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

Ex vivo maintenance of human stem cells is crucial for many clinical applications. Current culture methods rely on optimized combinations of cytokines. Although these conditions provide some level of stem cell support, they primarily induce proliferation and differentiation, resulting in reduced repopulation capacity. The recently identified legume lectin FRIL has been shown to preserve human cord blood progenitors up to a month in suspension culture without medium changes. To test whether FRIL also preserves human SCID repopulating stem cells (SRC), we cultured human CD34(+) cord blood cells in medium containing FRIL, with or without subsequent exposure to cytokines, and tested their repopulating potential. We report that FRIL maintains SRC between 6 and 13 days in culture. Incubation of CD34(+) cells with FRIL results in significantly lower numbers of cycling cells compared with cytokine-stimulated cells. CD34(+) cells first cultured with FRIL for 6 days and subsequently exposed to cytokines for an additional 4 days generated significantly more mononuclear and progenitor cells and higher levels of engraftment in NOD/SCID mice compared with CD34(+) cells cultured with FRIL alone. Similar results were obtained with CD34(+)CD38(-/low) cells, including expansion of SRC that were cultured in FRIL followed by cytokine stimulation. Moreover, CD34(+) cells precultured with FRIL successfully engrafted primary and more importantly secondary recipients with lymphoid and myeloid cells, providing further support that FRIL maintains SRC for prolonged periods.FRIL's ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications.
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PMID:The plant lectin FRIL supports prolonged in vitro maintenance of quiescent human cord blood CD34(+)CD38(-/low)/SCID repopulating stem cells. 1088 Jul 59

The major limitations of Moloney murine leukemia virus (MoMLV)-based vectors for human stem cell applications, particularly those requiring bone marrow (BM) stem cells, include their requirement for mitosis and retroviral receptor expression. New vectors based upon lentiviruses such as HIV-1 exhibit properties that may circumvent these problems. We report that novel third-generation, self-inactivating lentiviral vectors, expressing enhanced green fluorescent protein (EGFP) and pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G), can efficiently transduce primitive human repopulating cells derived from human BM and cord blood (CB) tested by the SCID-repopulating cell (SRC) assay. Highly purified CD34+ CD38- CB or BM cells were efficiently transduced (4-69%) and stably expressed in EGFP for 40 days in culture following infection for only 24 h without fibronectin, polybrene, or cytokines. Nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice transplanted with transduced cells from either CB or BM donors were well engrafted, demonstrating maintenance of SRC during the infection procedure. Serially obtained femoral BM samples indicated that the proportion of EGFP+ cells within both myeloid and lymphoid lineages was maintained or even increased over time, averaging 42.3 +/- 6.6% for BM donors and 23.3 +/- 7.2% for CB at 12 weeks. Thus, the third-generation lentivectors readily transduce human CB and BM stem cells, under minimal conditions of ex vivo culture, where MoMLV-based vectors are ineffective. Since CB is inappropriate for most therapeutic applications, the efficient maintenance and transduction of BM-derived SRC during the short infection procedure are notable advantages of lentivectors.
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PMID:Transduction of human CD34+ CD38- bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors. 1093 81

The low frequency of transplantable hematopoietic stem cells in adult human bone marrow (BM) and other differences from cord blood stem cells have impeded studies to optimize the retroviral transduction of stem cells from adult sources. To address this problem, first a cytokine combination was defined that would both maximize the kinetics of adult BM CD34(+)CD38(-) cell mitogenesis and minimize the period of prestimulation required for the transduction of these cells by a MSCV-GFP/neo(r) virus in tissue culture dishes in the absence of fibronectin. Three days of stimulation with flt3-ligand, Steel factor, interleukin (IL)-3, and hyper-IL-6 proved both necessary and sufficient to obtain 83% +/- 2% GFP(+) CD34(+)CD38(-) cells, 75% +/- 10% G418-resistant clonogenic progenitors, and 50% +/- 20% transduced long-term culture-initiating cells as recovered 48 hours after a single exposure to virus. Moreover, this was accompanied by a several-fold increase in viral receptor (pit-1) messenger RNA transcripts in the target cells. Using this prestimulation protocol, repeated daily exposure to new virus (3x) did not alter the proportion of transduced cells over that obtained with a single exposure. Adult human BM cells able to engraft immunodeficient (NOD/SCID-beta(2)M(-/-)) mice were also efficiently transduced (10%-20% GFP(+) human lymphoid and myeloid cells present 6-8 weeks after transplant) using a 6-day prestimulation and infection protocol. A clinically useful efficiency of retrovirus-mediated gene transfer to transplantable adult human BM stem cells can thus be obtained with a protocol that allows their semisynchronous activation into cycle and concomitant increased expression of virus receptor transcripts before virus exposure.
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PMID:Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin. 1100 95

Recent studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). LSCs have been documented for nearly all AML subtypes and have been phenotypically described as CD34+/CD38- or CD34+/HLA-DR-. Given the potentially critical role of these primitive cells in perpetuating leukemic disease, we sought to further investigate their molecular and cellular characteristics. Flow cytometric studies using primary AML tissue showed that the interleukin-3 receptor alpha chain (IL-3Ralpha or CD123) was strongly expressed in CD34+/CD38- cells (98 +/- 2% positive) from 16 of 18 primary specimens. Conversely, normal bone marrow derived CD34+/CD38- cells showed virtually no detectable expression of the CD123 antigen. To assess the functional role of IL-3Ralpha positive cells, purified CD34+/CD123+ leukemia cells were transplanted into immune deficient NOD/SCID mice. These experiments showed that CD123+ cells were competent to establish and maintain leukemic populations in vivo. To begin to elucidate a biological role for CD123 in leukemia, primary AML samples were analyzed with respect to signal transduction activity in the MAPK, Akt, and Stat5 pathways. Phosphorylation was not detected in response to IL-3 stimulation, thereby suggesting CD123 is not active in conventional IL-3-mediated signaling. Collectively, these data indicate that CD123 represents a unique marker for primitive leukemic stem cells. Given the strong expression of this receptor on LSCs, we propose that targeting of CD123 may be a promising strategy for the preferential ablation of AML cells.
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PMID:The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. 1102 53

In view of the limited potential for rapid hematological recovery after transplantation of umbilical cord blood cells (UCB) in adults, we have attempted to expand CD34+ selected hemopoietic stem cells (HSC) and progenitors in 2-week cultures of whole graft pools in the presence or absence of serum and stromal layers, and with various cytokine combinations including (1) FL + TPO; (2) FL + TPO plus SCF and/or IL6; or (3) SCF + IL6. Both in the input material and cultured grafts we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ CD38- subset by phenotyping. The highest fold-increase obtained for the number of nucleated cells (nc), CD34+, CD34+ CD38 cell numbers and CFC content was, respectively, 102 +/- 76, 24 +/- 19, 190 +/- 202 and 53 +/- 37 for stroma-free and 315 +/- 110, 25 +/- 3, 346 +/- 410 and 53 +/- 43 for stroma-supported cultures. CAFC week type 6 was maximally 11-fold expanded both under stroma-free and stroma-supported conditions. The FBMD-1 stromal cells supported a modest expansion of CD34+ CD38- cells (27 +/- 18-fold) and nc (6 +/- 4-fold), while a loss of CFC and CAFC subsets was observed. The stromal cells synergized with FL + TPO to give the highest expansion of hemopoietic progenitors. Stromal support could be fully replaced by complementing the FL + TPO stimulated cultures with SCF + IL6. FL + TPO were required and sufficient to give a 10- to 20-fold expansion of the ability of CD34+ UCB cells in 2-week cultures to engraft the BM of NOD/SCID mice. Stromal support, or complementation of the medium with SCF + IL6, did not significantly improve the in vivo engraftment potential. If the SRA and CAFC week 6 assays are accepted as tentative estimates of in vivo engrafting stem cells in humans, our findings may assist in the preparation of UCB grafts to meet the requirements for improved repopulation in the clinical setting.
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PMID:Successful short-term ex vivo expansion of NOD/SCID repopulating ability and CAFC week 6 from umbilical cord blood. 1106 30

Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34(+)CD38(-/low)CXCR4(+) cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34(-)CD38(-/low)Lin(-) cells were not detected. Moreover, whereas freshly isolated CD34(+)CD38(+/high) cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34(+)CD38(+) cells. Homing of enriched human CD34(+) cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34(+)CD38(-/low)CXCR4(+) cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34(+) cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Galpha(i) proteins, inhibited SDF-1-mediated in vitro transwell migration but not adhesion or in vivo homing of CD34(+) cells. Homing of human CD34(+) cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34(+)CD38(-/low)CXCR4(+) cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.
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PMID:Rapid and efficient homing of human CD34(+)CD38(-/low)CXCR4(+) stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2m(null) mice. 1134 60

Efflux of Hoechst 33342 from normal hematopoietic cells identifies a "side population" (SP(+)) of negatively staining cells that, in the mouse, are largely CD34(-) and are enriched for primitive progenitors. To further characterize human SP(+) cells, blood or bone marrow from 16 patients with acute myeloid leukemia (AML) was analyzed for their presence, immunophenotype, and cytogenetic and functional properties, and for the relation between SP phenotype and multidrug resistance-1 (MDR-1) expression. The mean percentages of SP(+) and MDR(+) cells was 8.1% (range, 0.5%-29.9%) and 12.8% (range, 0%-54.8%), respectively, with no correlation between the 2 values. The percentages of SP(+) cells that were CD34(+)CD38(-), CD34(+)CD38(+), or CD34(-) were 12% (range, 0.4%-50%), 25% (range, 0.5%-96%), and 63% (range, 4%-99%). Cytogenetically abnormal cells were always detected in the SP(-)CD34(+)CD38(-) and SP(+)CD34(-) fractions, and abnormal colonies (CFC), long-term culture-initiating cells (LTC-IC), and nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mouse leukemia-IC were detected in the former fraction. No progenitors were detected among SP(+)CD34(-) cells in any of these assays from 9 of 10 samples. In contrast, exclusively normal cells were detected in the SP(+)CD34(+)CD38(-) fraction from 9 of 15 samples, and CFC, LTC-IC, and multilineage engraftment in NOD/SCID mice from this subpopulation were also cytogenetically normal in 6 of 8, 6 of 7, and 2 of 2 cases studied, respectively. In contrast to murine studies, primitive progenitors are enriched among SP(+)CD34(+)CD38(-) cells from patients with AML. The molecular basis for Hoechst dye efflux is uncertain because it does not appear to be related to MDR-1 expression. (Blood. 2001;97:3882-3889)
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PMID:Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34(+)CD38(-) progenitor cells from patients with acute myeloid leukemia. 1138 30

The mechanism of hematopoietic stem cell migration and repopulation is not fully understood. Murine fetuses that lack the chemokine stromal-derived factor one (SDF-1null) or its receptor CXCR4 (CXCR4null) have multiple defects that are lethal, including impaired bone marrow hematopoiesis. These results suggest a major role for SDF-1/CXCR4 interactions in murine stem cell homing from the fetal liver into the bone marrow and its repopulation during development. SDF-1 is highly conserved between different species. Human and murine SDF-1 are cross-reactive and differ in one amino acid. Recently, we reported that SDF-1 and CXCR4 are essential for homing and repopulation of immune-deficient NOD/SCID and B2mnull NOD/SCID mice by human stem cells. In addition, immature human CD34+ cells and primitive CD34+/CD38-/low cells, which do not migrate toward a gradient of SDF-1 in vitro, and do not home and repopulate in vivo the murine bone marrow, can become functional repopulating cells by short-term 16-48 hr in vitro stimulation with cytokines such as SCF and IL-6 prior to transplantation. These cytokines increase surface CXCR4 expression, migration toward SDF-1, and in vivo homing and repopulation. We discuss the pleiotropic roles of SDF-1/CXCR4 interactions in human stem cell migration, development, and repopulation in transplanted immune-deficient mice.
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PMID:Mechanism of human stem cell migration and repopulation of NOD/SCID and B2mnull NOD/SCID mice. The role of SDF-1/CXCR4 interactions. 1145 29

The characteristics of hematopoietic progenitor and stem cell (HPC/HSC) populations in mammals vary according to their ontogenic stage. In humans, HPC/HSCs from umbilical cord blood (CB) are increasingly used as an alternative to HPC/HSCs from adult bone marrow (BM) for the treatment of various hematologic disorders. How the hematopoietic activity of progenitor and stem cells in CB differs from that in adult BM remains unclear, however. We compared CD34+ cells, a hematopoietic cell population, in CB with those in adult BM using phenotypic subpopulations analyzed by flow cytometry, the colony-forming activity in methylcellulose clonal cultures, and the repopulating ability of these cells in NOD/Shi-scid (NOD/SCID) mice. Although the proportion of CD34+ cells was higher in adult BM than in CB mononuclear cells, the more immature subpopulations, CD34+ CD33- and CD34+ CD38- cells, were present in higher proportions in CD34+ CB cells. Clonal culture assay showed that more multipotential progenitors were present in CD34+ CB cells. When transplanted into NOD/SCID mice. CD34+ adult BM cells could not reconstitute human hematopoiesis in recipient BM, but CD34+ CB cells achieved a high level of engraftment, indicating that CD34+ CB cells possess a greater repopulating ability. These results demonstrated that human hematopoiesis changes with development from fetus to adult. Furthermore, CD34+ CB cells contained a greater number of primitive hematopoietic cells, including HSCs, than did adult BM, suggesting the usefulness of CD34+ CB cells not only as a graft for therapeutic HSC transplantation but also as a target cell population for ex vivo expansion of transplantable HSCs and for gene transfer in gene therapy.
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PMID:Hematopoietic capability of CD34+ cord blood cells: a comparison with CD34+ adult bone marrow cells. 1150 59

Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+ CD38- cell numbers was, respectively, 1197 and 30,937 for stroma-free and 4066 and 117,235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+ CD38- CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+ CD38-, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.
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PMID:Stromal support augments extended long-term ex vivo expansion of hemopoietic progenitor cells. 1151 95


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