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Query: UMLS:C0085110 (SCID)
 11,041 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CD34(+)Thy-1(+)Lin- cells are enriched for primitive hematopoietic progenitor cells (PHP), as defined by the cobblestone area-forming cell (CAFC) assay, and for bone marrow (BM) repopulating hematopoietic stem cells (HSC), as defined by the in vivo SCID-hu bone assay. We evaluated the effects of different cytokine combinations on BM-derived PKH26-labeled CD34(+)Thy-1(+)Lin- cells in 6-day stroma-free cultures. Nearly all (>95%) of the CD34(+)Thy-1(+)Lin- cells divided by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and flk2/flt3 ligand (FL). The resulting CD34(hi) PKHlo (postdivision) cell population retained a high CAFC frequency, a mean 3.2-fold increase of CAFC numbers, as well as a capacity for in vivo marrow repopulation similar to freshly isolated CD34(+)Thy-1(+)Lin- cells. Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast, cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory factor contained a mean of 75% of undivided cells at day 6. These CD34(hi) PKHhi cells retained a high frequency of CAFC, whereas the small population of CD34(hi) PKHlo postdivision cells contained a decreased frequency of CAFC. These data suggest that use of a combination of TPO, KL, and FL for short-term culture of CD34(+)Thy-1(+)Lin- cells increases the number of postdivision PHP, measured as CAFC, while preserving the capacity for in vivo engraftment.
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PMID:Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy-1+Lin- cells with preserved ability to engraft SCID-hu bone. 945 50

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34+ Thy-1+ cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34+ Thy-1+ CFSElo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34+ Thy-1+ cells (a mean of 1/8.4). In contrast, CD34+ cells that had lost Thy-1 expression during culture (CD34+ Thy-1 CFSElo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1-expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34+ Thy-1+ fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34+ Thy-1+ cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1+ cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34+ cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34+ Thy-1+ cells divided under the conditions tested, promoting susceptibility to retroviral transduction.
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PMID:CD34+ cells from mobilized peripheral blood retain fetal bone marrow repopulating capacity within the Thy-1+ subset following cell division ex vivo. 1037 88

The effects of Flk2/Flt3 ligand (FL) administration on human hematopoiesis were investigated using SCID-hu mice transplanted with human fetal bone fragments. Treatment with recombinant human FL induced significant increases in the frequencies of the high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in steady-state human bone marrow. FL also promoted the expansion of high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in the human bone marrow during the recovery phase after irradiation, which was evident in increases in the frequencies as well as in the absolute numbers of colony-forming cells. Furthermore, higher percentages of CD33+ CD15- cells were found in the marrows treated with FL as compared to that of controls, indicating that FL hastened the recovery of at least some aspect of myelopoiesis after irradiation. These results indicate that FL induces the expansion of primitive hematopoietic progenitor cells in vivo and, therefore, may be useful in treating patients to promote an early hematopoietic recovery after cytoablative therapies.
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PMID:Administration of Flk2/Flt3 ligand induces expansion of human high-proliferative potential colony-forming cells in the SCID-hu mouse. 1037 92

Umbilical cord blood has provides an alternative source to bone marrow for transplantation in children and some adults. However it has been thought to be necessary to expand hematopoietic stem cells in cord blood for adult-size transplantation. Flow-cytometric analysis revealed that most immature hematopoietic progenitors express gp130 but not interleukin-6 receptor (IL-6R). We established an ex vivo expansion system of human hematopoietic stem/progenitor cells using soluble IL-6R (sIL-6R). A combination of sIL-6R/IL-6 complex and SCF expanded CFU-Mix approximately 60-fold in both serum-containing and serum-free cultures by day 14. Addition of anti-gp130 mAbs and anti-IL-6R mAb to the above cultures dose-dependently inhibited the expansion of progenitors, suggesting that gp130 signalling initiated by the sIL-6R/IL-6 complex is important for significant expansion of human hematopoietic stem/progenitor cells. Addition of thrombopoetin and/or Flk2/Flt3 ligand (FL) to culture with sIL-6R/IL-6/SCF augmented the expansion of not only hematopoietic progenitors assayable in clonal culture but also hematopoietic stem cells estimated by NOD/SCID mice. Recent evidence shows that hematopoietic stem cells first occur and expand in aorta-gonad-mesonephros(AGM) region at 10 to 11 days post coitum(dpc) in murine, which suggests that AGM region at this stage provides a microenvironment suitable for development for hematopoietic stem cells. We reported here on a novel stromal cell line derived from the AGM region at day 10.5 dpc, which supported for 6 weeks generation of human multipotential hematopoietic progenitors from cord blood CD34+38- primitive hematopoietic cells in a co-culture system. This cell line is expected to elucidate molecular mechanisms regulating early hematopoiesis, and pave the way for developing strategies for ex vivo expansion of human transplantable hematopoietic stem cells.
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PMID:[Cord blood transplantation and ex vivo expansion of hematopoietic stem cells]. 1047 32

The efficiency of retroviruses as transducing agents has been appreciated for many years, particularly for hematopoietic cell targets for which alternative strategies applicable to adherent cells are not effective. Advances in vector design, pseudotyping, and infection conditions have eliminated the need to cocultivate the target cells with virus-producing cells. Nevertheless, improvements are still needed for many applications, including those with a therapeutic or clinical cell-tracking objective. In this study we show that more positively charged surfaces, including those designed for the culture of anchorage-dependent cells, allow measurable levels of adhesion by different pseudotypes of retroviruses, which can result in increased gene transfer efficiencies to a variety of target cells including normal primary human hematopoietic cells as well as human leukemic cell lines and rat and murine fibroblasts. In the experiments with primary human cells, equal aliquots of enriched CD34+ cord blood cells were first stimulated for 2 days with cytokines (Flt3 ligand, Steel factor, IL-3, IL-6, and G-CSF) and then exposed for 4 days to a green fluorescent protein (GFP)- and Neo(r)-encoding retrovirus produced in PG13 cells. Both the final yield (approximately 300% relative to initial numbers), and the proportion (approximately 60%) of transduced CD34+ cells, colony-forming cells, and long-term culture-initiating cells were the same for cells infected either in tissue culture dishes or in fibronectin-coated petri dishes. Similar proportions (approximately 10%) and absolute yields of GFP+ human cells were also found in multilineage engrafted NOD/SCID mice assessed 6 to 8 weeks after being transplanted with these two types of transduced, but unselected, cells. These findings suggest a new and simpler approach for achieving high gene transfer efficiencies to hematopoietic cells.
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PMID:High-efficiency retroviral transduction of mammalian cells on positively charged surfaces. 1064 38

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.
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PMID:Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. 1074 63

Human CD34(-) hematopoietic stem cells (HSCs) have been identified as potential precursors of CD34(+) HSCs by using xenogeneic transplantation systems. However, the properties of CD34(+) cells generated from CD34(-) cells have not been precisely analyzed due to the lack of an in vitro system in which CD34(+) cells are continuously produced from CD34(-) cells. We conducted this study to determine whether CD34(+) cells generated in vitro from CD34(-) cells have long-term multilineage reconstitution abilities. Lin(-)CD34(-) population isolated from human cord blood was cultured in the presence of murine bone marrow stroma cell line, HESS-5, and human cytokines, thrombopoietin, Flk2/Flt3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin 3 (IL-3), and IL-6. They were analyzed weekly for their surface markers expressions, colony-forming cells, long-term culture initiating cells (LTC-IC), and SCID repopulating cells (SRC) abilities up to 30 days of culture. In this culture system, more than 10(7) CD34(+) cells can be continuously generated from 10(4) CD34(-) cells over 30 days. These CD34(+) cells produce colony-forming units, LTC-IC, and SRC with multi-lineage differentiation, all of which are characteristic features of hematopoietic stem/progenitor cells. These findings suggest that CD34(-) HSCs have extensive potential for the generation of CD34(+) HSCs in vitro. This system provides a novel and potentially useful procedure to generate CD34(+) cells for clinical transplantation and gene therapy.
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PMID:Extensive generation of human cord blood CD34(+) stem cells from Lin(-)CD34(-) cells in a long-term in vitro system. 1088 Jul 55

This article presents our studies on the adenoviral transduction efficiency, level of transgene expression, cell cycle status, and multilineage reconstitution ability of human CD34+ hematopoietic cells transduced under proliferating and survival growth conditions. Bone marrow and umbilical cord blood CD34+ cells were cultured in serum-free medium under survival conditions with thrombopoietin (Tpo) alone, or under proliferating conditions with Tpo, c-Kit ligand (KL), and Flt3 ligand (FL). Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) gene under the control of the PGK-1 promoter were used to transduce CD34+ cells. Approximately 10% of CD34+ cells were EGFP+ under both culture conditions. In contrast, up to 50% of CD34+CD38- cells were EGFP+, whereas a maximum of 8% of CD34+CD38(high) cells were EGFP+ (p < 0.001). Both colony-forming unit cells (CFU-C) and 5-week long-term culture-initiating cells (LTC-ICs) were efficiently transduced. Under survival conditions, a substantial fraction of transduced CD34+ cells remained quiescent. The nondividing CD34+EGFP+ cells contained LTC-ICs capable of reconstituting longterm culture for as long as 10 weeks. CD34+EGFP+ cells also retained the ability to engraft and multilineage-reconstitute NOD/SCID mice. These observations demonstrate that primitive human hematopoietic progenitor cells can be efficiently transduced by adenoviral vectors.
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PMID:Efficient adenoviral vector transduction of human hematopoietic SCID-repopulating and long-term culture-initiating cells. 1089 Jul 41

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.
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PMID:In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34(+) cord blood cells. 1114 69

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL, is a new member of the TNF family. It can specifically induce apoptosis in a variety of human tumors. To investigate the possibility of employing the TRAIL gene for systemic cancer therapy, we constructed a recombinant gene encoding the soluble form of the human Flt3L gene (hFlex) at the 5' end and the human TRAIL gene at the 3' end. Such design allows the TRAIL gene product to be secreted into the body circulation. We have also demonstrated that the addition of an isoleucine zipper to the N-terminal of TRAIL greatly enhanced the trimerization of the fusion protein and dramatically increased its anti-tumor activity. The fusion protein reached the level of 16-38 microg/ml in the serum after a single administration of the recombinant gene by hydrodynamic-based gene delivery in mice. A high level of the fusion protein correlated with the regression of a human breast tumor established in SCID mice. No apparent toxicity was observed in the SCID mouse model. In addition, the fusion protein caused an expansion of the dendritic cell population in the C57BL/6 recipient mice, indicating that the hFlex component of the fusion protein was functional. Thus, the hFlex-TRAIL fusion protein may provide a novel approach, with the possible involvement of dendritic cell-mediated anti-cancer immunity, for the treatment of TRAIL-sensitive tumors.
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PMID:Regression of human mammary adenocarcinoma by systemic administration of a recombinant gene encoding the hFlex-TRAIL fusion protein. 1127 79


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