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.     

Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells

OBJECTIVE: To support immune reconstitution after cord blood transplantation, immunotherapy using gene-modified dendritic cells (DCs), the most potent antigen-presenting cells, can be a powerful strategy for preventing infection and recurrence. To investigate the applicability of lentiviral vector-transduced DCs compared to retroviral vectors, we transduced umbilical cord blood (CB) CD34(+) cells, then expanded and differentiated them into DCs. MATERIALS AND METHODS: We transduced CB CD34(+) cells by vesicular stomatitis virus G-protein pseudotyped self-inactivating lentiviral vector or retroviral vectors carrying the enhanced green fluorescent protein gene. The cells were expanded in the stroma-dependent culture system and transferred to the culture condition for developing DCs. The efficiency of transduction and expression of the transgene in severe combined immunodeficiency (SCID) mice-repopulating cells (SRCs) and DCs were compared between lentiviral vector and retroviral vectors. Induced DCs were cocultured with allogeneic or autologous T cells to test the ability to present antigens. RESULTS: CB CD34(+) cells transduced by lentiviral vector and expanded ex vivo sustained stable transgene expression and multipotentiality by assessing SRCs assay and clonogenic assay of bone marrow cells from the transplanted mice. DCs derived from these cells expressed green fluorescent protein and surface markers CD1a, CD80, and HLA-DR and showed potent allo-stimulatory activity as well as nontransduced DCs did. On the other hand, we did not detect transgene expression in SRCs and DCs transduced by retroviral vectors. CONCLUSION: Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.     

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.     

Differentiation and maintenance of mast cells from CD34(+) human cord blood cells

OBJECTIVE: Establishment of a stable umbilical cord blood CD34(+) (UCB CD34(+)) cell culture system and identification of the cells in the cobblestone area differentiated from UCB CD34(+) long-term culture cells. MATERIALS AND METHODS: Human UCB CD34(+) cells were cultured on MS-5 mouse stroma cells in the presence of stem cell factor (SCF), flt-3 ligand (FL), and thrombopoietin (TPO) for 4 to 16 weeks. Cells in the culture medium and in the cobblestone area were collected and characterized by flow cytometry and microscopy. RESULTS: CD34(+) cells were stably expanded by culturing on MS-5 stroma cells in the presence of SCF, FL, and TPO for more than 4 months. Cells highly expressing CD117 (c-kit) appeared in the cobblestone area after 2 weeks and stably expanded. Isolation of cells highly expressing CD117 by fluorescence-activated cell sorter (FACS) revealed the cells were tryptase-positive and Fc epsilon receptor 1-negative mast cells. They showed typical mast cell morphology and released histamine upon stimulation by substance P or compound 48/80 in vitro. CONCLUSION: Human UCB CD34(+) cells were stably expanded on MS-5 stroma cells in the presence of SCF, FL, and TPO. Under this condition, multipotent CD34(+) cells and mast cells differentiated from UCB CD34(+) cells were expanded in the cobblestone area. The expanded mast cells showed histamine release after substance P or compound 48/80 stimulation. These human mast cells will be useful as a source of human cells for evaluating the allergic effects of drugs.     

Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells.

This study reports a lentiviral gene transfer protocol for efficient transduction of adult human peripheral blood (PB)-derived CD34+ NOD/SCID-repopulating cells (SRCs) using vesicular stomatitis virus-G protein (VSV-G)-pseudotyped lentiviruses encoding for enhanced green fluorescence protein (eGFP). Lentiviral stocks were concentrated by anion exchange chromatography, and transduction was performed under serum-free conditions at a multiplicity of infection (MOI) between 3 and 50. Similar transduction efficiencies were achieved in the presence and absence of cytokines. Transduction of PB-derived CD34+ cells at a MOI of 3 resulted in gene transfer efficiencies into SRCs of 9.2% and 12.0% in the absence and presence of cytokines, respectively. Using improved lentiviral vectors, transduction frequency varied between 42.0% (MOI 10) and 36.0% (MOI 50) with multilineage transgene expression within SRC-derived myeloid and lymphoid cells. The protocol described can be adapted for clinical application of lentiviral gene transfer into PB-derived CD34+ cells from adult patients.     

Similar myeloid recovery despite superior overall engraftment in NOD/SCID mice after transplantation of human CD34(+) cells from umbilical cord blood as compared to adult sources

Umbilical cord blood (UCB), bone marrow (BM) and mobilized peripheral blood (mPB) are used as sources of hematopoietic stem cells for transplantation. The NOD/SCID mouse model was used to compare the lineage-specific repopulating potential of CD34(+) cells derived from these sources. Six to 8 weeks after transplantation, blood, BM, spleen, liver and thymus, were harvested, and analyzed by flow cytometry using CD34, CD45, myeloid, and lymphoid lineage-specific antibodies. Fifty percent engraftment of human cells in bone marrow of mice was estimated to be reached with 0.55 x 10(6) CD34(+) UCB cells or with 7.9 x 10(6) CD34(+) cells from adult sources, illustrating a 10-fold superiority of UCB CD34(+) cells to engraft NOD/SCID mice. Lineage-specific characterization of engrafted human cells showed that the high engraftment potential of CD34(+) cells from UCB was due to a preferential B cell development (2-81%). In contrast, comparable percentages of myeloid cells were found following transplantation of CD34(+) cells from UCB, BM and mPB (1-38%), and occurred at significant levels only at relatively high doses. Since the CD34 content of UCB transplants is usually at least one log lower than of transplant from adult sources, these results correspond to the clinical findings with UCB transplantation showing a relatively high overall engraftment, but delayed myeloid recovery.     

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.     

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).     

Immunophenotypic and functional characterization of CD33(+)CD34(+) cells in human cord blood of preterm neonates

OBJECTIVE: To characterize CD33(+)CD34(+) cells, a major population in human cord blood (CB) CD34(+) cells of preterm neonates. MATERIALS: The proportion of CD33(+) cells was analyzed on CB CD34(+) cells from preterm and full-term neonates. CD33(+)CD34(+) cells were purified by cell sorting and analyzed on their clonogenic activity, proliferative activity in short-time liquid suspension culture, and GATA-2 mRNA expression by RT-PCR and Southern blot. RESULTS: The absolute numbers and proportion of CD34(+) cells in mononuclear cells inversely correlated with gestational age. CD33 was expressed on a majority of CB CD34(+) cells of preterm neonates but on only a minor population of them in full-term neonates. In addition, CD33 was dominantly expressed on CD38(-)CD34(+) cells or CD117(low)CD34(+) cells in CB of preterm neonates. CD33(+)CD34(+) cells of preterm cord blood had high proliferative and reproducible potentials compared with CD33(-)CD34(+) cells. CD33(+)CD34(+) cells as well as CD33(-)CD34(+) cells from preterm CB highly expressed GATA-2, in contrast to those from BM. CONCLUSIONS: These results suggest that CD33(+)CD34(+) cells, which are a major population in CB CD34(+) cells of preterm neonates, do not simply represent relatively mature myeloid lineage hematopoietic progenitor cells as those in adult BM CD34(+) cells, and may contain hematopoietic stem cells or primitive progenitor cells as in fetal liver.     

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.     

Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.     

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.     

Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells

The proliferation and expansion of human hematopoietic stem cells (HSC) in ex vivo culture was examined with the goal of generating a suitable clinical protocol for expanding HSC for patient transplantation.HSC were derived from umbilical cord blood (UCB) and adult patient peripheral blood stem cell collections. HSC were stimulated to proliferate ex vivo by a combination of two growth factors, flt-3 ligand (FL) and thrombopoietin/c-mpl ligand (TPO/ML), and assessed for expansion by flow cytometry.Ex vivo expansion cultures of UCB were maintained for prolonged periods (up to 16 weeks), and sufficient HSC were generated for adult transplantation. In contrast to UCB, FL + TPO/ML did not significantly increase CD34(+) peripheral blood stem cell (PBSC) numbers.UCB-HSC can be expanded in culture to numbers theoretically adequate for safe, rapid engraftment of adult patients. Additional studies are needed to establish the functional activity of expanded UCB-HSC.     

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.     

Kinetics of engraftment of CD34(-) and CD34(+) cells from mobilized blood differs from that of CD34(-) and CD34(+) cells from bone marrow

OBJECTIVE: Mobilized peripheral blood (PB) progenitors are increasingly used in autologous and allogeneic transplantation. However, the short- and long-term engraftment potential of mobilized PB or bone marrow (BM) has not been directly compared. Although several studies showed that BM-derived Lin(-)CD34(-) cells contain hemopoietic progenitors, no studies have addressed whether Lin(-)CD34(-) cells from mobilized PB contain hemopoietic progenitors. Here, we compared the short- and long-term engraftment potential of CD34(+) cells and Lin(-)CD34(-) cells in BM and PB of normal donors who received 5 days of granulocyte colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 35 x 10(3) CD34(+) or Lin(-)CD34(-) cells from G-CSF mobilized BM and PB of normal donors were transplanted in 60-day-old fetal sheep. Animals were evaluated 2 and 6 months after transplantation for human hemopoietic cells. In addition, cells recovered after 2 months from fetal sheep were serially passaged to secondary and tertiary recipients to assess long-term engrafting cells. RESULTS: Mobilized PB CD34(+) cells supported earlier development of human hemopoiesis than BM CD34(+) cells. When serially transferred to secondary and tertiary recipients, earlier exhaustion of human hematopoiesis was seen for PB than BM CD34(+) cells. A similar degree of chimerism was seen for Lin(-)CD34(-) cells from PB or BM in primary recipients. We again observed earlier exhaustion of human hemopoiesis with serial transplantation of PB than BM Lin(-)CD34(-) cells. CONCLUSIONS: Differences exist in the short- and long-term repopulating ability of cells in PB and BM from G-CSF mobilized normal donors, and this is independent of the phenotype. Studies are ongoing to examine if this reflects intrinsic differences in the repopulating potential between progenitors from PB and BM, or a lower frequency of long-term repopulating cells in PB than BM CD34(+) and Lin(-)CD34(-) cells, that may not be apparent if larger numbers of cells are transplanted.     

Thymic repopulation by CD34(+) human cord blood cells after expansion in stroma-free culture

Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34(+)CD38(-) or CD34(+)CD38(+) cells for up to 5 weeks in stroma-free cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-repopulating ability of expanded cells in vitro and in vivo. After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34(+)CD38(-) CB cells generated T cells and natural killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34(+)CD38(-) CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34(+)CD38(-) CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.     

SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection

Precise analysis of human CD34-negative (CD34(-)) hematopoietic stem cells (HSCs) has been hindered by the lack of a simple and reliable assay system of these rare cells. Here, we successfully identify human cord blood-derived CD34(-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) technique. Lineage-negative (Lin(-)) CD34(-) cells did not show SRC activity by conventional tail-vein injection, possibly due to their low levels of homing receptor expression and poor SDF-1/CXCR4- mediated homing abilities, while they clearly showed a high SRC activity by IBMI. They generated CD34(+) progenies not only in the injected left tibia but also in other bones following migration. Moreover, they showed slower differentiating and reconstituting kinetics than CD34(+) cells in vivo. These in vivo-generated CD34(+) cells showed a distinct SRC activity after secondary transplantation, clearly indicating the long-term human cell repopulating capacity of our identified CD34(-) SRCs in nonobese diabetic (NOD)/SCID mice. The unveiling of this novel class of primitive human CD34(-) SRCs by IBMI will provide a new concept of the hierarchy in the human HSC compartment and has important implications for clinical HSC transplantation as well as for basic research of HSC.     

Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/gammacnull mice model

Establishment of an assay capable of generating all classes of human lymphocytes from hematopoietic stem cells (HSCs) will provide new insight into the mechanism of human lymphopoiesis. We report ontogenic, functional, and histologic examination results of reconstituted human lymphocytes in NOD/SCID/ gammacnull mice after the transplantation of human cord blood (CB) CD34+ cells. After transplantation, human B, natural killer (NK), and T cells were invariably identified in these mice, even though no human tissues were cotransplanted. Immature B cells resided mainly in bone marrow (BM), whereas mature B cells with surface immunoglobulins were preferentially found in spleen. NK cells were identified in BM and spleen. T cells were observed in various lymphoid organs, but serial examinations after transplantation confirmed human T lymphopoiesis occurring in the thymus. These human lymphocytes were also functionally competent. Human immunoglobulin M (IgM), IgA, and IgG were detected in the sera of these mice. T cells showed a diverse repertoire of T-cell-receptor Vbeta (TCR Vbeta) chains, proliferated in response to phytohemagglutinin, and were cytotoxic against cell lines. NK activity was demonstrated using the K562 cell line. Immunohistochemical analysis revealed that human lymphocytes formed organized structures in spleen and thymus that were analogous to those seen in humans. In the thymus, CD4 and CD8 double-positive T cells were predominant and coexpressed CD1a and Ki-67, thereby supporting the notion that T lymphopoiesis was taking place. NOD/SCID/ gammacnull mice provide a unique model to investigate human lymphopoiesis without the cotransplantation of human tissues.