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Hematopoietic Repopulating Ability of Cord Blood CD34⁺ Cells in NOD/Shi-scid Mice

^a Department of Clinical Oncology and
^b Department of Molecular Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan;
^c Central Institute for Experimental Animals, Kawasaki, Japan;
^d Department of Pediatrics, Kyoto University, Kyoto, Japan

Key Words. Hematopoietic repopulating ability • Cord blood • CD34⁺ cells • NOD/Shi-scid mice • Hematopoietic stem cells • Hematopoietic stem cell transplantation

Correspondence: Kohichiro Tsuji, M.D., Department of Clinical Oncology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Telephone: +81-3-5449-5397; Fax: +81-3-5449-5428; e-mail: tsujik{at}ims.u-tokyo.ac.jp

	ABSTRACT

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Although umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable hematopoietic stem cells (HSC), information on the hematopoietic repopulating ability of CB HSC is still limited. We recently established a xenotransplantation system in NOD/Shi-scid mice to evaluate human stem cell activity. In the present study, we transplanted 5 to 10 x 10⁴ CB CD34⁺ cells into six NOD/Shi-scid mice treated with anti-asialo GM1 antiserum to investigate the hematopoietic repopulating ability of CB. The BM of all recipients contained human CD45⁺ cells 10 to 12 weeks after the transplantation (43.8 ± 17.7%). Clonal culture of the recipient BM cells revealed the formation of various types of human hematopoietic colonies, including myelocytic, erythroid, megakaryocytic, and multilineage colonies, indicating that CB HSC can differentiate into hematopoietic progenitors of various lineages. However, the extent of the differentiation and maturation differed with each lineage. CD13⁺/CD14⁺/CD33⁺ myelocytic cells were mainly repopulated in BM and peripheral blood (PB). While CD41⁺ megakaryocytic cells and platelets were present, few glycophorin A⁺CD71⁺ or hemoglobin -containing erythroid cells were detected. CD19⁺ B cells were the most abundantly repopulated in NOD/Shi-scid mice, but their maturational stage differed among the hematopoietic organs. Most of the BM CD19⁺ cells were immature B cells expressing CD10 but not surface immunoglobulin (Ig) M, whereas more mature CD19⁺CD10^– surface IgM⁺ B cells were predominantly present in spleen and PB. CD3⁺ T cells were not detected even in the recipient thymus. The transplantation to the NOD/Shi-scid mouse may provide a useful tool for evaluating the repopulating ability of transplantable human HSC.

	INTRODUCTION

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Clinically transplantable hematopoietic stem cells (HSC) should prove to have a long-term repopulating ability. Until recently, most of the human HSC studies aimed at clinical application have used in vitro assays for CD34⁺ cells, colony-forming cells in clonal culture, cobblestone area-forming cells, and long-term culture-initiating cells [1-5], but these surrogate assays have been shown not to correctly reflect stem cell activity. However, the recent development of assays measuring the ability to reconstitute human hematopoiesis in ontologically or genetically immunodeficient animals has enabled investigators to evaluate the stem cell activity [6-8]. The NOD/LtSz-scid mouse strain was reported to have a variety of immunological abnormalities such as T- and B-cell deficiency, defective natural killer (NK) cell activity, macrophage dysfunction, and absence of circulating complements [9]. Recently, we also established a xenotransplantation system for human HSC, using NOD/Shi-scid mice, which showed a similar phenotype except the defective NK cell activity [10]. When treated with anti-asialo GM1 antiserum to delete NK cells, the NOD/Shi-scid mice exhibited efficient engraftment of human HSC [11, 12].

Umbilical cord blood (CB) is increasingly being used as an alternative to bone marrow (BM) as a source of transplantable HSC for treatment of various hematological disorders [13-15]. However, information on the hematopoietic repopulating ability of CB HSC is still limited. In the present study, we transplanted CB CD34⁺ cells into NOD/Shi-scid mice treated with anti-asialo GM1 antiserum to investigate the hematopoietic repopulating ability of CB. The result demonstrated that CB CD34⁺ cells could reconstitute the multilineages of human hematopoiesis in NOD/Shi-scid mice, although the extent of the differentiation and maturation was different in each lineage of cells. This mouse model may prove a useful preclinical tool for assaying human HSC.

	MATERIALS AND METHODS

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Mice
Experimental NOD/Shi-scid mice [10] were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). The NOD/Shi-scid mice were kept in microisolator cages on laminar flow racks in a clean experimental room. They were maintained on an irradiated, sterile diet and given autoclaved, acidified water.

CD34⁺ Cell Purification
CB was obtained during normal full-term deliveries after obtaining informed consent. Mononuclear cells (MNC) were separated by Ficoll-Hypaque density gradient centrifugation after depletion of phagocytes with Silica (Immuno Biological Laboratories; Fujioka, Japan). The MNC were resuspended at 3 to 5 x 10⁷ cells/ml in phosphate-buffered saline (PBS) and mixed with Dynabeads M-450 CD34 (Dynal AS; Oslo, Norway; http://www.dynal.no), with a bead-to-cell ratio of 1:1. The cell-beads suspension was resuspended and incubated at 4°C for 30 min with gentle rotation. After the incubation, the cell-beads volume was expanded and placed in a DYNAL MPC (magnetic particle concentrator) to collect the Dynabeads M-450 CD34-rosetted cells. The rosetted cells were incubated with DETACHaBEAD CD34 (Dynal) at room temperature for 45 min for the detachment of Dynabeads M-450 CD34 from the positively selected cells. The released CD34⁺ cells were collected by placing the tube in the MPC, and their purity was evaluated by flow cytometric analysis. Approximately 95% of the separated cells were CD34⁺.

Transplantation into NOD/Shi-scid Mice
Xenotransplantation of the purified CD34⁺ cells was performed by a modification of the method previously described [11]. Briefly, 5 to 10 x 10⁴ CD34⁺ cells were injected into 8- to 10-week-old NOD/Shi-scid mice irradiated with 240 rads (⁶⁰Co) of total irradiation through the tail vein. Because the NK cell activity of the NOD/Shi-scid mice we used is not completely impaired [10], the recipient mice were injected intraperitoneally with 400 µl of PBS containing 20 µl of anti-asialo GM1 antiserum (Wako; Osaka, Japan) immediately before the cell transplantation to delete NK cells. Identical treatments were performed on days 11, 22, and 33 postinfusion of experimental cells. Mice were killed in a CO₂ chamber 10 to 12 weeks after the transplantation. Peripheral blood (PB) was collected by puncture of the tail vein or from pools formed in the chest cavity after severing the dorsal aorta into heparinized tubes. For preparation of platelets, PB was drawn into 40-mM D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK; Sigma; St Louis, MO) and gently mixed. Platelet rich plasma (PRP) was prepared by centrifuging the whole PB at 200 g for 20 min and aspirating PRP [16]. Femurs and tibiae were collected and aspirated with PBS containing 5% fetal bovine serum ([FBS]; Hyclone; Logan, UT; http://www.hyclone.com) to liberate BM cells. Cell suspensions were then filtered through a sterile 40-µm cell strainer (#2340; Falcon; Lincoln Park, NJ) to get rid of clumps and debris, and processed for flow cytometric analysis and clonal culture assay. The spleen and thymus from each mouse was harvested and strained through a sterile 70-µm cell strainer (#2350; Falcon) into 5% FBS-containing PBS to collect cells.

Flow Cytometric Analysis of Transplanted NOD/Shi-scid Mice
Surface markers on human hematopoietic cells reconstituted in BM, spleen, PB, and thymus of NOD/Shi-scid mice were analyzed by flow cytometry using FACSCalibur (Becton Dickinson; Mountain View, CA; http://www.bd.com) as described [11]. Briefly, after depletion of erythrocytes with Lysing Solution (Nichirei Co.; Tokyo, Japan), 5 x 10⁵ mouse BM, spleen, PB, and thymus cells suspended with 200 µl of PBS containing 2% FBS were stained with monoclonal antibodies (mAb). All antibody incubations were carried out for 30 min on ice. The presence of human hematopoietic cells was determined by detection of cells positively stained with fluorescein isothiocyanate (FITC)-conjugated anti-human CD45 in flow cytometric analysis. Successful engraftment by human hematopoietic cells was defined by the presence of at least 1% of human CD45⁺ cells in NOD/Shi-scid mouse BM or PB cells 10 to 12 weeks after the transplantation. In all recipients whose BM cells contained more than 1% CD45⁺ cells, human ALU sequences were detected in DNA extracted from the BM by polymerase chain reaction (PCR) analysis as mentioned below. Specific subsets of human hematopoietic cells were quantified by gating on human CD45-phycoerythrin-cyanine 5-succinimidylester (PE-Cy5)-positive cells and then assessing staining with anti-human CD34-FITC, CD10-FITC, CD3-FITC, immunoglobulin (Ig) M-FITC, CD8-FITC, CD33-PE, CD19-PE, CD13-PE, CD14-PE, CD4-PE, and CD41-PE. The expression of human CD41 was also assessed with NOD/Shi-scid mouse platelets in PRP. Nucleated human erythroid cells were assessed by quantifying human CD71-FITC/glycophorin A (GpA)-PE in NOD/Shi-scid mouse BM. All antibodies were from Becton Dickinson (San Jose, CA) except for anti-human CD3-FITC, CD41-PE, GpA-PE, CD45-PE-Cy5 (Immunotech; Marseille, France), and IgM-FITC (Dako; Osaka, Japan; http://www.dako.dk). For each mouse analyzed, an aliquot of cells was also stained with mouse IgG conjugated to FITC, PE, or PE-Cy5 as an isotype control.

Immunostaining
Immunostaining with the alkaline phosphatase anitalkaline phosphatase (APAAP) method using mAb of anti-human hemoglobin was performed as described previously [17]. Briefly, cytocentrifuged samples from PB were fixed with buffered formalin-acetone at 4°C, washed with Tris buffer saline (Wako), and preincubated with normal rabbit serum to saturate the Fc receptors on the cell surface. After washing, the samples were successively incubated with mouse mAb and rabbit anti-mouse IgG Ab (Medical and Biological Laboratories; Nagoya, Japan), and then reacted with calf intestinal alkaline phosphatase-mouse monoclonal antialkaline phosphatase complex (Dako). Alkaline phosphatase activity was detected with naphthol AS-TR phosphate sodium salt (Sigma) and fast red TR salt (Sigma) in pH 7.6, 40 mmol/l barbital buffer (Wako) containing levamisole (Sigma) to inhibit nonspecific alkaline phophatase activity. Positive cells were stained with reddish granules.

Clonal Culture
BM cells of engrafted NOD/Shi-scid mice were incubated in triplicate at a concentration of 1.5 x 10⁵ cells/ml in methylcellulose culture as previously reported [18, 19]. One milliliter of a mixture containing cells, -medium (Flow Laboratories; Rockville, MD), 0.9% methylcellulose (Shinetsu Chemical Co.; Tokyo, Japan; http://www.shinetsu.co.jp), 15% FBS, 1% deionized fraction V bovine serum albumin (Sigma), 5 x 10^–5 M mercaptoethanol (Eastman Organic Chemicals; Rochester, NY), and a combination of cytokines including human stem cell factor (SCF; 100 ng/ml), interleukin 6 (IL-6) (100 ng/ml), IL-3 (20 ng/ml), GM-CSF (10 ng/ml), and erythropoietin (EPO; 2 U/ml) were plated in each 35-mm standard nontissue culture dish (Nunc; Roskilde, Denmark; http://www.nalgenunc.com). Recombinant human IL-3, EPO, and GM-CSF were generous gifts from Kirin Brewery (Tokyo, Japan; http://www1.kirin.co.jp/english/r_d/pha/index.html). Recombinant human SCF and IL-6 were kindly provided by Amgen Biologicals (Thousand Oaks, CA; http://www.amgen.com), and Tosoh Co. (Ayase, Japan; http://www.tosoh.com), respectively. The dishes were incubated at 37°C in a humidified atmosphere flushed with 5% CO₂ in air. Colonies were scored at day 14 of culture according to criteria reported previously [20]. The abbreviations used for the colony types are as follows: G = granulocyte colonies; M = macrophage colonies; GM = granulocyte/macrophage colonies; E = erythroid bursts; and MIX = mixed hematopoietic colonies. G and M colonies were included in GM colonies in some experiments.

BM cells of engrafted NOD/Shi-scid mice were separately assessed for their content of human megakaryocytic (MK) colony-forming cells by plating aliquots in serum-free agarose culture containing human thrombopoietin (50 ng/ml), IL-6 (10 ng/ml), and IL-3 (10 ng/ml), using the MegaCult^TM-C Kit (#4971; StemCell Technologies Inc.; Vancouver, Canada; http://www.escult.com) [21]. After 10 to 12 days of incubation at 37°C in a humidified atmosphere flushed with 5% CO₂, 5% O₂, and 90% N₂, colonies containing CD41 (GPIIbIIIa)-positive megakaryocytes were identified by APAAP staining of cultures fixed in 1:3 methanol in acetone.

Quantitation of Human Immunoglobulin Level
Serum was collected from the chest cavity from individual engrafted mice and levels of human Ig were determined by enzyme-linked immunosorbent assay [22]. Plates were coated with goat anti-human Ig (IgG, IgM) Ab (Cappel; Durham, NC). Alkaline phosphatase-labeled goat anti-human Ig (IgG, IgM) Ab (Cappel) was used as the second layer. Human Ig levels were determined from human Ig standard (The Binding Site; Birmingham, UK; http://www.bindingsite.co.uk) curves run with each assay.

PCR Analysis
The detection of human ALU sequences in DNA was performed by PCR analysis [11, 23]. DNA extracted from BM cells of recipient mice or the colonies in clonal culture was subjected to PCR amplification using a pair of oligonucleotide primers: ALU-5, CACCTGTAATCCCAGCAGTTT-3; ALU-3, CGCGATCTCGGCTCACTGCA. Samples were denatured at 94°C for 4 min, then amplified by rounds consisting of 94°C for 1 min (denaturing), 55°C for 45 sec (annealing), and 72°C for 1 min (extension) for 21 cycles. Products were separated on a 3.0% agarose gel, stained with ethidium bromide, and photographed.

Statistical Analysis
Data are presented as the mean ± standard deviation. The statistical significance of the data was determined by Student's t-test. The significance level was set at 0.05.

	RESULTS

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Engraftment of Human Hematopoietic Cells in BM of NOD/Shi-scid Mice
To study the repopulating ability of CB CD34⁺ cells in NOD/Shi-scid mice treated with anti-asialo GM1 antiserum, six recipients sublethally irradiated were intravenously injected with 5 to 10 x 10⁴ CB CD34⁺ cells. Ten to 12 weeks after the transplantation, BM cells were harvested and analyzed by flow cytometry for the presence of human CD45⁺ cells. Successful engraftment was obtained in all of six recipients whose BM cells contained 43.8 ± 17.7% of human CD45⁺ cells. Figure 1A shows a representative result of flow cytometric analysis of BM cells in a recipient engrafted with 5 x 10⁴ CB CD34⁺ cells.

View larger version (20K): [in this window] [in a new window]   Figure 1. Flow cytometric analysis of a representative human/mouse chimera. Cord blood CD34+ cells (5 x 104) were transplanted into NOD/Shi-scid mice treated with anti-asialo GM1 antiserum, and the proportions of human CD45+ cells in recipient bone marrow (A), spleen (B), and peripheral blood (C) were analyzed by flow cytometry 12 weeks after the transplantation.

View larger version (43K): [in this window] [in a new window]   Figure 2. The expression of lineage markers on human CD45+ cells reconstituted in bone marrow (BM) of a NOD/Shi-scid mouse. The expression of CD10, CD19, CD3, CD33, CD13, CD14, CD41, CD57, and CD34 on human CD45+ cells (A), and the expression of CD71 and GpA (B) on nucleated cells were analyzed with BM cells of the recipient mouse engrafted with 5 x 104 cord blood CD34+ cells.

The human CD45⁺ cells in each hematopoietic organ were further tested for the expression of human lineage-specific markers by flow cytometry. Figure 3 shows the analysis of BM, spleen, and PB cells of the recipient presented in Figure 1. CD 34 was detected on human CD45⁺ cells in the three hematopoietic organs, but the percentages were low in the spleen and PB (0.87 ± 0.52% and 2.1 ± 0.91%, respectively) as compared with BM (9.9 ± 1.4%, p < 0.01). CD33⁺/CD13⁺/CD14⁺ myeloid cells were consistently found in the BM and PB (8.0 ± 1.6%/6.2 ± 2.1%/6.5 ± 0.6% and 9.1 ± 0.95%/8.1 ± 0.9%/5.0 ± 0.8%, respectively). However, the percentage of CD33⁺/CD13⁺/CD14⁺ cells in the spleen (2.0 ± 0.2%/2.3 ± 1.0%/1.1 ± 0.5%) were much lower than in the BM and PB (p < 0.05).

View larger version (41K): [in this window] [in a new window]   Figure 3. Development of cord blood CD34+ cells in the hematopoietic tissues of NOD/Shi-scid mice. The expression of CD34, CD10, immunoglobulin M (IgM), CD3, CD33, CD19, CD13, CD14, and CD57 on human CD45+ cells from bone marrow, spleen, and peripheral blood of the recipient mouse presented in Figure 1.

View larger version (14K): [in this window] [in a new window]   Figure 4. Circulating CD41+ platelets in peripheral blood (PB) of a NOD/Shi-scid mouse engrafted with 5 x 104 cord blood (CB) CD34+ cells. Platelet rich plasma isolated from PB of the mice untransplanted (A) and transplanted with 5 x 104 CB CD34+ cells (B) were stained with CD41-FITC (fluorescein isothiocyanate).

Colony-Forming Activity of Human Hematopoietic Cells Engrafted in NOD/Shi-scid Mice
Since BM cells of the recipient mice contained a number of human CD34⁺ cells, the colony-forming activity of the reconstituted cells was examined. Table 1 shows the result of methylcellulose culture of 1.5 x 10⁵ BM cells of the untransplanted NOD/Shi-scid mouse and a recipient engrafted with 5 x 10⁴ CB CD34⁺ cells whose BM cells contained 30.4% CD45⁺ cells and 8.4% CD45⁺CD34⁺ cells. No colonies were formed from BM cells of the untransplanted mouse although a small number of mouse macrophage clusters and erythroid bursts were detected. By contrast, BM cells of the engrafted recipients produced various types of human hematopoietic colonies including GM colonies, E bursts, and MIX colonies (Fig. 5A). DNA extracted from the colonies expressed human ALU sequences on PCR analysis (Fig. 6). The 1.5 x 10⁵ BM cells also produced 6 ± 3 MK colonies consisting of CD41⁺ megakaryocytes in serum-free agarose culture (Fig. 5B). This result indicates that HSC in CB CD34⁺ cells can differentiate into various hematopoietic progenitors including myelocytic, erythroid, megakaryocytic, and multipotential progenitors in NOD/Shi-scid mice.

View larger version (244K): [in this window] [in a new window]   Figure 5. In situ appearance of a representative human hematopoietic mixed colony growing in methylcellulose cultures at day 14 of culture (A) and human CD41+ megakaryocytes (MK) in MK colonies at day 11 of serum- free agarose culture (B) of bone marrow cells from a NOD/Shi-scid mouse engrafted with cord blood CD34+ cells.

View larger version (50K): [in this window] [in a new window]   Figure 6. The expression of human ALU sequences in DNA extracted from human MIX (mixed hematopoietic), G (granulocyte), GM (granulocyte/macrophage), M (macrophage) colonies, and E (erythroid) bursts formed from bone marrow (BM) cells of NOD/Shi-scid mice engrafted with cord blood (CB) CD34+ cells. DNA extracted from BM cells of NOD/Shi-scid mice untransplanted and human hematopoietic colonies formed from fresh CB CD34+ cells were used as negative (Neg) and positive (Pos) controls, respectively. The arrow shows that the sequence amplified by two primers is of 221 base pairs.

	DISCUSSION

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BM cells of recipient mice were able to form various types of human hematopoietic colonies in clonal culture, indicating that CB HSC can differentiate into hematopoietic progenitors of various lineages in NOD/Shi-scid mice. However, the extent of their differentiation and maturation differed with each lineage. The CD34⁺ cells repopulated in NOD/Shi-scid mice contained human megakaryocytic progenitors, and CD41⁺ megakaryocytic cells were detected in recipient BM. In addition, we demonstrated the presence of CD41⁺ platelets in PB of NOD/Shi-scid mice for the first time. These findings indicate that human megakaryocytic progenitors can differentiate and mature to produce platelets in NOD/Shi-scid mice. However, previous reports described the slow recovery of platelets in recipient PB in therapeutic CB transplantation as compared with BM transplantation [25]. Therefore, the development of CB megakaryocytic progenitors, which were derived from fetal hematopoiesis, may be regulated by a mechanism different from that in adult hematopoiesis in vivo.

The development of human erythropoiesis occurred in a manner different from that of megakaryopoiesis in NOD/Shi-scid mice. In erythropoiesis, EPO has been shown to be the principal factor for the maturation of erythroid cells, which are cross-sensitized between mouse and human [26, 27]. Nevertheless, no or few late stage human erythroid cells containing hemoglobin or GpA⁺CD71⁺ erythroblasts were found, although a substantial number of erythroid progenitors were detected in BM. Cashman et al. reported that very few GpA⁺ late-stage erythrocytes in NOD/LtSz-scid mice engrafted with CB cells were found even when EPO and other human-specific cytokines were injected [28]. Thus, the erythropoiesis in NOD/scid mice may be compromised because the hematopoietic microenvironment is unsuitable for the maturation of human erythroid cells. Alternatively, some factor(s) other than EPO may be required for the maturation of human fetal erythroid cells in vivo.

CD19⁺ B cells were the most abundantly repopulated in NOD/Shi-scid mice, but their maturational stage differed among the hematopoietic organs. While most of the B cells in BM belonged to the CD19⁺CD10⁺ immature population not expressing sIgM, CD19⁺CD10^–sIgM⁺ cells, a more mature B-cell population, predominated in spleen and PB. Thus, BM is the primary site of human B-cell development in NOD/Shi-scid mice, similar to the situation in humans. However, despite the fact that a number of CD19⁺ cells were reconstituted, the production of human immunoglobulin was not observed in the present study, suggesting that B cells cannot differentiate into immunoglobulin-producing plasma cells in NOD/Shi-scid mice. Christianson et al. and Vormoor et al. detected human immunoglobulin in the serum of NOD/LtSz-scid mice transplanted with human PB MNC and scid mice transplanted with human CB MNC, respectively [29, 30]. This discrepancy may be due to T cells present in PB and CB MNC, or a difference in microenvironment between NOD/Shi-scid mice and NOD/LtSz-scid or scid mice.

In contrast to B cells, T cells were detected only rarely in NOD/Shi-scid mice engrafted with CB CD34⁺ cells as was reported in NOD/LtSz-scid mice transplanted with CB or BM [31]. Inasmuch as Robin et al. recently showed that human CD34⁺CD19^– BM cells isolated from NOD/LtSz-scid recipients transplanted with CB CD34⁺ cells expressed T-cell potential in fetal thymus organ culture, human T-cell progenitors may be unable to migrate into mouse thymus in vivo [32].

In summary, CB CD34⁺ cells had the capability to reconstitute human hematopoiesis in NOD/Shi-scid mice treated with anti-asialo GM1 antiserum. The pattern of the reconstitution was similar to that in NOD/LtSz-scid mice reported previously [17, 31]. In both mice, multilineage reconstitution occurs, but the extent of the differentiation and maturation differed in each hematopoietic lineage. Xenotransplantation in NOD/Shi-scid mouse may provide a useful tool comparable with the NOD/LtSz-scid mouse model for evaluating the repopulating ability of transplantable human HSC.

	ACKNOWLEDGMENTS

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	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kobayashi M, Laver JH, Kato T et al. Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with steel factor and/or interleukin-3. Blood 1996;88:429-436.[Abstract/Free Full Text]

2. Petzer AL, Hogge DE, Landsdorp PM et al. Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium. Proc Natl Acad Sci USA 1996;93:1470-1474.[Abstract/Free Full Text]

3. Hao QL, Shah AJ, Thiemann FT et al. A functional comparison of CD34⁺CD38^– cells in cord blood and bone marrow. Blood 1995;86:3745-3753.[Abstract/Free Full Text]

4. Luens KM, Travis MA, Chen BP et al. Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34⁺Thy-1⁺Lin^– cells with preserved ability to engraft SCID-hu bone. Blood 1998;91:1206-1215.[Abstract/Free Full Text]

5. Bernstein ID, Andrews RG, Zsebo KM. Recombinant human stem cell factor enhances the formation of colonies by CD34⁺ and CD34⁺lin^– cells, and the generation of colony-forming cell progeny from CD34⁺lin^– cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Blood 1991;77:2316-2321.[Abstract/Free Full Text]

6. 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]

7. Bhatia M, Wang JCY, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci USA 1997;94:5320-5325.[Abstract/Free Full Text]

8. Gan OI, Murdoch B, Larochelle A et al. Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells. Blood 1997;90:641-650.[Abstract/Free Full Text]

9. Shultz LD, Schweitzer PA, Christianson SW et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol 1995;154:180-191.[Abstract]

10. Koyanagi Y, Tanaka Y, Kira J et al. Primary human immunodeficiency virus type 1 viremia and central nervous system invasion in a novel hu-PBL-immunodeficient mouse strain. J Virol 1997;71:2417-2424.[Abstract]

11. Xu MJ, Tsuji K, Ueda T et al. Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood 1998;92:2032-2040.[Abstract/Free Full Text]

12. 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]

13. Gluckman E, Broxmeyer HA, Auerbach AD et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989;321:1174-1178.[Medline]

14. Wagner JE, Kernan NA, Steinbuch M et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995;346:214-219.[CrossRef][Medline]

15. Rubinstein P, Carrier C, Scaradavou A et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998;339:1565-1577.[Abstract/Free Full Text]

16. Yang FC, Tsuji K, Oda A et al. Differential effects of human granulocyte colony-stimulating factor (hG-CSF) and thrombopoietin on megakaryopoiesis and platelet function in hG-CSF receptor-transgenic mice. Blood 1999;94:950-958.[Abstract/Free Full Text]

17. Sui X, Tsuji K, Tajima S et al. Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34⁺ cells. J Exp Med 1996;183:837-845.[Abstract/Free Full Text]

18. Cashman JD, Lapidot T, Wang JC et al. Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice. Blood 1997;89:4307-4316.[Abstract/Free Full Text]

19. Lapidot T, Pflumio F, Doedens M et al. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 1992;255:1137-1141.[Abstract/Free Full Text]

20. Nakahata T, Ogawa M. Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors. J Clin Invest 1982;70:1324-1328.

21. Hogge D, Fanning S, Bockhold K et al. Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay. Br J Haematol 1997;96:790-800.[CrossRef][Medline]

22. Sakamaki S, Matsunaga T, Kuga T et al. Long-term survival and differentiation of human peripheral blood CD34⁺ cells in SCID mice. Int J Hematol 1997;65:375-384.[CrossRef][Medline]

23. Cesano A, Hoxie JA, Lange B et al. The severe combined immunodeficient (SCID) mouse as a model for human myeloid leukemias. Oncogene 1992;7:827-836.[Medline]

24. Boggs DR. The total marrow mass of the mouse: a simplified method of measurement. Am J Hematol 1984;16:277-286.[Medline]

25. Nishihira H, Toyoda Y, Miyazaki H et al. Growth of macroscopic human megakaryocyte colonies from cord blood in culture with recombinant human thrombopoietin (c-mpl ligand) and the effects of gestational age on frequency of colonies. Br J Haematol 1996;92:23-28.[CrossRef][Medline]

26. Krantz SB. Erythropoietin. Blood 1991;77:419-434.[Free Full Text]

27. Liboi E, Carroll M, D'Andrea AD et al. Erythropoietin receptor signals both proliferation and erythroid-specific differentiation. Proc Natl Acad Sci USA 1993;90:11351-11355.[Abstract/Free Full Text]

28. Cashman J, Bockhold K, Hogge DE et al. Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood. Br J Haematol 1997;98:1026-1036.[CrossRef][Medline]

29. Christianson SW, Greiner DL, Hesselton RA et al. Enhanced human CD4⁺ T cell engraftment in beta2-microglobulin-deficient NOD-scid mice. J Immunol 1997;158:3578-3586.[Abstract]

30. Vormoor J, Lapidot T, Pflumio F et al. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 1994;83:2489-2497.[Abstract/Free Full Text]

31. Hogan CJ, Shpall EJ, McNulty O et al. Engraftment and development of human CD34(⁺)-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice. Blood 1997;90:85-96.[Abstract/Free Full Text]

32. Robin C, Pflumio F, Vainchenker W et al. Identification of lymphomyeloid primitive progenitor cells in fresh human cord blood and in the marrow of nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice transplanted with human CD34⁺ cord blood cells. J Exp Med 1999;189:1601-1610.[Abstract/Free Full Text]

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