Similar repopulating capacity of mitotically active and resting umbilical cord blood CD34(+) cells in NOD/SCID mice

It was hypothesized that during mammalian development, the extensive need for hematopoietic cells requires equal contribution to blood cell production from both quiescent and cycling hematopoietic stem cells (HSCs) while maintaining the stem cell pool. To investigate this hypothesis, the engraftment potential of umbilical cord blood (UCB) CD34(+) cells residing in either G(0) (G(0)CD34(+) cells) or G(1) (G(1)CD34(+) cells) phases of the cell cycle was assessed in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. Whereas the level of chimerism in mice transplanted with UCB G(0)CD34(+) cells was 69.9% +/- 24.0%, mice receiving equal numbers of G(1)CD34(+) cells harbored 46.7% +/- 21.3% human cells 8 weeks posttransplantation. Both groups of cells sustained multilineage differentiation and the production of CD34(+) cells in recipient animals. The relationship between the number of transplanted G(0)CD34(+) or G(1)CD34(+) cells and the level of chimerism was analyzed by a general linear models procedure. Although the initial level of chimerism following transplantation of G(0)CD34(+) cells was higher than that sustained by G(1)CD34(+) cells, the increment in the degree of chimerism obtained with each additional 10(3) cells of either phenotype was identical, suggesting that the reconstitution potential of these 2 types of cells was similar. Of interest is that human cells recovered from primary recipients of both G(0)CD34(+) and G(1)CD34(+) cells engrafted in secondary NOD/SCID recipients, albeit at a substantially lower level, confirming the primitive nature of UCB CD34(+) cells residing in G(1).     

Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables.

Retroviral transduction of human hematopoietic stem cells is still limited by lack of information about conditions that will maximize stem cell self-renewal divisions in vitro. To address this, we first compared the kinetics of entry into division of single human CD34+CD38- cord blood (CB) cells exposed in vitro to three different flt3-ligand (FL)-containing cytokine combinations. Of the three combinations tested, FL + hyperinterleukin 6 (HIL-6) yielded the least clones and these developed at a slow rate. With either FL + Steel factor (SF) + HIL-6 + thrombopoietin (TPO) or FL + SF + interleukin 3 (IL-3) + IL-6 + granulocyte-colony-stimulating factor (G-CSF), >90% of the cells that formed clones within 6 days undertook their first division within 4 days, although not until after 24 hours. These latter two, more stimulatory, cytokine combinations then were used to assess the effect of duration of cytokine exposure on the efficiency of transducing primitive CB cells with a gibbon ape leukemia virus-pseudotyped murine retroviral vector containing the enhanced green fluorescent protein (GFP) cDNA and the neomycin resistance gene. Fresh lin- CB cells exposed once to medium containing this virus plus cytokines on fibronectin-coated dishes yielded 23% GFP+ CD34+ cells and 52-57% G418-resistant CFC when assessed after 2 days. Prestimulation of the target cells (before exposing them to virus) with either the four or five cytokine combination increased their susceptibility. In both cases, the effect of prestimulation assessed using the same infection protocol was maximal with 2 days of prestimulation and resulted in 47-54% GFP+ CD34+ cells and 67-69% G418-resistant CFC. Repeated daily addition of new virus (up to three times), with assessment of the cells 2 days after the last addition of fresh virus, gave only a marginal improvement in the proportion of transduced CD34+ cells and CFC, but greatly increased the proportion of transduced LTC-IC (from 40% to >99%). Transplantation of lin- CB cells transduced using this latter 6-day protocol into NOD/SCID mice yielded readily detectable GFP+ cells in 10 of 11 mice that were engrafted with human cells. The proportion of the regenerated human cells that were GFP+ ranged from 0.2-72% in individual mice and included both human lymphoid and myeloid cells in all cases. High-level reconstitution with transduced human cells was confirmed by Southern blot analysis. These findings demonstrate that transplantable hematopoietic stem cells in human CB can be reproducibly transduced at high efficiency using a 6-day period of culture in a retrovirus-containing medium with either FL + SF + HIL-6 + TPO or FL + SF + IL-3 + IL-6 + G-CSF in which virus is added on the third, fourth, and fifth day.     

High marrow seeding efficiency of human lymphomyeloid repopulating cells in irradiated NOD/SCID mice

Transplantable human hematopoietic stem cells (competitive repopulating units [CRU]) can be quantitated based on their ability to produce large populations of lymphoid and myeloid progeny within 6 weeks in the marrow of intravenously injected, sublethally irradiated NOD/SCID mice. It is shown that the proportions of total injected human fetal liver and cord blood CRU in the marrow of mice 24 hours after transplantation are 5% and 7%, respectively, as determined by limiting-dilution assays in other primary and secondary NOD/SCID mice. The similarity in these 2 seeding efficiency values suggests that mechanisms regulating the ability of human hematopoietic stem cells to enter the marrow from the blood, at least in this xenotransplant model, do not change between fetal life and birth. In addition, it appears that previously reported human stem cell frequencies and their in vivo self-renewal activity measured in NOD/SCID mice have been markedly underestimated. (Blood. 2000;96:3979-3981)     

Enhanced engraftment of umbilical cord blood-derived stem cells in NOD/SCID mice by cotransplantation of a second unrelated cord blood unit

OBJECTIVE: Umbilical cord blood (UCB) is considered as an attractive alternative source of hematopoietic stem cells for allogeneic stem cell transplantations in patients who lack human leukocyte antigen (HLA)-matched donors. However, the low cell dose adversely affects hematopoietic recovery and therefore limits application of UCB transplantation in adults. Transplantation of multiple UCB units could be a strategy to overcome cell dose limitations. MATERIALS AND METHODS: To investigate the effect of double cord transplantation, nonobese diabetic/severe combined immunodeficient mice were transplanted with human hematopoietic progenitor cells (CD34(+)) derived from two UCB units with HLA disparity. Human cell engraftment and donor origin was determined by flow cytometry. RESULTS: Double CB transplantation resulted in increased engraftment levels in the bone marrow and peripheral blood in comparison with recipients of a single unit. Because this effect could be due to the higher cell dose (2.10(5) vs 1.10(5) cells), double CB transplantation was compared with single units containing equal cell numbers (2.10(5)). In some cases, engraftment levels in recipients of single units containing 2.10(5) cells were significantly higher than after transplantation of 1.10(5) cells. These engraftment levels were similar to those observed after double CB transplantation. Chimerism analysis indicated that increased engraftment in recipients of two units was predominantly derived from one unit, whereas in other cases the contribution of the two units was similar. CONCLUSION: These results indicate that engraftment may be enhanced by addition of a second unrelated CB that might be attributed to a cell dose effect or due to a graft-facilitating effect.     

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.     

Adapted NOD/SCID model supports development of phenotypically and functionally mature T cells from human umbilical cord blood CD34(+) cells

The NOD-LtSZ scid/scid (NOD/SCID) repopulation assay is the criterion for the study of self-renewal and multilineage differentiation of human hematopoietic stem cells. An important shortcoming of this model is the reported absence of T-cell development. We studied this aspect of the model and investigated how it could be optimized to support T-cell development. Occasionally, low-grade thymic engraftment was observed in NOD/SCID mice or Rag2(-/-)gamma(c)(-/-) mice. In contrast, the treatment of NOD/SCID mice with a monoclonal antibody against the murine interleukin-2R beta, (IL-2R beta) known to decrease natural killer cell activity, resulted in human thymopoiesis in up to 60% of the mice. T-cell development was phenotypically normal and resulted in polyclonal, mature, and functional CD1(-) TCR alpha beta (+) CD4(+) or CD8(+) single-positive T cells. In mice with ongoing thymopoiesis, peripheral T cells were observed. TREC analysis showed that T cells with a naive phenotype (CD45RA(+)) emerged from the thymus. In approximately half of these mice, the peripheral T cells included a pauciclonal outgrowth of CD45RO(+) cells. These data suggest that all elements of a functional immune system were present in these animals.     

Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood

Time course studies of sublethally irradiated non-obese mice with severe combined immunodeficiency (NOD/SCID mice) transplanted intravenously with 10⁷ human cord blood cells showed a rapid and parallel regeneration of human erythroid, granulopoietic, megakaryopoietic and B-lymphoid progenitors, as well as more primitive subpopulations of CD34⁺ cells (defined by their multi-lineage in vitro colony-forming ability, coexpression of Thy-1, or functional activity in long-term culture-initiating cell [LTC-IC] assays), in the marrow, spleen and blood. Maximum numbers of human cells were reached within 6 weeks and were then sustained for another 18–20 weeks. ³H-thymidine suicide studies showed all types of in vitro clonogenic human progenitors tested and the human LTC-IC to be proliferating in vitro throughout this period. A 2-week course of injections of human Steel factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin given just prior to assessment of the mice had no effect on any of these human engraftment parameters. 4–6 weeks post-transplant, the marrow of primary NOD/SCID recipients contained human cells that were able to regenerate lymphopoiesis and/or myelopoiesis in secondary irradiated NOD/SCID mice. These findings establish a baseline for the kinetics of engraftment, multi-lineage differentiation and self-renewal of human cord blood stem cells in this xenogeneic transplant model and thus set the stage for future studies of their regulation in vivo .     

Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice

In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34(+) cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45(+) cells expressed the transgene 90 to 120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45(+) cells expressing the EGFP transgene at this time. The transplantation of purified EGFP(+) cells increased the proportion of CD45(+) cells positive for EGFP expression to 57. 7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, respectively. Interestingly, the transplantation of EGFP(-) cells purified at 24 hours after infection also generated a significant engraftment of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.     

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.     

Cocultivation of umbilical cord blood CD34^＋ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells

AIM:To establish a novel coculture system for ex vivoexpansion of umbilical cord blood(UCB)hematopoieticprogenitors using thrombopoietin(TPO)/Flt-3 ligand(FL)-transduced human marrow-derived mesenchymalstem cells(tfhMSCs)as feeder.METHODS:UCB CD34~ cells were isolated and culturedusing four culture systems in serum-containing or serum-free medium.Suitable aliquots of cultured cells wereused to monitor cell production,clonogenic activity,and long-term culture-initiating culture(LTC-IC)output.Finally,the severe-combined immunodeficient(SCID)mouse-repopulating cell(SRC)assay was performed toconfirm ability of the cultured cells to reconstitute long-term hematopoiesis.RESULTS:There were no significant differences in thenumber of total nucleated cells among different culturesystems in serum-containing medium during 21-dculture.However,on d 14,the outputs of CD34~ cells,CFU-C and CFU-GEMM in ffhMSCs coculture system weresignificantly enhanced.LTC-IC assay demonstrated thatthe tfhMSCs coculture system had the most powerfulactivity.The severe-combined immunodeficient(SCID)mouse repopulating cell(SRC)assay confirmed extensiveability of the expanded cells to reconstitute long-termhematopoiesis.Furthermore,PCR analysis demonstratedthe presence of human hematopoietic cells in the bonemarrow and peripheral blood cells of NOD/SCID mice. CONCLUSION:The TPO/FL-transduced hMSCs,incombination with additive cytokines,can effectivelyexpand hematopoietic progenitors from UCB in vitro andthe tfhMSCs coculture system may be a suitable systemfor ex vivo manipulation of primitive progenitor cellsunder contact culture conditions.     

Efficient assay for evaluating human thrombopoiesis using NOD/SCID mice transplanted with cord blood CD34+ cells

A suitable model for the preclinical study of human platelet production in vivo has not been available. NOD/SCID mice were characterized as representing an efficient engraftment model for human hematopoietic stem cells, which resulted in the production of human platelets. Here, we evaluated in vivo human thrombopoiesis and ex vivo human platelet functions in NOD/SCID mice transplanted with human cord blood (CB) CD34(+) cells. Human platelets and human CD45(+) cells appeared in peripheral blood of NOD/SCID mice from 4 wk after transplantation. Human platelets produced in these mice showed CD62P expression and the activation of GPIIb/IIIa on human platelets on stimulation with an agonist. PEG-rHuMGDF (0, 0.5 and 5 microg/kg/d s.c.) was injected for 14 d into mice that had been confirmed to produce human platelets stably. The number of human platelets increased about twofold at 0.5 microg/kg/d and about fivefold at 5 microg/kg/d after 14 d. Withdrawal of PEG-rHuMGDF administration caused the human platelet count to return to the pretreatment level. Further, re-administration of PEG-rHuMGDF induced a similar human thrombopoietic response as it did on initial administration. These results suggest that NOD/SCID mice engrafted with human CB CD34(+) cells will be useful for the study of human platelet production in vivo.     

Platelet-derived growth factor promotes ex vivo expansion of CD34+ cells from human cord blood and enhances long-term culture-initiating cells,non-obese diabetic/severe combined immunodeficient repopulating cells and formation of adherent cells

Platelet-derived growth factor (PDGF) is a major mitogen for connective tissue cells. In this study, we investigated the effects and mechanism of PDGF on the ex vivo expansion of cord blood CD34+ cells. Our data demonstrated that among various cytokine combinations of thrombopoietin (TPO), interleukin 1 beta (IL-1beta), IL-3, IL-6 and Flt-3 ligand (Flt-3L), TPO + IL-6 + Flt-3L was most efficient in promoting the expansion of CD34+ cells, CD34+CD38- cells, mixed-lineage colony-forming units (CFU-GEMM) and long-term culture-initiating cells (LTC-IC) by 21.7 +/- 5.00-, 103 +/- 27.9-, 10.7 +/- 7.94- and 6.52 +/- 1.51-fold, respectively, after 12-14 d of culture. The addition of PDGF increased the yield of these early progenitors by 45.0%, 66.5%, 45.1% and 79.8% respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice. The expression of PDGF receptor (PDGFR)-beta was not detectable in fresh CD34+ cells but was upregulated after culture for 3 d. PDGF also enhanced the development of adherent cells/clusters that expressed the endothelial markers VE-cadherin and CD31. These findings suggest that PDGF is an effective cytokine for the ex vivo expansion of early stem and progenitor cells. The mechanism could be mediated by PDGFR-beta on committed CD34+ progenitor cells and/or secondary to the stimulation of autologous, stromal feeder cells.     

Direct comparison by limiting dilution analysis of long-term culture- initiating cells in human bone marrow, umbilical cord blood, and blood stem cells

Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two- stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.     

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.     

Evaluation of TCR Vbeta subfamily T cell expansion in NOD/SCID mice transplanted with human cord blood hematopoietic stem cells

Examination of the T cell receptor (TCR) gene repertoire is important in the analysis of the immune status of models, because clonal expansion of T cells permits the identification of specific antigen responses of T cells. Little is known about T-cell immunity in the humanized NOD/SCID mouse model. TCR Vbeta repertoire usage and clonality were analyzed to investigate the distribution and clonal expansion of TCR Vbeta subfamily T cells in NOD/SCID mice transplanted with human cord blood (CB) hematopoietic stem cells. The NOD/SCID mice were sublethally irradiated ((60)Co, 300cGy) to eliminate residual innate immunity in the host. The experimental mice were transplanted intravenously with CB CD34(+) cells sorted by MACS. After 6 weeks, RNA was obtained from peripheral blood, bone marrow and thymus of the study animals. The gene expression and clonality of the TCR Vbeta repertoire were determined by RT-PCR and GeneScan techniques. A restricted range of TCR Vbeta usage was exhibited in the bone marrow of mice, which included TCR Vbeta 1, 2, 9, 13 and 19. Further, oligoclonal expression of some TCR Vbeta subfamilies (Vbeta9, 13, 19) was identified by GeneScan technique. To investigate the reason for oligoclonal expansion of the TCR Vbeta subfamily T cells from CB in mouse models, the T-cell culture with tissue-antigen of NOD/SCID mouse was performed in vitro. The cells from peripheral blood mononuclear cells and bone marrow, spleen, thymus in NOD/SCID mice were frozen and thawed, and used as tissue-antigen. CB mononuclear cells were separately cultured with the component from those murine cells for 15-20 days. Oligoclonal expression or oligoclonal trend of some TCR Vbeta subfamilies (Vbeta10, 11 and Vbeta2, 15, 16, 19) was detected in T cells after stimulation with tissue-antigen of NOD/SCID mouse. Interestingly, a similar clonal expansion of the TCR Vbeta11 subfamily was found in T cells cultured with peripheral blood, bone marrow and spleen respectively. The TCR Vbeta subfamily T cells could be reconstituted in humanized NOD/SCID mouse transplanted with CD34(+) cells from CB. The restricted expression and clonal expansion of some CB T cell clones may be induced by tissue-antigens of NOD/SCID mice.     

Stem cell factor improves SCID-repopulating activity of human umbilical cord blood-derived hematopoietic stem/progenitor cells in xenotransplanted NOD/SCID mouse model

Poor in vivo homing capacity of hematopoietic stem/progenitor cells (HS/PCs) from umbilical cord blood (UCB) can be reversed by short-term ex vivo manipulation with recombinant human stem cell factor (rHuSCF). This study was designed to evaluate the effect of ex vivo manipulation of UCB-derived HS/PCs with rHuSCF on human cell engraftment rates in xenotransplanted NOD/SCID mouse model. The human cell engraftment rates in xenotransplanted primary and secondary NOD/SCID mice were characterized using four-color flow cytometric analysis and progenitor assay. Grafts of rHuSCF-treated UCB CD34(+) cells resulted in significantly higher levels of human cell engraftment than that of nontreated ones in both xenotransplanted primary and secondary NOD/SCID recipients. Fresh UCB CD34(+) cells did not express either of the matrix metalloproteinase (MMP) family members MMP-2 or MMP-9. rHuSCF-treated UCB CD34(+) cells expressed significant levels of MMP-2 and MMP-9. Pretreatment of UCB CD34(+) cells with the specific MMP inhibitor completely blocked human cell engraftment in xenotransplanted NOD/SCID recipients. Our results indicate that ex vivo manipulation of human HS/PCs with rHuSCF might provide an optimal approach to develop more effective stem cell-based therapies in situations where engraftment is delayed due to limiting HS/PCs number, for example, UCB transplantation.     

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.     

Multilineage engraftment of refrozen cord blood hematopoietic progenitors in NOD/SCID mice

Seven cord blood (CB) units were tested for their capacity to repopulate irradiated NOD/SCID mice after one or two successive cryopreservation procedures. In primary transplants with frozen or refrozen CB cells we observed equivalent human colonies and percentages of human CD45+ cells, with multilineage engraftment. In secondary transplants flow cytometry and polymerase chain reaction for the a satellite region of chromosome 17 showed equivalent levels of human engraftment. Since CB units have, to date, mainly been stored in individual bags, our results suggest new options for optimizing the timing of infusions of expanded and non-expanded progenitors in transplants.