Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34(+) cells in NOD/SCID mice

OBJECTIVE: Mesenchymal stem cells (MSC) have been implicated as playing an important role in hematopoietic stem cell engraftment. We identified and characterized a new population of MSC derived from human fetal lung. In cotransplantation experiments, we examined the homing of MSC as well as the effect on engraftment of human umbilical cord blood (UCB)-derived CD34(+) cells in NOD/SCID mice. MATERIALS AND METHODS: Culture-expanded fetal lung-derived CD34(+) cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. Irradiated (3.5 Gy) NOD/SCID mice (n = 51) were transplanted intravenously with 0.03 to 1.0 x 10(6) UCB CD34(+) cells in the presence or absence of 1 x 10(6) culture-expanded fetal lung-derived MSC, irradiated CD34(-) cells, B cells, or with cultured MSC only. RESULTS: Culture-expanded fetal lung CD34(+) cells were identified as MSC based on phenotype (CD105(+), SH3(+), SH4(+), CD160(+)) and their multilineage potential. Cotransplantation of low doses of UCB CD34(+) cells and MSC resulted in a three-fold to four-fold increase in bone marrow engraftment after 6 weeks, whereas no such effect was observed after cotransplantation of irradiated CD34(-) or B cells. Homing experiments indicated the presence of MSC in the lung, but not in the bone marrow, of NOD/SCID mice. CONCLUSIONS: We identified a population of MSC derived from human fetal lung. Upon cotransplantation, MSC, but not irradiated CD34(-) or B cells, promote engraftment of UCB CD34(+) cells in bone marrow, spleen, and blood by mechanisms that may not require homing of MSC to the bone marrow.     

Mutant N-ras preferentially drives human CD34+ hematopoietic progenitor cells into myeloid differentiation and proliferation both in vitro and in the NOD/SCID mouse

OBJECTIVES: Ras oncogene mutations are the most frequently observed genetic abnormality (20-40% of patients) in acute myeloid leukemia (AML), and in the preleukemic conditions myelodysplastic syndrome (MDS) and myeloproliferative disorder (MPD). We have previously shown that mutant N-ras (N-rasm) can induce myeloproliferative disorders and apoptosis in a murine reconstitution system. In the present study we investigated the effect of N-rasm in human primary hematopoietic progenitor cells (HPC). METHODS: Cord blood CD34+ hematopoietic progenitor cells (HPC) were transduced with retroviral vectors containing green fluorescence protein (GFP) alone, or in combination with N-rasm. Cells were then cultured in vitro with a cytokine supplement or cocultured with murine stroma MS-5 cells. The in vivo behavior of transduced cells was examined in the NOD/SCID mouse model. RESULTS: N-rasm-transduced cells exhibited greater proliferative capacity; a higher frequency of granulocyte-macrophage colony-forming unit (CFU-GM); and an increase in myelomonocytic lineage cells with a concomitant decrease in lymphoid and erythroid cells. Analysis of transduced HPC in NOD/SCID mice revealed higher bone marrow engraftment by N-rasm HPC and increased numbers of myeloid lineage cells. CONCLUSIONS: The results demonstrate that N-rasm in HPC induces myeloproliferation both in vitro and in the NOD/SCID mouse model as a primary event that does not appear to be dependent on cooperating transforming events.     

Decreased homing of retrovirally transduced human bone marrow CD34+ cells in the NOD/SCID mouse model

OBJECTIVE: Many clinical gene therapy trials have described poor engraftment of retrovirally transduced CD34(+) cells. Because engraftment is dependent upon successful homing of graft cells to the bone marrow (BM), we examined whether retroviral-mediated gene transfer (RMGT) induces a homing defect in CD34(+) cells. METHODS: Homing of fluorescently labeled human BM CD34(+) cells transduced with three separate retroviral vectors (MFG-eGFP, LNC-eGFP, and LXSN) was assessed in nonobese diabetic/severe combined immunodeficient mice. RESULTS: Homing of transduced CD34(+) cells was significantly decreased 20 hours after transplantation compared with freshly isolated control and cultured untransduced control cells. Specifically, homing of GFP(+) cells in the graft was preferentially decreased thus skewing the contribution of transduced cells to engraftment. Transduced cells were not selectively trapped in other organs and BM-homed transduced cells did not undergo apoptosis at a higher rate than untransduced cells. Adhesion molecule expression and binding activity was not altered by RMGT. This homing defect was reversed when transduced cells were cultured over CH-296 for 2 additional days with SCF only. CONCLUSION: These data suggest that RMGT of hematopoietic cells may compromise their homing potential and implicate transduction-induced reduced homing in the observed low engraftment of retrovirally transduced CD34(+) cells. These results may have a direct clinical application in gene therapy protocols.     

Human CD34+ cell preparations contain over 100-fold greater NOD/SCID mouse engrafting capacity than do CD34- cell preparations.

OBJECTIVE: The CD34 cell surface marker is used widely for stem/progenitor cell isolation. Since several recent studies reported that CD34(-) cells also have in vivo engrafting capacity, we quantitatively compared the engraftment potential of CD34(+) vs CD34(-) cell preparations from normal human placental/umbilical cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PBSC) specimens, using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. METHODS: CD34(+) and CD34(-) cell preparations were purified by four different approaches in 14 individual experiments involving 293 transplanted NOD/SCID mice. In most experiments, CD34(+) cells were depleted twice (CD34(=)) in order to obtain efficient depletion of CD34(+) cells from the CD34(-) cell preparations. RESULTS: Dose-dependent levels of human hematopoietic cells were observed after transplantation of CD34(+) cell preparations. To rigorously assess the complementary CD34(-) cell preparations, cell doses 10- to 1000-fold higher than the minimum dose of the CD34(+) cell preparations necessary for engraftment were transplanted. Nevertheless, of 125 NOD/SCID mice transplanted with CD34(-) cell preparations purified from the same starting cells, only six mice had detectable human hematopoiesis, by flow cytometric or PCR assay. CONCLUSIONS: CD34(-) cells provide only a minor contribution to hematopoietic engraftment in this in vivo model system, as compared to CD34(+) cells from the same samples of noncultured human cells. Hematopoiesis derived from actual CD34(-) cells is difficult to distinguish from that due to CD34(+) cells potentially contaminating the preparations.     

Ex vivo culture of human CD34+ cord blood cells with thrombopoietin (TPO) accelerates platelet engraftment in a NOD/SCID mouse model

OBJECTIVES: Hematopoietic recovery, in particular platelet reconstitution, can be severely delayed after transplantation with cord blood (CB) stem cells (SC). Expansion of CB SC may be one way to improve the recovery, but there is concern that ex vivo expansion compromises the repopulating ability of SC. METHODS: We used a short-term expansion protocol with TPO as single growth factor. The expanded cells were tested in the NOD/SCID mouse model and both platelet recovery and repopulation capacity were examined and compared with unexpanded CD34+ CB cells of the same CB donor. RESULTS: Platelet recovery started 1 week earlier in mice transplanted with TPO-expanded CD34+ cells and at days 5 and 8 after transplantation, 6.2 +/- 2.6 and 13.9 +/- 6.7 plt/microL were observed, respectively. At similar time intervals 0.0 and 1.5 +/- 0.2 plt/microL respectively were detected in mice receiving the unmanipulated CD34+ grafts. This was accompanied by a higher number of CFU-Mk in the bone marrow (BM) 7 days after transplantation. Moreover, the BM engraftment and the lineage differentiation of human cells at 6 weeks after transplantation was similar, suggesting that long-term engraftment was not compromised by the expansion procedure. CONCLUSION: Ex vivo expansion with TPO as single growth factor results in an accelerated platelet recovery in NOD/SCID mice and appears not to affect the long-term repopulation capacity.     

Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34(+) cells transplanted into conditioned NOD/SCID recipients

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34(+) cells, or their G(0) (G(0)CD34(+)) or G(1) (G(1)CD34(+)) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34(+) cells harbored similar numbers of CD34(+) and CD34(-) cells, indicating that CD34(+) cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G(2)+M than in recipients of G(0)/G(1) cells (34.6% +/- 5.9% and 17.1% +/- 6.3%, respectively; P <.01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.     

Regulation of human short-term repopulating cell (STRC) engraftment in NOD/SCID mice by host CD122+ cells

OBJECTIVE: NOD/SCID and NOD/SCID B2m(null) mice are used for the in vivo study of human hematopoietic stem cells (HSC). A previously unrecognized HSC in cord blood, termed short-term repopulating cell (STRC), has been identified using NOD/SCID B2m(null) mice. However, only low levels of STRC engraft in NOD/SCID mice, presumably due to their higher levels of NK cell activity. The objective of these studies was to deplete NK cells both by genetic manipulation of the hosts and by antibody depletion of cell populations that may regulate engraftment with human STRC. METHODS: C57BL/6-SCID mice and immunodeficient NOD mice genetically deleted in NK cell activity were injected intravenously with human cord blood cells to quantify STRC engraftment. Cohorts of these mice were also treated with anti-NK1.1 or anti-CD122 (IL-2r beta-chain) antibodies. RESULTS: Human STRC fail to engraft in C57BL/6-SCID mice treated with anti-NK1.1 or with anti-CD122 antibody that targets mouse NK and myeloid cells. NOD/SCID mice, NOD-Rag1(null) mice, and NOD-Rag1(null)Pfp(null) mice that are genetically deleted in NK cell cytotoxic activity support only low levels of STRC engraftment. In contrast, STRC engraft at high levels in all three strains of immunodeficient NOD mice treated with anti-CD122 antibody. CONCLUSION: Injection of anti-CD122 antibody leads to high levels of STRC engraftment in immunodeficient NOD mice, but not in C57BL/6-SCID mice. These data document that depletion of NK cells is required, and that additional murine host innate immune factors, presumably myeloid cells, are important in regulating human STRC engraftment.     

Homing defect of cultured human hematopoietic cells in the NOD/SCID mouse is mediated by Fas/CD95

OBJECTIVE: To determine the bone marrow homing efficiency (20 hours) of cultured compared to noncultured umbilical cord blood (UCB)-derived human hematopoietic cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse, and to explain the difference in homing between these populations. METHODS: Human UCB CD34+ cells were cultured for up to 5 days, reselected, and used for transplantation, phenotype analysis, and functional studies, including adhesion and trans-endothelial migration assays. Seeding of CD34+ cells was measured after labeling of cells with 111-Indium, while homing of colony-forming cells (CFC) and SCID-repopulating (SRC) cells was determined using functional assays. RESULTS: Short-term culture was associated with a decrease in the 20-hour homing of CD34+ cells, CFC, and SRC to the BM. Although cultured compared to noncultured cells showed increased expression and function (adhesion/migration) of several cell adhesion molecules described to play a role in homing and engraftment, culture also induced expression of Fas/CD95 and rendered cells more susceptible to apoptosis. Finally, we demonstrate that the level of Fas/CD95 on cultured cells was inversely related to the ability of CFC to home to the BM, and that the homing of cultured CFC could be restored by incubating cells prior to transplantation with Fas/CD95-blocking mAb ZB4. CONCLUSION: These data implicate Fas/CD95 in the homing defect of cultured human hematopoietic cells in the NOD/SCID transplant model and suggest that prevention of apoptosis may be an important strategy to improve engraftment of ex vivo-manipulated HSC in a clinical setting.     

A strategy for enhancing the engraftment of human hematopoietic stem cells in NOD/SCID mice

To overcome the limitations of allogeneic hematopoietic stem cell transplantation (HSCT), we conducted a study to identify a strategy for enhancing hematopoietic stem cell (HSC) engraftment during HSCT. Co-transplantation experiments with mesenchymal stem cells (MSCs) derived from adult human tissues including bone marrow (BM), adipose tissue (AT), and umbilical cord blood (CB) were conducted. We showed that AT-MSCs and CB-MSCs enhanced the engraftment of HSCs as effectively as BM-MSCs in NOD/SCID mice, suggesting that AT-MSCs and CB-MSCs can be used as alternative stem cell sources for enhancing the engraftment and homing of HSCs. CB-MSCs derived from different donors showed different degrees of efficacy in enhancing the engraftment of HSCs. The most effective CB-MSCs showed higher proliferation rates and secreted more MCP-1, RANTES, EGF, and VEGF. Our results suggest that AT-MSCs and CB-MSCs could be alternative stem cell sources for co-transplantation in HSCT. Furthermore, in terms of MSCs’ heterogeneity, characteristics of each population of MSCs are considerable factors for selecting MSCs suitable for co-transplantation with HSC.     

Human NK cell development in NOD/SCID mice receiving grafts of cord blood CD34+ cells

Definition of the cytokine environment, which regulates the maturation of human natural killer (NK) cells, has been largely based on in vitro assays because of the lack of suitable animal models. Here we describe conditions leading to the development of human NK cells in NOD/SCID mice receiving grafts of hematopoietic CD34+ precursor cells from cord blood. After 1-week-long in vivo treatment with various combinations of interleukin (IL)-15, flt3 ligand, stem cell factor, IL-2, IL-12, and megakaryocyte growth and differentiation factor, CD56+CD3- cells were detected in bone marrow (BM), spleen, and peripheral blood (PB), comprising 5% to 15% of human CD45+ cells. Human NK cells of NOD/SCID mouse origin closely resembled NK cells from human PB with respect to phenotypic characteristics, interferon (IFN)-gamma production, and cytotoxicity against HLA class 1-deficient K562 targets in vitro and antitumor activity against K562 erythroleukemia in vivo. In the absence of growth factor treatment, CD56+ cells were present only at background levels, but CD34+CD7+ and CD34-CD7+ lymphoid precursors with NK cell differentiation potential were detected in BM and spleen of chimeric NOD/SCID mice for up to 5 months after transplantation. Our results demonstrate that limitations in human NK cell development in the murine microenvironment can be overcome by treatment with NK cell growth-promoting human cytokines, resulting in the maturation of IFN-gamma-producing cytotoxic NK cells. These studies establish conditions to explore human NK cell development and function in vivo in the NOD/SCID mouse model.     

Engraftment kinetics of human CD34+ cells from cord blood and mobilized peripheral blood co-transplanted into NOD/SCID mice

We have reported short periods of post transplant neutropenia in human patients co-transplanted with cord blood (CB) and low numbers of haploidentical mobilized peripheral blood (MPB) CD34+ cells. To investigate the effect that the proportion of MPB to CB cells may have on engraftment kinetics, we have co-transplanted fixed numbers of human CB CD34+ cells mixed with different numbers of MPB CD34+ cells into NOD/SCID mice. We periodically quantified the proportion of human cells and the relative contribution of MPB and CB cells to the human engraftment on marrow aspirates. At the lowest MPB/CB ratios (5 : 1, 10 : 1), the contribution of CB cells predominated at all time points analyzed, and in three out of four experiments MPB cell contributions progressively decreased from day +15. At higher MPB/CB ratios, MPB cells had a more important contribution to both early and late engraftment, with the highest cell ratio resulting in only marginal CB cell engraftment. Therefore, our results showed greater potential, on a per cell basis, of human CB vs MPB cells for competitive sustained engraftment in the xenogeneic model used, which was only abrogated by the co-infusion of very high numbers of MPB cells.     

Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34 cells after ex vivo expansion

OBJECTIVE: The relationship between phenotype and function in ex vivo-cultured human hematopoietic stem cells (HSC) remains poorly understood. We investigated the effects of a short-term serum-free culture on the relationship between stem cell phenotype, cell division history, and function in human CD34(+) cells. METHODS: G-CSF-mobilized peripheral CD34(+) cells were cultured for 4 days with stem cell factor, flt-3 ligand, and thrombopoietin. The phenotype (CD34, CD38, HLA-DR, c-kit), cell division history, colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and NOD/SCID repopulating activities were evaluated at Day 0 and 4. RESULTS: We observed a loss of CD38, HLA-DR, and c-kit surface expression resulting in a drastic increase in CD34(+)CD38(-), CD34(+)HLA-DR(-), and CD34(+)c-kit(-/low) cells at Day 4. In contrast, the frequency of Thy-1(+) cells was maintained. We observed a 1.3-fold expansion of CFC, a 4.8-fold increase in LTC-IC, and an overall maintenance of the NOD/SCID repopulating cell activity. CD34(+)CD38(-) and CD34(+)HLA-DR(-) cells detected at Day 4 displayed the most active pattern of division (4 to 5 divisions) whereas 60% of CD34(+)Thy-1(+) cells divided 0 to 2 times during the same period. At Day 4, the NOD/SCID repopulating activity was associated with Thy-1(+) cells with no more than 2 divisions. CONCLUSIONS: Our results show that the relationship between stem cell phenotype and function is dramatically altered in cultured CD34(+) cells. Thy-1 expression and cell division history appear to be superior to CD38, HLA-DR, and c-kit, or to homing molecules (CXCR4, VLA-4) as predictors of the repopulating activity of cultured peripheral CD34(+) cells.     

Human CD34+ hematopoietic cells transduced by retrovirus-mediated interferon alpha gene maintains regeneration capacity and engraftment in NOD/SCID mice.

To achieve long-term expression of human interferon alpha-5 (IFNalpha) gene in the bone marrow (BM) hematopoietic microenvironment, replication-deficient retroviral vector LSN-IFNalpha was used to deliver the IFNalpha gene into human BM CD34+ cells. After fibronectin-facilitated transduction, a fraction of CD34+ cells was plated in methylcellulose medium with or without G418 to assess transduction efficiency and the effect of IFNalpha gene transfer on colony formation. Colony-forming assay in the presence of G418 (400 microg/mL) revealed that 41% CFU-GM colonies are G418 resistant after infection with LSN-IFNalpha retrovirus. There was no significant difference in CFU-GM/BFU-E colony formation among IFNalpha gene-transduced CD34+ cells, control vector (LXSN) transduced-CD34+ cells and nontransduced CD34+ cells. Another portion of CD34+ cells was grown in liquid medium to measure IFNalpha production. RIA revealed that IFNalpha gene-transduced CD34+ cells produced 72.2 +/- 15.4 U/mL (10(6) cells/24 hours) of IFNalpha compared with 8.3 +/- 2.1 U/mL and 4.3 +/- 1.2 U/mL in LXSN-transduced or nontransduced CD34+ cells, respectively. The remaining portion of transduced CD34+ cells was transplanted into immunodeficient (NOD/SCID) mice to allow analysis of long-term expression of IFNalpha. Transplantation of 1x10(6) CD34+ cells into sublethally irradiated NOD/SCID mice showed that IFNalpha and neo(r) mRNA were detectable in engrafted mouse BM cells for up to 6 months. We conclude that continual local expression of IFNalpha in transduced CD34+ cells does not impair either CD34+ cell growth and differentiation or engraftment and long-term survival in NOD/SCID mice.     

Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice

OBJECTIVE: For approximately 5% of autologous transplant recipients and a higher proportion of allogeneic transplant recipients, low level and delayed platelet engraftment is an ongoing problem. Mesenchymal stem cells (MSC), which can be derived from bone marrow as well as other organs, are capable of differentiation into multiple cell types and also support hematopoiesis in vitro. Because cotransplantation of marrow-derived stromal cells has been shown to enhance engraftment of human hematopoietic stem cells, we hypothesized that cotransplantation of MSC could enhance platelet and myeloid cell development. MATERIALS AND METHODS: We tested this hypothesis by transplantation of CD34-selected mobilized human peripheral blood stem cells (PBSC) into sublethally irradiated NOD/SCID mice with or without culture-expanded human MSC and evaluated human myeloid, lymphoid, and megakaryocytic engraftment with flow cytometry and in vitro cultures. RESULTS: We find that MSC cotransplantation enhances human cell engraftment when a limiting dose (<1 x 10(6)) of CD34 cells is administered. This enhancement is characterized by a shift in the differentiation of human cells from predominantly B lymphocytes to predominantly CD13(+), CD14(+), and CD33(+) myeloid cells with a corresponding increase in myeloid CFU in the marrow. Megakaryocytopoiesis is enhanced by MSC cotransplantation as assessed by an increase in both marrow CFU-MK and circulating human platelets. In contrast, MSC do not affect the percentage of human bone marrow cells that expresses CD34(+). CONCLUSIONS: Cotransplantation of human mesenchymal stem cells with CD34(+)-selected hematopoietic stem cells enhances myelopoiesis and megakaryocytopoiesis.     

Human dendritic cell subsets in NOD/SCID mice engrafted with CD34+ hematopoietic progenitors

Distinct human dendritic cell (DC) subsets differentially control immunity. Thus, insights into their in vivo functions are important to understand the launching and modulation of immune responses. We show that nonobese diabetic/LtSz-scid/scid (NOD/SCID) mice engrafted with human CD34+ hematopoietic progenitors develop human myeloid and plasmacytoid DCs. The skin displays immature DCs expressing Langerin, while other tissues display interstitial DCs. Myeloid DCs from these mice induce proliferation of allogeneic CD4 T cells in vitro, and bone marrow human cells containing plasmacytoid DCs release interferon-alpha (IFN-alpha) upon influenza virus exposure. Injection of influenza virus into reconstituted mice triggers IFN-alpha release and maturation of mDCs. Thus, these mice may provide a model to study the pathophysiology of human DC subsets.     

Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model

The reduced engraftment potential of hematopoietic stem/progenitor cells (HSPCs) after exposure to cytokines may be related to the impaired homing ability of actively cycling cells. We tested this hypothesis by quantifying the short-term homing of human adult CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals. We show that the loss of engraftment ability of cytokine-activated CD34+ cells is associated with a reduction in homing of colony-forming cells (CFCs) to bone marrow (BM) at 24 hours after transplantation (from median 2.8% [range, 1.9%-6.1%] to 0.3% [0.0%-0.7%]; n = 3; P < .01), coincident with an increase in CFC accumulation in the lungs (P < .01). Impaired BM homing of cytokine-activated cells was not restored by using sorted cells in G0G1 or by inducing cell cycle arrest at the G1/S border. Blocking Fas ligation in vivo did not increase the BM homing of cultured cells. Finally, we tested cytokine combinations or culture conditions previously reported to restore the engraftment of cultured cells but did not find that any of these was able to reverse the changes in homing behavior of cytokine-exposed cells. We suggest that these changes in homing and, as a consequence, engraftment result from the increased migratory capacity of infused activated cells, leading to the loss of selectivity of the homing process.     

Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice

OBJECTIVE: We previously demonstrated that cellular copper is involved in the regulation of proliferation and differentiation of hematopoietic progenitor cells. Modulation of cellular copper was achieved by supplementing the culture with a copper chelator that reduces cell copper content, or copper salts, which elevate the level of cellular copper. In the present study, we evaluated the effect of short-term (3-week) treatment with the copper chelator tetraethylenepentamine (TEPA) on short- and long-term (up to 11 weeks) ex vivo expansion of hematopoietic progenitors, as well as on their SCID engraftment potential. MATERIALS AND METHODS: Cord blood-derived purified CD34+ cells were grown in liquid medium supplemented with the cytokines stem cell factor, thrombopoietin, Flt3 ligand, and IL-6, and the chelator TEPA for the first 3 weeks and then for up to 11 weeks with cytokines alone. Control cultures were supplemented with cytokines alone for the entire culture duration. Cultured cells were characterized by immunophenotyping and cloning (CFUc). Transplantability was assayed by injection of repurified CD34+ cells into NOD/SCID mice. RESULTS: In the short term, TEPA supported increased percentages of early progenitors over control cultures incubated with cytokines alone (CD34(+)CD38-, p=0.001 and CD34(+)Lin-, p=0.016). In the long term, TEPA pretreated cultures showed prolonged expansion of CD34+ cells (p=0.01) and CFUc (p=0.002) compared with that of untreated cultures. The SCID engraftment potential of CD34+ cells repurified from the TEPA-treated cultures was higher compared with that of the control, i.e., only cytokine-treated cultures (p=0.03). CONCLUSION: TEPA enabled preferential proliferation of early progenitor cells with the phenotype CD34(+)CD38- and CD34(+)CD38- Lin- during the first weeks of culture, resulting in the observed increased long-term ex vivo expansion and engraftment capabilities.     

Reconstitution of functional human B lymphocytes in NOD/SCID mice engrafted with ex vivo expanded CD34(+) cord blood cells

OBJECTIVE: Functional capacity of B cells developed from ex vivo expanded hematopoietic stem cells has not been fully evaluated. Therefore, we investigated the antigen-specific antibody production in human B cells maturated from ex vivo expanded cord blood (CB) CD34(+) cells in NOD/Shi-scid (NOD/SCID) mice. MATERIALS AND METHODS: CB CD34(+) cells were cultured for 5 days in the presence of human cytokines and the murine stromal cell line HESS-5, and transplanted into irradiated NOD/SCID mice. These mice, reconstituted with human hematopoietic cells, were challenged with T-cell-independent (TI) or T-cell-dependent (TD) antigens after CD19(+) cells appeared at 6 weeks. RESULTS: Three months later, anti-dinitrophenol (DNP)-specific antibody was detected in both mice immunized with DNP-Ficoll (TI) and those immunized with DNP-keyhole limpet hemocyanin or DNP-ovalbumin (TD). The anti-DNP antibody was mainly immunoglobulin M, but a small amount of immunoglobulin G also was detected. In the spleen, the majority of CD19(+) cells expressed mature B-cell markers such as CD40, immunoglobulin M, immunoglobulin D, cytoplasmic Cmu, and light chains kappa, and lambda. CONCLUSIONS: These results indicate that human B cells develop from CD34(+) cells in NOD/SCID mice to produce antigen-specific antibody with in vivo primary stimulation. This system provides a powerful and versatile tool for studying the entire process of human B-lymphocyte development and producing specific human monoclonal antibodies.