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Query: UNIPROT:P10721 (c-kit)
 6,575 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Factors that may improve retroviral transduction of primitive human hematopoietic cells were studied using MFG-based vectors containing a LacZ gene and produced either by a murine (psi-Crip) or a human (Tasaf) cell line. Cord blood (CB) or bone marrow (BM) CD34+ cells were stimulated and transduced in the presence of three cytokines (interleukin 3 [IL-3], IL-6, and stem cell factor [SCF; c-Kit Ligand]). In the supernatant infection protocol, hematopoietic progenitor cells as measured by X-Gal staining of colony-forming unit cells (CFU-Cs) were transduced more effectively with Tasaf (20%) than with psi-Crip (8%). In contrast, there was no difference between these two cell lines in a coculture protocol. However, gene transfer into more primitive CD34+CD38- subsets and in LTC-IC-derived colonies was low. The use of a large number of cytokines including FLT3-L and PEG-rhMGDF increased the transduction efficiency into CD34+CD38(-)-derived CFU-Cs (35% by PCR) or LTC-ICs (10%). A virus pseudotyped with gibbon ape leukemia virus (GALV) envelope further improved gene transfer to 60 and 48% for LacZ+ CFU-C- and LTC-IC-derived colonies, respectively. These conditions of transduction allowed multilineage engraftment of primitive cord blood cells in NOD-SCID mice. Moreover, 10% (at least) of the human hematopoietic cells recovered from the marrow of these immunodeficient animals were transduced. These data suggest that the efficiency of transduction of human hematopoietic primitive cells can be significantly improved by judicious combinations of recombinant cytokines and high retroviral titers.
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PMID:Retrovirus-mediated gene transfer into human CD34+38low primitive cells capable of reconstituting long-term cultures in vitro and nonobese diabetic-severe combined immunodeficiency mice in vivo. 968 21

Although the hematopoietic activities of human CD34+ bone marrow (BM) and cord blood (CB) cells have been well characterized, the phenotype of nonobese-diabetic severe combined immunodeficient (NOD/SCID) mice repopulating cells (SRCs) in CB and BM has not yet been fully examined. To address this issue, various hematopoietic activities were compared in terms of total and CD34+ CB and BM cells. Clonal culture of fluorescence-activated cell sorter (FACS) CD34+ CB and BM cells revealed a higher incidence of colony-forming cells with greater proliferation capacity in CB over BM CD34+ cells. CB CD34+ cells also demonstrated higher secondary plating efficiency over BM cells. In addition, we demonstrated that mice transplanted with CB mononuclear cells (MNCs) showed significantly higher levels of chimerism than those transplanted with BM MNCs. However, recipients of FACS-sorted CD34+ CB cells showed significantly lower levels of chimerism than those that received total CB MNCs, suggesting a role of facilitating cells in the CD34- cell population. To further analyze the role of CD34- cells, the NOD/SCID repopulating ability of FACS-sorted CB CD34-c-kit+Lin- and CD34-c-kit-Lin- cells were examined. However, SRCs were not detected in those cells. Taken together, these data suggest that CB is a better source of hematopoietic stem cells and that there are cells in the CD34- fraction that facilitate repopulation of hematopoiesis in the NOD/SCID environment.
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PMID:Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells. 1052 63

A hierarchy of progenitor cells is thought to exist in human acute myeloid leukemia (AML), with only the most primitive cells capable of proliferating to maintain the malignant clone. To further characterize this AML cell hierarchy, we evaluated the coexpression of CD34 and c-kit (CD117) on cells that are capable of long-term proliferation in vitro and in vivo.AML cells were sorted for coexpression of CD34 and c-kit (CD117) using two c-kit monoclonal antibodies (mAbs), clones 95C3 and 104D2. Sorted subfractions were evaluated for the ability to produce colony-forming units (CFU) for up to 8 weeks in suspension culture (SC) and for the capacity to repopulate NOD/SCID mice. When expression of c-kit on blood cells from 19 AML patients at diagnosis was compared using both mAbs, expression defined by 104D2 (34% +/- 6% c-kit(+)) was somewhat higher than that defined using 95C3 (18% +/- 4%). AML cells were sorted for coexpression of CD34 and c-kit using both c-kit mAbs, and the subfractions were assayed in vitro and in vivo. Whereas the majority of AML blast cells lacked expression of CD34, most AML cells capable of proliferating to produce CFU after 4 to 8 weeks in SC were CD34(+)/c-kit(-). Cultures of sorted CD34(+)/c-kit(-) cells, supplemented with steel factor, were composed of a large proportion (18% to 87%) of CD34(+)/c-kit(+) cells after 1 week, suggesting that either c-kit expression was upregulated or CD34(+)/c-kit(+) cells were produced. Moreover, the CD34(+)/c-kit(-) subfraction was found to be capable of responding to steel factor alone to produce CFU after 4 weeks in SC. In most AML patients tested (11/15), the only sorted subfraction capable of engrafting NOD/SCID mice was CD34(+)/c-kit(-). The CD34(+)/c-kit(+) subfraction from only 2 of the 15 patients and CD34(-) cells from 3 patients also engrafted the NOD/SCIDs. Only the CD34(+)/c-kit(+) subfraction of normal bone marrow engrafted. These studies suggest that primitive AML cells capable of long-term proliferation in vitro and NOD/SCID repopulation differ from primitive normal progenitor cells in their lack of surface expression of c-kit.
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PMID:Primitive acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo lack surface expression of c-kit (CD117). 1088 Jul 52

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

There has been great interest in the ex vivo expansion of human long-term repopulating hematopoietic stem cells (LTR-HSCs) for a variety of clinical applications such as umbilical cord blood transplantation. The glucoprotein130 signal, activated by a complex of interleukin 6 (IL-6) and soluble IL-6 receptor (IL-6/sIL-6R), acts dramatically in synergy with the c-Kit or Flk2/Flt3 signal to expand immature human HSCs. We demonstrate a significant ex vivo expansion of human LTR-HSCs capable of repopulating in newly discovered nonobese diabetes/Shi-severe combined immunodeficient (NOD/Shi-SCID) mice. The proportion of human CD45+ cells in recipient marrow was 10 times higher in animals receiving the cultured cells with stem cell factor, Flk2/Flt3 ligand, thrombopoietin, and IL-6/sIL-6R than in those receiving comparable numbers of fresh cord blood CD34+ cells. The expansion rate provided by this combination was estimated to be 4.2-fold by a limiting dilution method. Addition of IL-3 to the culture with the cytokine combination abrogated the repopulating ability of the expanded cells. The culture method with the IL-6/sIL-6R complex and other cytokines may pave the way for ex vivo expansion of human transplantable HSCs suitable for clinical applications.
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PMID:Ex vivo expansion of human hematopoietic stem cells. 1137 56

The purpose of this study was to purify and characterize canine hematopoietic progenitor cells for surface antigen phenotype and reconstitution ability. Canine hematopoietic progenitor cells were isolated by density gradient sedimentation, lineage depletion with monoclonal antibodies, and fluorescence-activated cell sorting (FACS) for selection of cells with low-forward and right-angle scatter that were rhodamine 123 (Rh-123)(dull). Isolated cells were characterized for expression of CD34, c-kit, and Flt-3. A canine/murine xenograft model and a mixed-chimerism assay were used to examine the in vivo proliferative potential of isolated cells. The lineage-positive (Lin(+)) cells represented 80 +/- 11% (n = 22) of the input mononuclear cells. Lineage depletion resulted in a fourfold increase in colony-forming unit granulocyte/monocyte (CFU-GM), a 2.5-fold increase in burst-forming unit-erythroid (BFU-E), and a twofold increase in the number of Rh-123(dull) cells over nonlineage-depleted bone marrow mononuclear cells (BMMCs). Lineage depletion led to a 2.7-fold enrichment of CD34 cells, a 10.4-fold enrichment of c-kit cells, and a 10.8-fold enrichment of CD34/c-kit(+1) cells over total BMMCs. Nineteen percent of lineage-negative (Lin(-)) cells were positive for Flt-3. Injection of canine cells into irradiated (400 rads) NOD/SCID mice resulted in the detection of canine CD45(+) cells with BMMCs, Lin(-) cells, or Rh-123(dull) cells. Transplantation of purified Lin(-) cells in dog leukocyte antigen-matched littermates resulted in low-level engraftment for at least 10 weeks. The development of methods for purification and characterization of canine hematopoietic progenitor cells should enhance the utilization of the canine model for a variety of experimental and therapeutic purposes.
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PMID:Isolation and characterization of canine hematopoietic progenitor cells. 1137 63

Complex cytogenetic abnormalities confer dismal prognoses in myeloid malignancies. Even bone marrow transplantation from siblings or matched unrelated donors offer minimal chances for cure, suggesting that these cases are not only refractory to chemotherapy but also resist the graft-vs.-leukemia effect. We herein describe the first permanent, factor-independent c-kit(hi+) cell line CS-1 derived from an unrelated donor stem cell transplanted patient with relapsed acute myeloid leukemia (AML)-M5a of high-risk karyotype [monosomy 7, t(2;11)(q31;p13), t(10;12)(q24;q24)]. Having the same karyotype, CS-1 exhibits an autonomous growth pattern and responds to stem cell factor (SCF). CS-1 did not induce T cell activation in mixed-lymphocyte-tumor-cultures (MLTCs) and, when used as third party stimulators, decreased T cell proliferation in mixed-lymphocyte reactions (MLRs). Cytokines added exogenously or secreted from bystander T cells caused CS-1 to differentiate into dendritic cells (DCs). CS-1-derived DCs, in contrast to DCs originating from non-malignant CD34(+) progenitor cells, had virtually no T cell stimulatory effect, indicating that CS-1 is both immunosuppressive and poorly immunogenic. These properties may partially be due to the detected downregulation of costimulatory molecules and appear to involve a soluble factor. CS-1 cells injected subcutaneously (s.c.) to non-obese diabetes/severe combined immunodeficient (NOD/SCID) mice produced solid tumors, disseminating into bone marrow and spleen. The data show that transforming AML blasts with high-risk karyotype into DCs is insufficient to restore their immunogenicity and that the CS-1 cell line is useful to identify tumor-related immunosuppressive mechanisms in vitro and in vivo.
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PMID:CS-1, a novel c-kithi+ acute myeloid leukemia cell line with dendritic cell differentiation capacity and absent immunogenicity. 1267 85

The cytokine tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. Through its ability to promote ex vivo expansion and oncoretroviral transduction of primitive human hematopoietic progenitors, the flt3 ligand (FL) has emerged as a key stimulator of candidate human hematopoietic stem cells (HSCs). However, recent studies in the mouse suggest that though it is present on short-term repopulating cells, flt3 is not expressed on bone marrow long-term reconstituting HSCs, the ultimate target for the development of cell replacement and gene therapy. Herein we demonstrate that though only a fraction of human adult bone marrow and cord blood CD34+long-term culture-initiating cells (LTC-ICs) express flt3, most cord blood lymphomyeloid HSCs capable of in vivo reconstituting nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice are flt3+. The striking difference in flt3 and c-kit expression on mouse and candidate human HSCs translated into a corresponding difference in flt3 and c-kit function because FL was more efficient than SCF at supporting the survival of candidate human HSCs. In contrast, SCF is far superior to FL as a viability factor for mouse HSCs. Thus, the present data provide compelling evidence for a contrasting expression and response pattern of flt3 and c-kit on mouse and human HSCs.
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PMID:Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells. 1267 89

Umbilical cord blood (UCB) is a source of hematopoietic stem cells and other stem cells, and human UCB cells have been reported to contain transplantable hepatic progenitor cells. However, the fractions of UCB cells in which hepatic progenitor cells are rich remain to be clarified. In the present study, first, the fractionated cells by CD34, CD38, and c-kit were transplanted via portal vein of NOD/SCID mice, and albumin mRNA expression was examined in livers at 1 and 3 months posttransplantation. At 1 and 3 months, albumin mRNA expression in CD34+UCB cells-transplanted livers was higher than that in CD34- cells-transplanted livers. Albumin mRNA expression in CD34+CD38+ cells-transplanted livers was higher than that in CD34+CD38- cells-transplanted [corrected] liver at 1 month. However, it was much higher [corrected] in CD34+CD38- cell-transplanted livers at 3 months. Similar expression of albumin mRNA was obtained between CD34+CD38+c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, and between CD34+CD38-c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, respectively. Second, fluorescence in situ hybridization and immunohistochemistry were performed to examine whether UCB cells really transdifferentiated into hepatocytes or they only fused with mouse hepatocytes. In mouse liver sections, of 1.2% cells which had human chromosomes, 0.9% cells were due to cell fusion, whereas 0.3% cells were transdifferentiated into human hepatocytes. These results suggest that CD34+UCB cells are rich fractions in hepatic progenitor cells, and that transdifferentiation from UCB cells into hepatocytes as well as cell fusion simultaneously occur in this situation.
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PMID:Analyses to clarify rich fractions in hepatic progenitor cells from human umbilical cord blood and cell fusion. 1547 86

NOD mice exhibit defects in T cell functions that have been postulated to contribute to diabetes susceptibility in this strain. However, early T cell development in NOD mice has been largely unexplored. NOD mice with the scid mutation and Rag1 deficiency were analyzed for pre-T cell development in the NOD genetic background. These strains reveal an age-dependent, programmed breakdown in beta selection checkpoint enforcement. At 5-8 wk of age, even in the absence of TCRbeta expression, CD4+ and CD4+CD8+ blasts appear spontaneously. However, these breakthrough cells fail to restore normal thymic cellularity. The breakthrough phenotype is recessive in hybrid (NODxB6)F1-scid and -Rag1null mice. The breakthrough cells show a mosaic phenotype with respect to components of the beta selection program. They mimic normal beta selection by up-regulating germline TCR-Calpha transcripts, CD2, and Bcl-xL and down-regulating Bcl-2. However, they fail to down-regulate transcription factors HEB-alt and Hes1 and initially express aberrantly high levels of Spi-B, c-kit (CD117), and IL-7Ralpha. Other genes examined distinguish this form of breakthrough from previously reported models. Some of the abnormalities appear first in a cohort of postnatal thymocytes as early as the double-negative 2/double-negative 3 transitional stage. Thus, our results reveal an NOD genetic defect in T cell developmental programming and checkpoint control that permits a subset of the normal outcomes of pre-TCR signaling to proceed even in the absence of TCRbeta rearrangement. Furthermore, this breakthrough may initiate thymic lymphomagenesis that occurs with high frequency in both NOD-scid and -Rag1null mice.
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PMID:Deranged early T cell development in immunodeficient strains of nonobese diabetic mice. 1549 84


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