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

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

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.
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PMID:Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division. 1037 91

To delineate factors involved in NK cell development, we established an in vitro system in which lineage marker (Lin)-, c-kit+, Sca2+ bone marrow cells differentiate into lytic NK1.1+ but Ly49- cells upon culture in IL-7, stem cell factor (SCF), and flt3 ligand (flt3L), followed by IL-15 alone. A comparison of the ability of IL-7, SCF, and flt3L to generate IL-15-responsive precursors suggested that NK progenitors express the receptor for flt3L. In support of this, when Lin-, c-kit+, flt3+ or Lin-, c-kit+, flt3- progenitors were utilized, 3-fold more NK cells arose from the flt3+ than from the flt3- progenitors. Furthermore, NK cells that arose from flt3- progenitors showed an immature NK1.1dim, CD2-, c-kit+ phenotype as compared with the more mature NK1.1bright, CD2+/-, c-kit- phenotype displayed by NK cells derived from flt3+ progenitors. Both progenitors, however, gave rise to NK cells that were Ly49 negative. To test the hypothesis that additional marrow-derived signals are necessary for Ly49 expression on developing NK cells, flt3+ progenitors were grown in IL-7, SCF, and flt3L followed by culture with IL-15 and a marrow-derived stromal cell line. Expression of Ly49 molecules, including those of which the MHC class I ligands were expressed on the stromal or progenitor cells, as well as others of which the known ligands were absent, was induced within 6-13 days. Thus, we have established an in vitro system in which Ly49 expression on developing NK cells can be analyzed and possibly experimentally manipulated.
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PMID:Differentiation of NK1.1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor cells. 1045 5

Flt3 ligand (FL) is an important cytokine that affects the proliferation of hematopoietic stem cells and multipotent progenitors. In addition, FL seems to be strongly involved in the differentiation of B cells and macrophages. These two cell types are derived from separate hematopoietic lineages and display distinct surface markers, for instance, the pan-B cell marker B220 (CD45R) and the myelo/monocytic marker Mac-1 (CD11b), respectively. However, reports during several years have shown that some lineage markers can be coexpressed on factor-dependent progenitor cells as well as on some malignant leukemic clones. In the present study, we describe the ability of FL to induce the development and growth of Mac-1+ progenitor cells coexpressing B220 from c-kit+Lin- mouse bone marrow cells. FL was shown to be necessary but not sufficient for the development of Mac-1(-)B220+ cells, because certain other cytokines, in particular IL-6, had to be added to the cultures. An extended characterization of the cells revealed coexpression of other early B-cell markers, i.e., CD24, CD43, and c-kit. They expressed transcripts for c-fms, the receptor for macrophage-colony stimulating factor (M-CSF), and were able to develop into macrophages at high numbers, but not to other myeloid cells. By RT-PCR analysis we could also demonstrate expression of the B-cell associated genes Pax-5, Rag-2, and TdT. In contrast, Mac-1(+)B220- cells from the same cultures did not express any of the B-cell genes, and retained a broader myeloid differentiation capacity. Despite these B-cell associated features, Mac-1(+)B220- cells could not be induced towards B-cell progenitors. Our data suggest that FL triggers the activation of some B-cell associated genes in progenitor cells predestined to macrophage differentiation.
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PMID:Flt3 ligand induces the outgrowth of Mac-1+B220+ mouse bone marrow progenitor cells restricted to macrophage differentiation that coexpress early B cell-associated genes. 1056 Sep 12

Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34(+)CD38(-) phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34(+)CD38(-) BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34(+)CD38(-) progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.
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PMID:Distinct requirements for optimal growth and In vitro expansion of human CD34(+)CD38(-) bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells. 1059 54

We describe here that lineage phenotype- negative (Lin)(-)c-kit(+) hematopoietic progenitor cells (HPCs) from day 13 postcoitus (dpc) murine fetal liver (FL) can generate dendritic cell (DC) precursors when cultured in vitro in the presence of PA6 stromal cells plus granulocyte/macrophage colony-stimulating factor (GM-CSF) + stem cell factor (SCF) + Flt3 ligand (Flt3L) for 12 to 14 days, and develop into mature DCs when stimulated with GM-CSF plus mouse tumor necrosis factor alpha (mTNFalpha) for an additional 3 to 5 days. A transwell culture system showed that the generation of DC precursors depended on the support of PA6 cell-secreted soluble factor(s). The mature DCs derived from 13 dpc FL Lin(-)c-kit(+) HPCs showed characteristic morphology and function of DCs and expressed high levels of Ia, CD86, and CD40 molecules, low levels of DEC205, E-cadherin, and F4/80 molecules, but barely detectable CD11c antigen. Once FL-derived HPCs were cultured without GM-CSF, NK1.1(+) cells developed in the presence of PA6 cells + SCF + Flt3L. These NK1.1(+) cells could develop into DC precursors at an earlier stage of differentiation by reculturing with PA6 cells + SCF + Flt3L + GM-CSF, but they would be irreversibly committed to NK cell precursors without GM-CSF after 3 days, suggesting that GM-CSF plays a critical role in controlling the transition of DC and NK cell precursors from 13 dpc FL-derived Lin(-)c-kit(+) HPCs. This study represents the first success in generating mature DCs in vitro from murine FL HPCs. (Blood. 2000;95:138-146)
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PMID:Development of dendritic cells in vitro from murine fetal liver-derived lineage phenotype-negative c-kit(+) hematopoietic progenitor cells. 1060 96

Dendritic cells (DCs) are essential for the presentation of antigens in the primary immune response. To examine the generation of DCs from hemopoietic stem cells in the bone marrow (BM), lineage-negative (Lin-)/CD71- bone marrow cells (BMCs) from C57BL/6 mice were separated into major histocompatibility complex (MHC) class Ihigh/ c-kit(low) and MHC class Ihigh/c-kit(low)(phenotypically c-kit-negative, but c-kit message only detected by reverse transcriptase-polymerase chain reaction) populations. A large number of cells with the morphological, phenotypical, and functional characteristics of DCs was generated from both c-kit(low) and c-kit(low) populations when cultured with a combination of cytokines (GM-CSF, tumor necrosis factor-a [TNF-a], interleukin 7 [IL-7], IL-3, stem cell factor [SCF], and flt3 ligand); the cytokine combination studies revealed that SCF and IL-3 in addition to GM-CSF and TNF-a are essential for DCs to be generated from these primitive populations. To our surprise most (>80%) generated cells expressed high levels of DC surface markers such as DEC205 and MHC class II, and they were potent stimulators in the primary allogeneic T cell activation. The development of DCs from c-kit(<low) cells was slower than that from c-kit(low) cells. These results indicate that c-kit(<low) cells are more primitive than c-kit(low) cells, although both c-kit*(low) cells and c-kit(<low) cells can differentiate into DCs. It should be noted that the combination of these cytokines selectively induces DCs from both c-kit(<low) and c-kit(low) cells in vitro, suggesting that the ex vivo expansion of DCs using these primitive cells would be applicable to immunotherapy.
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PMID:Development of mouse dendritic cells from lineage-negative c-kit(low) pluripotent hemopoietic stem cells in vitro. 1066 72

Dendritic cells (DCs) are powerful antigen-presenting cells. Because DCs are rare cells, methods to produce them in vitro are valuable ways to study their biologic properties and to generate cells for immunotherapy. This study defines the antigen-presenting properties of DCs generated in vitro from CD34+ cells of patients with breast cancer. The combination of cytokines flt3 ligand + c-kit ligand + granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-4 (IL-4) + tumor necrosis factor-alpha (TNF-alpha) was used to maximize the output of mature DCs in the culture of CD34+ cells while minimizing the production of monocytes. Cells grew and differentiated into DCs as measured by a time-dependent upregulation of cell surface antigens major histocompatibility complex class II, CD1a, CD80, CD86, CD40, and CD4, so that 40% +/- 9% (n = 6) of cells in culture at day 15 were CD1a+CD14-. Markers were acquired in the same sequence as on monocytes induced to differentiate with GM-CSF + IL-4. Differentiation was marked by a time-dependent increase in allostimulatory function, which, at its peak, was more potent than in cultures of DCs generated from monocytes with GM-CSF + IL-4, but was comparable on a cell-to-cell basis to that of mature monocytes cultured in flt3-ligand + c-kit-ligand + GM-CSF + IL-4 + TNF-alpha. Both CD34+ cell-derived and monocyte-derived DCs were able to process and to present tetanus toxoid and keyhole limpet hemocyanin to autologous T cells and to present major histocompatibility class I-binding peptides to CD8+ cytotoxic T lymphocytes inducing interferon-gamma production. Altogether, these results suggest that DCs generated from CD34+ cells of patients with breast cancer with flt3 ligand, c-kit ligand, GM-CSF, IL-4, and TNF-alpha are competent antigen-presenting cells, particularly for CD8+ cytotoxic T lymphocytes, and resemble mature monocyte-derived DCs in the assays described here.
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PMID:Dendritic cells generated from CD34+ progenitor cells with flt3 ligand, c-kit ligand, GM-CSF, IL-4, and TNF-alpha are functional antigen-presenting cells resembling mature monocyte-derived dendritic cells. 1068 37

The combined analysis of the expression of receptor tyrosine kinases c-Kit and Flt3/Flk-2 and of the human CD25 gene expressed as a transgene under the regulation of the mouse lambda5 promoter in the bone marrow of 1-week-old mice allows us to identify three stages of B lymphocyte development before the CD19(+)c-Kit(+) pre-B-I cells. Single-cell PCR analysis of the rearrangement status of the Ig heavy chain alleles allows us to order these early stages of B cell development as follows: (i) B220(+)CD19(-)c-Kit(lo)Flt3/Flk-2(hi)lambda5(-), (ii) B220(+)CD19(-)c-Kit(lo)Flt3/Flk-2(hi)lambda5(+) and (iii) B220(+)CD19(+)c-Kit(lo)Flt3/Flk-2(lo)lambda5(+) before B220(+)CD19(+)c-Kit(lo)Flt3/Flk-2(-)lambda5(+) pre-B-I cells. All these progenitors are clonable on stromal cells in the presence of IL-7 and can differentiate to CD19(+)c-Kit(-) B-lineage cells. A combination of stem cell factor, Flt3 ligand and IL-7 was also able to support the proliferation and differentiation of the progenitors in a suspension culture. Furthermore, the analyses indicate that the onset of D(H)J(H) rearrangements precedes the expression of the lambda5 gene. These progenitor populations were characteristic of juvenile mice and could not be detected in the bone marrow of adult mice. Hence the expression pattern, and probably the function, of the receptor tyrosine kinases in early B cell differentiation appears to be different in juvenile and adult mice.
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PMID:Identification of CD19(-)B220(+)c-Kit(+)Flt3/Flk-2(+)cells as early B lymphoid precursors before pre-B-I cells in juvenile mouse bone marrow. 1070 Apr 66

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


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