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Query: UMLS:C0751781 (NOD)
 6,696 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) recipients. These SCID-repopulating cells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34(+) CD38(-) cells. The highly purified SRC were capable of extensive proliferation in NOD/SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400, 000 progeny 6 weeks after the transplant. Detailed flow cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34(+) CD38(-) cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and/or expanding these cells during in vitro culture.
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PMID:Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. 914 35

On the subject of acute myeloid leukemia (AML), there is little consensus about the target cell within the hematopoietic stem cell hierarchy that is susceptible to leukemic transformation, or about the mechanism that underlies the phenotypic, genotypic and clinical heterogeneity. Here we demonstrate that the cell capable of initiating human AML in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice) - termed the SCID leukemia-initiating cell, or SL-IC - possesses the differentiative and proliferative capacities and the potential for self-renewal expected of a leukemic stem cell. The SL-ICs from all subtypes of AML analyzed, regardless of the heterogeneity in maturation characteristics of the leukemic blasts, were exclusively CD34++ CD38-, similar to the cell-surface phenotype of normal SCID-repopulating cells, suggesting that normal primitive cells, rather than committed progenitor cells, are the target for leukemic transformation. The SL-ICs were able to differentiate in vivo into leukemic blasts, indicating that the leukemic clone is organized as a hierarchy.
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PMID:Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. 921 98

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic-scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34(-)CD19(+) (B-lymphoid) and CD34(+) (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains approximately 5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38(-) and CD38(+) subsets of CD34(+) CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34(+)CD38(-) human CB cells in serum-free medium containing flt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant, P < 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.
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PMID:Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. 927 12

We report here on a novel stromal cell line, AGM-S3, derived from the aorta-gonad-mesonephros (AGM) region of a 10.5 days postcoitum (dpc) mouse embryo. The AGM-S3 cells promoted production of hematopoietic progenitors and day-12 spleen colony-forming cells from Lin-c-Kit+Sca-1(+) murine primitive hematopoietic cells. They also supported for 6 weeks generation of human multipotential progenitors from cord blood CD34(+)CD38(-) primitive hematopoietic cells. Human long-term repopulating hematopoietic stem cells (LTR-HSC) with the potential to reconstitute hematopoiesis in NOD/SCID mice were maintained on AGM-S3 cells for at least 4 weeks. Flow cytometric analysis showed that CD13, vascular cellular adhesion molecule-1, and Sca-1 were expressed on AGM-S3 cells. Because stem cell factor, interleukin-6 (IL-6), and oncostatin M, but not IL-3, IL-11, leukemia- inhibitory factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, thrombopoietin, and Flk2 ligand were detected in reverse transcription-polymerase chain reaction analysis of AGM-S3 cells, the cells seem to express species-cross reactive molecule(s) other than the cytokines examined and which act on primitive hematopoietic progenitor/stem cells. This cell line is expected to elucidate molecular mechanisms regulating early hematopoiesis and pave the way for developing strategies for expansion of human transplantable HSC.
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PMID:Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. 973 Oct 61

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here, we identify a newly discovered human repopulating cell, distinct from previously identified repopulating cells, that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells, or CD34neg-SRC. CD34neg-SRC are restricted to a Lin-CD34-CD38- population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR, Thy-1 and CD34). In contrast to CD34+ subfractions, Lin-CD34-CD38- cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations, providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation.
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PMID:A newly discovered class of human hematopoietic cells with SCID-repopulating activity. 973 90

Purified CD34(+) and CD34(+)CD38(-) human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296-supported transduction, CD34(+) and CD34(+)CD38(-) UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 10(5) input equivalent transduced CD34(+) or 5 x 10(3) input equivalent CD34(+)CD38(-) UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34(+) cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45(+) cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area-forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34(+) cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34(+) and highly purified CD34(+)CD38(-) UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.
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PMID:Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice. 983 3

Acute myeloid leukemia (AML) occurs as the result of malignant transformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML cells are capable of proliferation in vitro, suggesting that AML cells may be organized in a hierarchy, with only the most primitive of these cells capable of maintaining the leukemic clone. To further investigate this hypothesis, we have evaluated a strategy for purifying these primitive cells based on surface antigen expression. As an in vitro endpoint, we have determined the phenotype of AML progenitor cells which are capable of producing AML colony-forming cells (CFU) for up to 8 weeks in suspension culture (SC) and compared the phenotype with that of cells which reproduce AML in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activated cell sorted (FACS) for coexpression of CD34 and CD71, CD38, and/or HLA-DR and the subfractions were assayed in vitro and in vivo at various cell doses to estimate purification. While the majority of primary AML CFU lacked expression of CD34, most cells capable of producing CFU after 2 to 8 weeks in SC were CD34(+)/CD71(-). HLA-DR expression was heterogeneous on cells producing CFU after 2 to 4 weeks. However, after 6 to 8 weeks in SC, the majority of CFU were derived from CD34(+)/HLA-DR- cells. Similarly, the majority of cells capable of long-term CFU production from SC were CD34(+)/CD38(-). Most cells that were capable of engrafting NOD/SCID mice were also CD34(+)/CD71(-) and CD34(+)/HLA-DR-. Engraftment was not achieved with CD34(+)/CD71(+) or HLA-DR+ subfractions, however, in two patients, both the CD34(+) and CD34(-) subfractions were capable of engrafting the NOD/SCID mice. A three-color sorting strategy combining these antigens allowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cells in one experiment. Phenotyping studies suggest even higher purification could be achieved by combining lack of CD38 expression with the CD34(+)/CD71(-) or CD34(+)/HLA DR- phenotype. These results suggest that most AML cells capable of long-term proliferation in vitro and in vivo share the CD34(+)/CD71(-)/HLA-DR- phenotype with normal stem cells. Our data suggests that in this group of patients the leukemic transformation has occurred in a primitive progenitor, as defined by phenotype, with some degree of subsequent differentiation as defined by functional assays.
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PMID:Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71(-)/HLA-DR-. 983 39

The identification of molecules that regulate human hematopoietic stem cells has focused mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs) may play a critical role in the specification of hematopoietic tissue from the mesodermal germ layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified primitive human hematopoietic cells from adult and neonatal sources. Populations of rare CD34(+)CD38(-)Lin- stem cells were isolated from human hematopoietic tissue and were found to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor beta, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34(+)CD38(-) phenotype and repopulation capacity. In contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34(+) CD38(-)Lin- cells, whereas at higher concentrations BMP-4 extended the length of time that repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a new pathway for controlling human stem cell development and a powerful model system for studying the biological mechanism of BMP action using primary human cells.
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PMID:Bone morphogenetic proteins regulate the developmental program of human hematopoietic stem cells. 1019 Sep 5

In contrast to myeloid and B-lymphoid differentiation, which take place in the marrow environment, development of T cells requires the presence of thymic stromal cells. We demonstrate in this study that human CD34+, CD34+ CD38+ and CD34+ CD38(low) cells from both cord blood and adult bone marrow reproducibly develop into CD4+ CD8+ T cells when introduced into NOD-SCID embryonic thymuses and further cultured in organotypic cultures. Such human/mouse FTOC fetal thymic organ culture) thus represents a reproducible and sensitive system to assess the T-cell potential of human primitive progenitor cells. The frequency of T-cell progenitors among cord-blood-derived CD34+ cells was estimated to be 1/500. Furthermore, the differentiation steps classically observed in human thymus were reproduced in NOD-SCID FTOC initiated with cord blood and human marrow CD34+ cells: immature human CD41(low) CD8- sCD3- TCR alphabeta- CD5+ CD1a+ T cells were mixed with CD4+ CD8+ cells and more mature CD4+ CD8- TCR alphabeta+ cells. However, in FTOC initiated with bone marrow T progenitors, <10% double-positive cells were observed, whereas this proportion increased to 50% when cord blood CD34+ cells were used, and most CD4+ cells were immature T cells. These differences may be explained by a lower frequency of T-cell progenitors in adult samples, but may also suggest differences in the thymic signals required by bone marrow versus cord blood T progenitors. Finally, since cytokine-stimulated CD34+ CD38(low) cells retained their ability to generate T cells, these FTOC assays will be of value to monitor, when combined with other biological assays, the influence of different expansion protocols on the potential of human stem cells.
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PMID:Identification of human T-lymphoid progenitor cells in CD34+ CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures. 1019 44

Evidence has been provided recently that shows that high concentrations of cytokines can fulfill functions previously attributed to stromal cells, such as promote the survival of, and led to a net increase in human primitive progenitors initiating long-term cultures in vitro (LTC-IC) or engrafting NOD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in vivo. These data prompted us to re-evaluate whether stromal cells will further alter the properties of primitive progenitor cells exposed to cytokines. Single CD34(+)CD38(low) and CD38(neg) cells were incubated 10 days in serum-containing or serum-free medium in the presence or in the absence of murine marrow-derived stromal cells (MS-5). Recombinant human cytokines stem cell factor (SCF), pegylated-megakaryocyte growth and differentiation factor (PEG-MGDF), FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were systematically added at various concentrations (10 to 300 ng/mL). Cell proliferation and LTC-IC potential were evaluated in each clone after 10 days. A striking and consistent observation was the retention of a high LTC-IC potential in clones exposed to cytokines in the presence of stromal feeders, whereas clones exposed to cytokines alone in the absence of stromal feeders rapidly lost their LTC-IC potential as they proliferated. This was reflected both by the higher proportion of wells containing LTC-IC and by the high numbers of CFC produced after 5 weeks in clones grown with MS-5 during the first 10 days. We further showed by analyzing multiple replicates of a single clone at day 10 that MS-5 cells promoted a net increase in the LTC-IC compartment through self-renewal divisions. Interestingly, these primitive LTC-IC were equally distributed among small and large clones, as counted at day 10, indicating that active proliferation and loss of LTC-IC potential could be dissociated. These observations show that, in primitive cells, stromal cells counteract differentiation events triggered by cytokines and promoted self-renewal divisions. Furthermore, the almost identical distribution of the size of the clones with or without MS-5 suggests that proliferation and function of human primitive cells may be independently regulated by external signals, and that the former is primarily under the control of cytokines.
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PMID:Murine stromal cells counteract the loss of long-term culture-initiating cell potential induced by cytokines in CD34(+)CD38(low/neg) human bone marrow cells. 1039 20


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