem cells. J Immunol 1996;156:3207¨C3214.
+ y: N, m! P" N. m" i, n
Ogawa M, Matsuzaki Y, Nishikawa S et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991;174:63¨C71.  U" V0 y; @5 ]+ B
$ l3 L: b: T. d' v1 K0 e; r
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¨C770.

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¨C2517.

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¨C182.

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¨C48.0 S+ c+ S- i4 l
' O8 O0 R6 T, r+ t
Kondo M, Weissman IL, Akashi K. Ientification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 1997;91:661¨C672.

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¨C165.' a9 X1 f2 W5 \- B: r+ f/ w- l. [# L

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¨C3167.! G2 u6 O0 a+ [) \
- X! J2 g" H  U  E' X! T# L
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¨C429.+ D2 r! R8 |! Q: y
% T# t& }$ @, }/ b4 Q$ G- ]6 h
Gunji Y, Nakamura M, Osawa H et al. Human primitive hematopoietic progenitor cells are more enriched in KITlow cells than in KIThigh cells. Blood 1993;82:3283¨C3289.- L& b$ r7 O' ?1 {4 {9 G2 f

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¨C1519.+ c+ _/ F# ]& J  b) t6 P6 g
2 j2 M3 b5 N. P( c1 |6 H
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¨C4142.2 I7 t7 ^2 f$ [( r. z
/ i5 o; {0 ^3 j/ _4 S: y
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¨C530.' H( h% X0 }+ e( [, N$ l: {
! e8 h! \1 }" m
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¨C624.+ n& K8 J5 F3 C6 k) M" Z8 M( n; d' t
8 i% ?$ Q. c: x  L
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¨C9841.

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¨C1021.

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