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

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1 Hematopoietic Repopulating Ability of Cord Blood CD34 + Cells in NOD/Shi-scid Mice TAKAHIRO UEDA, a HIROSHI YOSHINO, a KIMIO KOBAYASHI, c MARIKO KAWAHATA, c YASUHIRO EBIHARA, a MAMORU ITO, c SHIGETAKA ASANO, a,b TATSUTOSHI NAKAHATA, d KOHICHIRO TSUJI a 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 ABSTRACT 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 CB CD34 + cells into six NOD/Shiscid 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 α-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. Stem Cells 2000;18: INTRODUCTION 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, colonyforming 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]. Correspondence: Kohichiro Tsuji, M.D., Department of Clinical Oncology, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo , Japan. Telephone: ; Fax: ; tsujik@ims.utokyo.ac.jp Received February 16, 2000; accepted for publication March 27, AlphaMed Press /2000/$5.00/0 STEM CELLS 2000;18:

2 Ueda, Yoshino, Kobayashi et al. 205 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/Shiscid 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 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 cells/ml in phosphate-buffered saline (PBS) and mixed with Dynabeads M-450 CD34 (Dynal AS; Oslo, Norway; 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 CD34 + cells were injected into 8- to 10-week-old NOD/Shi-scid mice irradiated with 240 rads ( 60 Co) 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 antiasialo 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 CO 2 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; 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; as described [11]. Briefly, after depletion of erythrocytes with Lysing Solution (Nichirei Co.; Tokyo, Japan), 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,

3 206 Repopulation by CD34 + Cells in NOD/Shi-scid Mice 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; 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 antihuman hemoglobin α 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 phosphatasemouse 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 cells/ml in methylcellulose culture as previously reported [18, 19]. One milliliter of a mixture containing cells, α-medium (Flow Laboratories; Rockville, MD), 0.9% methylcellulose (Shinetsu Chemical Co.; Tokyo, Japan; 15% FBS, 1% deionized fraction V bovine serum albumin (Sigma), 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; Recombinant human IL-3, EPO, and GM-CSF were generous gifts from Kirin Brewery (Tokyo, Japan; Recombinant human SCF and IL-6 were kindly provided by Amgen Biologicals (Thousand Oaks, CA; and Tosoh Co. (Ayase, Japan; respectively. The dishes were incubated at 37 C in a humidified atmosphere flushed with 5% CO 2 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 serumfree agarose culture containing human thrombopoietin (50 ng/ml), IL-6 (10 ng/ml), and IL-3 (10 ng/ml), using the MegaCult -C Kit (#4971; StemCell Technologies Inc.; Vancouver, Canada; [21]. After 10 to 12 days of incubation at 37 C in a humidified atmosphere flushed with 5% CO 2, 5% O 2, and 90% N 2, 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; 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.

4 Ueda, Yoshino, Kobayashi et al. 207 RESULTS 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 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 CB CD34 + cells. The expression of lineage markers on human CD45 + cells reconstituted in BM was analyzed in the six recipients. Figure 2A shows a representative BM profile of a mouse recipient. The most prominent population consisted of CD19 + B-lymphoid cells (85.9 ± 7.1%), which contained immature CD19 + CD10 + cells (78.0 ± 8.1%). The recipients also possessed CD13 + /CD33 + myeloid cells (11.1 ± 7.9%/11.5 ± 6.0%), CD14 + monocytic cells (5.4 ± 2.7%), CD41 + megakaryocytic cells (1.7 ± 0.4%), and CD34 + immature cells (11.3 ± 6.2%), but neither CD3 + T cells nor CD57 + NK cells in BM CD45 + cells. A small number of GpA + CD71 + erythroid cells were detected in BM cells of a recipient who possessed 30.4% CD45 + cells (Fig. 2B). Differentiation and Maturation of Each Lineage of Cells in Various Hematopoietic Organs To investigate the differentiation and maturation of CB CD34 + cells in various hematopoietic organs of NOD/Shi-scid mice, BM, spleen, and PB cells were analyzed in four recipients successfully engrafted. Spleen and PB cells, as well as BM cells, contained human CD45 + cells, but the proportions of CD45 + cells in spleen and PB were lower than that in BM in all of the four recipients (52.2 ± 15.2%, 22.9 ± 17.7%, and 17.3 ± 14.6% in BM, spleen, and PB, respectively, p < 0.05). Figure 1 shows a representative result of flow cytometric analysis of BM, spleen, and PB of the same recipient. 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). The presence of human platelets was examined by staining platelets in PRP with anti-human CD41 in a mouse which had 62.2% CD45 + cells in BM. Human CD41 + platelets occupied 35.5% of the platelets of the mouse PB (Fig. 4). The existence of mature erythrocytes was examined by the APAAP staining of cytospin samples with anti-human hemoglobin α antibody. In PB of a mouse which possessed 37% CD45 + cells, human erythroid cells possessing hemoglobin α were not detected (data not shown). (A) BM (B) Spleen (C) PB 30 Counts Counts % % Counts % M1 M1 M1 0 CD45 FITC 0 CD45 FITC 0 CD45 FITC Figure 1. Flow cytometric analysis of a representative human/mouse chimera. Cord blood CD34 + cells ( ) 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.

5 208 Repopulation by CD34 + Cells in NOD/Shi-scid Mice A Mouse IgG1 PE Mouse IgG1 FITC CD19 PE CD10 FITC CD34 FITC 7.2% 71.3% 17.9% 5.3% CD33 PE 7.5% CD13 PE 17.8% CD3 FITC CD14 PE 10.0% CD3 FITC CD41 PE 2.5% CD57 FITC B GpA PE 1.0% CD71 FITC 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 cord blood CD34 + cells. Human T cells, as defined by CD3 expression, could not be detected in BM, spleen, or PB. When examined with the cells in thymus of six recipients, human T cells could not be detected except in one mouse whose thymocytes contained 84.5% human CD45 + cells. Phenotypic analysis of the human thymocytes showed 90.8% CD4 + CD8 + cells and 5.5% CD4 + CD8 cells. In contrast to T cells, human CD19 + B cells were most predominant in all of BM, spleen, and PB (Fig. 3). When the CD19 + populations in BM, spleen, and PB were further analyzed for cell surface expressions of CD10 and IgM as evidence of B-cell development, all the mice examined showed a similar pattern of expression in the three hematopoietic compartments. In the BM, the majority of CD19 + cells expressed CD10 (95.3 ± 1.5%) and a low level of cell surface IgM (sigm) (22.7 ± 1.3%), an immature phenotype. On the other hand, approximately 90% of the CD19 + cells in the spleen and PB expressed sigm, representing a more mature B-cell population. The plasma of three mice which possessed greater than 10% human CD45 + cells in PB was tested for the presence of human IgG and IgM. Although

6 Ueda, Yoshino, Kobayashi et al. 209 BM Spleen PB CD33 PE 6.7% 8.2% 2.2% 1.6% CD34 FITC 10% 2.8% CD13 PE CD14 PE 6.9% 1.6% CD3 FITC 9.2% 4.3% 0.7% 4.4% CD3 FITC CD19 PE CD19 PE 3.4% 88% 68% 27% CD10 FITC 84% 0.4% 23% 83% 76% IgM FITC 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.

7 210 Repopulation by CD34 + Cells in NOD/Shi-scid Mice Counts A CD41 FITC Counts CD41 FITC Figure 4. Circulating CD41 + platelets in peripheral blood (PB) of a NOD/Shi-scid mouse engrafted with cord blood (CB) CD34 + cells. Platelet rich plasma isolated from PB of the mice untransplanted (A) and transplanted with CB CD34 + cells (B) were stained with CD41-FITC (fluorescein isothiocyanate). human CD45 + cells of the three mice contained 86.2 ± 5.0%, 88.2 ± 9.3% and 85.9 ± 1.7% CD19 + cells in BM, spleen, and PB, respectively, human IgG and IgM were not detectable 12 weeks after the transplantation. 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 BM cells of the untransplanted NOD/Shi-scid mouse and a recipient engrafted with 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 B (Fig. 5A). DNA extracted from the colonies expressed human ALU sequences on PCR analysis (Fig. 6). The 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. DISCUSSION The present study demonstrated that CB CD34 + cells have the capability to repopulate and expand in the hematopoietic microenvironment of NOD/Shi-scid mice. For instance, a recipient mouse shown in Figure 1 contained cells in two femurs and two tibiae, of which 30% and 4% were human CD45 + and CD45 + CD34 + cells, respectively. Thus, CD45 + cells and CD45 + CD34 + cells in the femurs and tibiae were generated from input CB CD34 + cells, showing an increase of at least 1,050-fold in CD45 + cells and 140-fold in CD45 + CD34 + cells, supposing that the femurs and tibiae represent 20% of the BM cavity of the whole body [24]. Human CD34 + cells were also detected in PB and spleen, but the percentages were very low as compared with those detected in BM. This observation suggests that human hematopoiesis primarily occurs in BM of NOD/Shi-scid mice. 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 Table 1. Formation of human hematopoietic colonies from bone marrow (BM) cells of NOD/Shi-scid mice engrafted with CB CD34 + cells Mice n of colonies per BM cells GM* E* MIX* MK** Untransplanted Transplanted with human CB CD34 + cells 25.7 ± ± ± ± 3.0 *The clonal cultures were incubated for 14 days with human stem cell factor, interleukin 3 (IL-3), GM-CSF, and erythropoietin. **The clonal cultures were incubated for 12 days with human IL-6, IL-3, and thrombopoietin. CD41 + megakaryocytes were identified by alkaline phosphatase antialkaline phosphatase staining. Values are means ± standard deviation from triplicate cultures. CB = cord blood; GM = granulocyte/macrophage colonies; E = erythroid bursts; MIX = mixed hematopoietic colonies; MK = megakaryocytes colonies.

8 Ueda, Yoshino, Kobayashi et al. 211 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 serumfree agarose culture (B) of bone marrow cells from a NOD/Shi-scid mouse engrafted with cord blood CD34 + cells. 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. 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/Shiscid 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 α 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,

9 212 Repopulation by CD34 + Cells in NOD/Shi-scid Mice 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/LtSzscid 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. 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