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.     

Human short-term repopulating stem cells are efficiently detected following intrafemoral transplantation into NOD/SCID recipients depleted of CD122+ cells

The nonobese diabetic/severe combined immune deficiency (NOD/SCID) xenotransplantation model has emerged as a widely used assay for human hematopoietic stem cells; however, barriers still exist that limit engraftment. We previously identified a short-term SCID-repopulating cell (SRC) following direct intrafemoral injection into NOD/SCID mice, whereas others characterized similar SRCs using NOD/SCID mice depleted of natural killer (NK) cell activity. To determine the model that most efficiently detects short-term SRCs, we compared human engraftment in 6 different xenotransplantation models: NOD/SCID-beta2-microglobulin-null mice, anti-CD122 (interleukin-2 receptor beta [IL-2Rbeta])-treated or unmanipulated NOD/SCID mice, each given transplants by intravenous or intrafemoral injection. Human cell engraftment was highest in intrafemorally injected anti-CD122-treated NOD/SCID mice compared to all other groups at 2 and 6 weeks after transplantation. These modifications to the SRC assay provide improved detection of human stem cells and demonstrate that CD122+ cells provide barriers to stem cell engraftment, a finding with potential clinical relevance.     

Natural killer cell depletion by anti-asialo GM1 antiserum treatment enhances human hematopoietic stem cell engraftment in NOD/Shi-scid mice

The scid mutation was backcrossed on to the NOD/Shi mouse background, resulting in the development of NOD/Shi-scid mice, which showed lack of mature lymphocytes, macrophage dysfunction and absence of circulating complement, but were not as impaired in natural killer (NK) cell activity as NOD/LtSz-scid mice. We then examined the effect of recipient NK cell depletion by anti-asialo GM1 antiserum on the repopulation of human cord blood (CB) hematopoietic stem cells (HSC) in NOD/Shi-scid mice to clarify the role of recipient NK cells in human HSC engraftment. The anti-asialo GM1 antiserum treatment significantly enhanced the engraftment of CB CD34+ cells, but did not affect the differentiation of the engrafted HSC into each hematopoietic lineage. The NK cell depletion was effective at early stages of the engraftment, but not 3 weeks after the transplantation. The anti-asialo GM1 antiserum treatment did not improve the engraftment by human HSC in scid mice which lack mature lymphocytes, but show neither macrophage dysfunction nor a reduction in circulating complement, indicating that macrophages and/or complement also have roles in HSC graft rejection. The present study indicates that the preconditioning targeting of recipient NK cells in addition to T cell suppression and myeloablation might prevent HSC graft failure, and that NOD/Shi-scid mice treated with anti-asialo GM1 antiserum could provide a useful tool for evaluating the repopulating ability of transplantable human HSC.     

An assay for long-term engrafting human hematopoietic cells based on newborn NOD/SCID/beta2-microglobulin(null) mice

OBJECTIVE: Of various types of xenograft assays, the use of NOD/SCID mice has been the most popular method for quantitating candidate human stem cells. Limitations of the assay include low levels of engraftment, except when large numbers of cells are injected, and the development of thymic lymphoma, which precluded observation of long-term engraftment. In order to establish an assay that allows long-term in vivo engraftment and higher engraftment levels by a reasonable number of human cells, we tested a model based on "conditioned newborn" NOD/SCID or NOD/SCID/beta2-microglobulin(null) (BMG(null)) mice. MATERIALS AND METHODS: Using human cord blood mononuclear cells, we tested various nonradiation conditioning regimens and cell injection routes. Conditioning with a combination of 5-fluorouracil (5FU) and anti-mouse c-kit blocking antibody (Ack-2) or a combination of busulfan (BU) and cyclophosphamide (CY) and the use of facial vein for the cell injection route yielded the highest levels of multilineage engraftment. RESULTS: At 4-5 months posttransplantation, the median of engraftment level in bone marrow with 5FU/Ack-2 and BU/CY regimens were 0.9% (range: 0.2-40.5%) and 2.1% (range: 0.3-2.4%) in NOD/SCID mice, and 11.3% (range: 0.7-38%) and 14.1% (range: 0.8-52%) in NOD/SCID/BMG(null) mice, respectively. Multilineage engraftment was demonstrated by flow cytometry and by the growth of multilineage colonies in methylcellulose culture. Secondary transplantation of the isolated human CD45(+) cells, also performed at 4-5 months posttransplantation, revealed engraftment at the levels of 1.5 +/- 0.42% at 2 months after secondary transplantation. CONCLUSION: Our assay may provide a quantitative method for analysis of human hematopoietic stem cells.     

Modeling normal and malignant human hematopoiesis in vivo through newborn NSG xenotransplantation

Various strains of immune-compromised mice have been developed to investigate human normal and malignant stem cells in vivo. NOD/SCID mice harboring complete null mutation of Il2rg (NSG mice) lack T cells, B cells, and NK cells, and support high levels of engraftment by human cord blood hematopoietic stem cells (CB HSCs) and acute myeloid leukemia stem cells (AML LSCs). In addition to achieving high levels of human hematopoietic cell engraftment, use of newborn NSG mice as recipients has enabled the investigation into how human CB HSCs generate mature immune subsets in vivo. Moreover, through establishing an in vivo model of human primary AML by xenotransplantation of human LSCs into newborn NSG mice, functional properties of human AML such as cell cycle, location, and self-renewal capacity can be examined in vivo. Newborn NSG xenogeneic transplantation model may facilitate the understanding of human normal and malignant hematopoiesis and contribute to the development of novel therapies against hematologic diseases.     

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.     

Engraftment of human CD34+ cells leads to widespread distribution of donor-derived cells and correction of tissue pathology in a novel murine xenotransplantation model of lysosomal storage disease

A novel murine system was developed to study the in vivo localization of xenotransplanted human cells and assess their therapeutic effect in an authentic model of disease. The beta-glucuronidase (GUSB) mutation of the mucopolysaccharidosis type VII (MPSVII) mouse was backcrossed onto the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation strain. The resulting NOD/SCID/MPSVII mice displayed the characteristic features of lysosomal storage disease because of GUSB deficiency and were also capable of engrafting human cells. Human CD34+ hematopoietic progenitor cells from healthy, GUSB+ donors engrafted NOD/SCID/MPSVII mice in a manner similar to that of standard NOD/SCID mice. Six to 12 weeks following transplantation, 1% to 86% of the host bone marrow was positive for human CD45. By using a GUSB-specific histochemical assay, human engraftment was detected with single-cell sensitivity not only in well-characterized hematopoietic tissues like bone marrow, spleen, lymph node, and thymus, but also in other nonhematopoietic organs like liver, kidney, lung, heart, brain, and eye. Quantitative measurements of GUSB activity confirmed this expansive tissue distribution. The GUSB-specific assays were validated for their accuracy in identifying human cells through colocalization of human CD45 expression with GUSB activity in tissues of mice receiving transplants. An analysis of the therapeutic effects of engrafted human cells revealed a reduction of pathologic storage material in host organs, including the bone, spleen, and liver. Such xenotransplantation experiments in the NOD/SCID/MPSVII mouse represent a powerful approach to both study the in vivo biology of human cells and gather preclinical data regarding treatment approaches for a human disease.     

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.     

Development of both human connective tissue-type and mucosal-type mast cells in mice from hematopoietic stem cells with identical distribution pattern to human body

The transplantation of primitive human cells into sublethally irradiated immune-deficient mice is the well-established in vivo system for the investigation of human hematopoietic stem cell function. Although mast cells are the progeny of hematopoietic stem cells, human mast cell development in mice that underwent human hematopoietic stem cell transplantation has not been reported. Here we report on human mast cell development after xenotransplantation of human hematopoietic stem cells into nonobese diabetic severe combined immunodeficient (NOD/SCID)/gamma(c)(null) (NOG) mice with severe combined immunodeficiency and interleukin 2 (IL-2) receptor gamma-chain allelic mutation. Supported by the murine environment, human mast cell clusters developed in mouse dermis, but they required more time than other forms of human cell reconstitution. In lung and gastric tract, mucosal-type mast cells containing tryptase but lacking chymase located on gastric mucosa and in alveoli, whereas connective tissue-type mast cells containing both tryptase and chymase located on gastric submucosa and around major airways, as in the human body. Mast cell development was also observed in lymph nodes, spleen, and peritoneal cavity but not in the peripheral blood. Xenotransplantation of human hematopoietic stem cells into NOG mice can be expected to result in a highly effective model for the investigation of human mast cell development and function in vivo.     

Characterization of the NOD/scid-[Tg]DR1 mouse expressing HLA-DRB1*01 transgene: a model of SCID-hu mouse for vaccine development

OBJECTIVE: We previously showed enhanced engraftment of human T cells in the transgenic NonObese Diabetic/severe combined immunodeficient (NOD/scid)-DR1 mice, compared to NOD/scid mice. We now characterize their immunobiology, innate immunity, and intrahepatic neonatal engraftment of cord blood mononuclear cells (CBMNC), and test immune responses of these chimeric mice to an experimental cancer vaccine. METHODS: Fluorescence in situ hybridization analysis, blood biochemistry, hematology, and fluorescein-activated cell sorting analyses of cellular subsets were performed on NOD/scid-DR1 mice, in comparison to parental NOD/scid mice. Innate immunity and lifespan were examined. Histology of engrafted tissues and short-term intrahepatic engraftment of CBMNC were performed. Intracellular interferon-gamma (IFN-gamma) production was assessed in mice immunized with cancer vaccine. RESULTS: The DR1 transgene was located on chromosome 5 and no significant changes were observed in blood chemistry, peripheral blood counts, lymphoid subsets, natural killer cell and lipopolysaccharide response, and antigen presentation in the NOD/scid-DR1 mice, compared to NOD/scid mice. Interestingly, NOD/scid-DR1 mice had a significantly longer lifespan (approximately 14 months) than NOD/scid mice (approximately 8.5 months). Engraftment with human cord blood cells resulted in slight changes in the architecture/structure of spleens. No correlation was found between DR1 genotype of the donor CBMNC and extent of engraftment of human T cells. Enhanced engraftment of human cells was observed with intrahepatic injections of CBMNC in neonatal NOD/scid DR1 mice. Intracellular IFN-gamma was detected in human cells, when chimeric mice were immunized with a cancer vaccine. CONCLUSION: NOD/scid-DR1 mice were similar in most of the physiological parameters as the NOD/scid mice, with the exception of longer lifespan. Intrahepatic engraftment of neonatal mice is the preferred protocol of xenotransplantation in this model and the engrafted human cells can respond to a cancer vaccine.     

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.     

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.     

Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses

Critical to the development of an effective HIV/AIDS model is the production of an animal model that reproduces long-lasting active replication of HIV-1 followed by elicitation of virus-specific immune responses. In this study, we constructed humanized nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/interleukin-2 receptor gamma-chain knockout (IL2Rgamma(null)) (hNOG) mice by transplanting human cord blood-derived hematopoietic stem cells that eventually developed into human B cells, T cells, and other monocytes/macrophages and 4 dendritic cells associated with the generation of lymphoid follicle-like structures in lymphoid tissues. Expressions of CXCR4 and CCR5 antigens were recognized on CD4+ cells in peripheral blood, the spleen, and bone marrow, while CCR5 was not detected on thymic CD4+ T cells. The hNOG mice showed marked, long-lasting viremia after infection with both CCR5- and CXCR4-tropic HIV-1 isolates for more than the 40 days examined, with R5 virus-infected animals showing high levels of HIV-DNA copies in the spleen and bone marrow, and X4 virus-infected animals showing high levels of HIV-DNA copies in the thymus and spleen. Furthermore, we detected both anti-HIV-1 Env gp120- and Gag p24-specific antibodies in animals showing a high rate of viral infection. Thus, the hNOG mice mirror human systemic HIV infection by developing specific antibodies, suggesting that they may have potential as an HIV/AIDS animal model for the study of HIV pathogenesis and immune responses.     

In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice

We have previously demonstrated that high levels of allogeneic, donor-derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34⁺ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45⁺ human cells. CD45⁺ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3⁺ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16-fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.     

Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor γ chain

Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human haemopoiesis and lymphoid cell function. However, the utility of existing mouse strains can be limited by shortened life-spans, spontaneous production of functional lymphocytes with ageing, and residual innate immunity leading to variable levels of engraftment. Mice with a deletion of the common cytokine receptor γ chain (γc) gene have reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2)-deficient strain produced mice doubly homozygous for the γc and RAG2 null alleles (γc⁻/RAG2⁻). These mice have a stable phenotype characterized by the absence of all T lymphocyte, B lymphocyte and NK cell function. Injection of human B-lymphoblastoid cells resulted in earlier fatal metastatic lymphoproliferative disease than in NOD/LtSz- scid controls. This was particularly evident in animals injected intravenously, possibly because of residual NK activity in NOD/LtSz- scid mice. Levels of engraftment with peripheral-blood-derived human lymphocytes were also increased and associated with higher CD4/CD8 ratios. These findings demonstrate that this new strain of immunodeficient mice has significant advantages over existing strains for engraftment of human cells, and may be useful for study of adoptive immunotherapy and novel therapies for GvHD and HIV infection.     

beta2 microglobulin-deficient (B2m(null)) NOD/SCID mice are excellent recipients for studying human stem cell function

Human SCID repopulating cells (SRC) are defined based on their functional ability to repopulate the bone marrow of NOD/SCID mice with both myeloid and lymphoid cell populations. The frequency of SRC in umbilical cord blood cells is 1 in 9.3 x 10(5) mononuclear cells. We report that as few as 8 x 10(4) human cord blood mononuclear cells transplanted into NOD/SCID/B2m(null )mice resulted in multilineage differentiation in the murine bone marrow, revealing a more than 11-fold higher SRC frequency than in NOD/SCID mice. Moreover, as few as 2 to 5 x 10(3) CD34(+) cells recovered from the bone marrow of primary transplanted NOD/SCID mice were sufficient for engrafting secondary NOD/SCID/B2m(null )mice with SRC, suggesting SRC self-renewal. Thus, by using NOD/SCID/B2m(null )mice as recipients, we established a functional assay for human stem cells capable of engrafting the bone marrow of primary and secondary transplanted immune-deficient mice with SRC, providing a model that better resembles autologous stem cell transplantation.     

Intra-bone marrow transplantation facilitates pauci-clonal human hematopoietic repopulation of NOD/SCID/beta2m(-/-) mice

OBJECTIVE: Intra-bone marrow transplantation (IBMT) has been shown to improve the limit of detection of primitive human SCID-repopulating cells (SRC) in NOD/SCID mice when compared to intravenous transplantation. We sought to further refine detection of SRC by comparing NOD/SCID mice to the more sensitive NOD/SCID/beta2m(-/-)strain as IBMT recipients of limiting numbers of purified primitive human hematopoietic cells. MATERIALS AND METHODS: Purified human Lin(-)CD34(+)CD38- cells at limiting doses were delivered by IBMT into NOD/SCID and NOD/SCID/beta2m(-/-) strains of recipient mice. Six weeks posttransplantation, injected and noninjected bones were analyzed separately for multilineage human hematopoietic chimerism. RESULTS: NOD/SCID/beta2m(-/-) mice are superior recipients for IBMT and show a trend toward increased levels of human hematopoietic engraftment. In addition, in contrast to NOD/SCID recipients, NOD/SCID/beta2m(-/-) mice were reconstituted with as few as five highly purified cells, indicative of pauci-clonal repopulation. Analysis of injected and noninjected bones demonstrated that engrafting cells were capable of in vivo migration and expansion. Although SRC hematopoietic reconstitution of NOD/SCID mice is commonly lymphoid-dominant, multilineage analysis of separate bone sites following IBMT of purified cells revealed that a subset of mice was repopulated with a myeloid-dominant graft in at least one bone site, revealing that SRC are developmentally heterogeneous among Lin(-)CD34(+)CD38- cells and capable of distinct differentiation potential. CONCLUSION: IBMT into NOD/SCID/beta2m(-/-) mice provides a highly sensitive experimental transplantation assay for the detection of human hematopoietic repopulating cells and demonstrates that Lin(-)CD34(+)CD38- cells are more highly enriched for human SRC than originally predicted.     

Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice

Here we report that a new nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse line harboring a complete null mutation of the common cytokine receptor gamma chain (NOD/SCID/interleukin 2 receptor [IL2r] gamma(null)) efficiently supports development of functional human hemato-lymphopoiesis. Purified human (h) CD34(+) or hCD34(+)hCD38(-) cord blood (CB) cells were transplanted into NOD/SCID/IL2rgamma(null) newborns via a facial vein. In all recipients injected with 10(5) hCD34(+) or 2 x 10(4) hCD34(+)hCD38(-) CB cells, human hematopoietic cells were reconstituted at approximately 70% of chimerisms. A high percentage of the human hematopoietic cell chimerism persisted for more than 24 weeks after transplantation, and hCD34(+) bone marrow grafts of primary recipients could reconstitute hematopoiesis in secondary NOD/SCID/IL2rgamma(null) recipients, suggesting that this system can support self-renewal of human hematopoietic stem cells. hCD34(+)hCD38(-) CB cells differentiated into mature blood cells, including myelomonocytes, dendritic cells, erythrocytes, platelets, and lymphocytes. Differentiation into each lineage occurred via developmental intermediates such as common lymphoid progenitors and common myeloid progenitors, recapitulating the steady-state human hematopoiesis. B cells underwent normal class switching, and produced antigen-specific immunoglobulins (Igs). T cells displayed the human leukocyte antigen (HLA)-dependent cytotoxic function. Furthermore, human IgA-secreting B cells were found in the intestinal mucosa, suggesting reconstitution of human mucosal immunity. Thus, the NOD/SCID/IL2rgamma(null) newborn system might be an important experimental model to study the human hemato-lymphoid system.     

Involvement of CXCR4 and IL-2 in the homing and retention of human NK and NK T cells to the bone marrow and spleen of NOD/SCID mice

Human natural killer (NK) and NK T cells play an important role in allogeneic bone marrow (BM) transplantation and graft-versus-leukemia (GVL) effect. The mechanisms by which these cells home to the BM and spleen are not well understood. Here we show that treatment of these cells with pertussis toxin and neutralizing antibodies to the chemokine receptor CXCR4 inhibited homing of the cells to the BM, but not the spleen, of NOD/SCID mice. The retention of NK and NK T cells within the spleen and BM was dependent on Galphai signaling and CXCR4 function. The chemokine receptors CXCR4 and CXCR3 are expressed predominantly on the cell surface of NK T cells. Following activation with interleukin-2 (IL-2), the levels of CXCR4 on NK and NK T cells decreased significantly. Treatment of cells with IL-2 inhibited their migration in response to CXCL12 and their homing and retention in the BM and spleen of NOD/SCID mice. In contrast to CXCR4, the expression levels of the chemokine receptor CXCR3 and the migration of cells in response to CXCL9 and CXCL10 increased after IL-2 treatment. Thus, down-regulation of CXCR4 and up-regulation of CXCR3 may direct the trafficking of cells to the site of inflammation, rather than to hematopoietic organs, and therefore may limit their alloreactive potential.     

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.