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  • Vogel, Wichard  (7)
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  • 1
    Language: English
    In: Annals of the New York Academy of Sciences, June 2007, Vol.1106, pp.262-71
    Description: The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)-derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271(+) population. The recognized CD271(+) populations were fractionated by fluorescence-activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU-F. The results showed that only the CD271(bright) but not the CD271(dim) population contained CFU-F. Two-color flow cytometry analysis revealed that only the CD271(bright) population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271(bright) population but no other BM cells. The new MSC-specific molecules included the platelet-derived growth factor receptor-beta (CD140b), HER-2/erbB2 (CD340), frizzled-9 (CD349), the recently described W8B2 antigen, as well as cell-surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9-3C2F1, and HEK-3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM-MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.
    Keywords: Cell Culture Techniques -- Methods ; Cell Separation -- Methods ; Mesenchymal Stem Cells -- Cytology
    ISSN: 0077-8923
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 2
    In: Annals of the New York Academy of Sciences, June 2007, Vol.1106(1), pp.262-271
    Description: :  The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)‐derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271+ population. The recognized CD271+ populations were fractionated by fluorescence‐activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU‐F. The results showed that only the CD271bright but not the CD271dim population contained CFU‐F. Two‐color flow cytometry analysis revealed that only the CD271bright population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271bright population but no other BM cells. The new MSC‐specific molecules included the platelet‐derived growth factor receptor‐β (CD140b), HER‐2/erbB2 (CD340), frizzled‐9 (CD349), the recently described W8B2 antigen, as well as cell‐surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9‐3C2F1, and HEK‐3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM‐MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.
    Keywords: Mesenchymal Stem Cells ; Bone Marrow ; Msc ; Prospective Isolation
    ISBN: 1573316768
    ISSN: 0077-8923
    E-ISSN: 1749-6632
    E-ISSN: 19306547
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  • 3
    In: STEM CELLS Translational Medicine, January 2013, Vol.2(1), pp.53-60
    Description: Advanced adult soft‐tissue sarcomas (STSs) are rare tumors with a dismal prognosis and limited systemic treatment options. STSs may originate from mesenchymal stem cells (MSCs). The goal of this study was to establish the expression pattern of MSC markers in sarcoma cell lines and primary tumor samples by flow cytometry. The data suggest a hierarchical cytoarchitecture of the most common adult type sarcomas and introduce W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers. Advanced adult soft‐tissue sarcomas (STSs) are rare tumors with a dismal prognosis and limited systemic treatment options. STSs may originate from mesenchymal stem cells (MSCs); the latter have mainly been isolated from adult bone marrow as plastic‐adherent cells with differentiation capacity into mesenchymal tissues. Recently, a panel of antibodies has been established that allows for the prospective isolation of primary MSCs with high selectivity. Similar to cancer stem cells in other malignancies, sarcoma stem cells may bear immunophenotypic similarity with the corresponding precursor, that is, MSCs. We therefore set out to establish the expression pattern of MSC markers in sarcoma cell lines and primary tumor samples by flow cytometry. In addition, fibroblasts from different sources were examined. The results document a significant amount of MSC markers shared by sarcoma cells. The expression pattern includes uniformly expressed markers, as well as MSC markers that only stained subpopulations of sarcoma cells. Expression of W5C5, W8B2 (tissue nonspecific alkaline phosphatase [TNAP]), CD344 (frizzled‐4), and CD271 marked subpopulations displaying increased proliferation potential. Moreover, CD271+ cells displayed in vitro doxorubicin resistance and an increased capacity to form spheres under serum‐free conditions. Interestingly, another set of antigens, including the bona fide progenitor cell markers CD117 and CD133, were not expressed. Comparative expression patterns of novel MSC markers in sarcoma cells, as well as fibroblasts and MSCs, are presented. Our data suggest a hierarchical cytoarchitecture of the most common adult type sarcomas and introduce W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers.
    Keywords: Mesenchymal Stem Cell ; Sarcoma Stem Cell ; Cancer Stem Cell ; Stem Cell Marker
    ISSN: 2157-6564
    E-ISSN: 2157-6580
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  • 4
    Language: English
    In: Haematologica, February 2003, Vol.88(2), pp.126-33
    Description: Mesenchymal stem cells (MSC) and neural progenitor cells (NPC) are pluripotent cells. The former can give rise to myocytes, chondrocytes, adipocytes, and osteogenic cells, while the latter can give rise to astrocytes, neurons, and oligodendrocytes. The aim of this study was to analyze and compare the antigen expression patterns of MSC and NPC. Human bone marrow-derived MSC and NPC were analyzed by flow cytometry and immunocytochemistry using a variety of unique monoclonal antibodies (57D2, W4A5, W8B2) generated in our laboratory. In addition, the expression profile of CD antigens and intracellular differentiation markers was analyzed. We show for the first time that CD10+, CD13+, CD61+, CD90+, CD105 (endoglin)+, CD45-, CD34-, and CD133- MSC also expressed CD109, CD140b (PDGF-RB), CD164, and CD172a (SIRPa). In addition, we found heterogeneity of MSC as demonstrated by the preferential expression of nestin and W8B2 antigen on distinct MSC subpopulations. Morphologically, these populations comprised small single cells and larger cells with polygonal appearance. NPC expressed high levels of CD56, CD90 and nestin and moderate levels of CD15, W4A5, and 57D2 antigens. In contrast, CD133 and CD172 were found only on NPC subpopulations. Our data demonstrate nestin expression in most NPC as well as in immature MSC subpopulations. MSC and NPC subpopulations can now be distinguished using our novel antibodies W8B2, 57D2, and W4A5.
    Keywords: Mesoderm -- Cytology ; Neurons -- Cytology ; Pluripotent Stem Cells -- Cytology
    ISSN: 0390-6078
    E-ISSN: 15928721
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 5
    Language: English
    In: Experimental Hematology, 2002, Vol.30(6), pp.537-545
    Description: OBJECTIVEMonoclonal antibody (mAb) 9C4 detects a surface antigen expressed on immature erythroid progenitor cells and epithelial tumor cell lines. The aim of this study was to identify the recognized surface antigen and to analyze a potential role of this molecule in early steps of erythropoiesis. MATERIALS AND METHODSA pituitary-derived retroviral cDNA library was used to generate viruses and infect NIH-3T3 fibroblasts. The transfected cells were stained with mAb 9C4; positive cells were sorted by FACS; and a clonal cell line binding mAb 9C4 was established. cDNA encoding the 9C4-binding protein was amplified by polymerase chain reaction and cloned. Reactivity of mAb 9C4 with human bone marrow (BM) cells was analyzed by flow cytometry. RESULTSSequence analysis of the isolated cDNA uncovered a 100% identity with the epithelial cellular adhesion molecule (Ep-CAM). Two-color flow cytometric analysis revealed that almost 100% of Ep-CAM(+) BM cells coexpressed CD105, E-cadherin, and high levels of CD71. Fractions of Ep-CAM(+) BM cells also were CD34(+) but lacked glycophorin A expression, suggesting that Ep-CAM(+) cells represent immature erythroid cells. Reverse transcriptase polymerase chain reaction analysis of BM mononuclear cells revealed that the 9C4(+) erythroblast population but not the 9C4(-) fraction expressed Ep-CAM mRNA. Peripheral blood CD34(+) cells induced in vitro to differentiate into the erythroid lineage showed strong Ep-CAM expression on days 3 to 7 of culture. The addition of Ep-CAM-specific mAbs 9C4 or KS1/4 to the culture resulted in two- to three-fold up-regulation of Ep-CAM protein expression. CONCLUSIONmAb 9C4 recognizes Ep-CAM, a molecule expressed in the early steps of erythropoiesis.
    Keywords: Medicine
    ISSN: 0301-472X
    E-ISSN: 1873-2399
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  • 6
    Language: German
    In: Medizinische Klinik, 1997, Vol.92(7), pp.410-414
    Description: Die Anwendung hämatopoetischer Wachstumsfaktoren und die Retransfusion patienteneigener, aus dem Blut gewonnener peripherer Blutstammzellen (PBSZ) hat in den letzten Jahren die Option zur Durchführung einer Hochdosischemotherapie (HDT) auch bei fortgeschrittenen soliden Tumoren ermöglicht. Da mit der Transfusion von peripheren Blutstammzellen die Myelosuppression beherrschbar wurde, bestimmt die nichthämatologische Toxizität den Grad der Dosiseskalation.In der Regel wurden Hochdosischemotherapien nach vorausgegangener dosisintensivierter Chemotherapie durchgeführt. Durch die vorgeschaltete Chemotherapie soll einerseits die Chemosensibilität der Tumorerkrankung nachgewiesen und andererseits eine Stammzellmobilisierung bewirkt werden. Früher wurde die Hochdosischemotherapie durch eine autologe Knochenmarktransplantation (AKMT), heute fast ausnahmslos durch die Retransfusion von peripheren Blutstammzellen als spezifische Supportivmaßnahme begleitet. Die peripheren Blutstammzellen werden durch die Apherese der mononukleären Zellfraktion am Zellseparator gesammelt.Nennenswerte Erfahrungen mit der Hochdosischemotherapie beim Erwachsenen liegen für das Mamma-, Hoden-, Ovarial-, kleinzellige Bronchialkarzinom (SCLC), Melanom, Glioblastom und Weichgewebssarkom vor. Die Studien belegen die Durchführbarkeit und Effektivität der Hochdosischemotherapie bei tolerabler Toxizität.Die Hochdosischemotherapie wird zukünftig eine zunehmende Bedeutung in dem primären Chemotherapiekonzept von soliden Tumoren bekommen.The availability of hemopoetic growth factors and the retransfusion of autologous peripheral blood stem cells (PBSC) have enabled high-dose chemotherapy (HDT) options for the treatment of advanced solid tumours, during recent years. Though the transfusion of PBSC can manage the myelosuppression, dose-escalation ist still limited by non-haematological toxicity.Usually, HDT has been given after a preceeding dose-intense chemotherapy that allowed the evaluation of chemosensitivity of the disease and resulted in a stem cell mobilization into the peripheral blood. The autologous bone marrow transplantation has almost completely been replaced by retransfusion of PBSC together with hemopoetic growth factors as specific supportive treatment. The PBSC are harvested by apheresis using a blood cell separator.Results on the efficacy of HDT are available for breast, testicular, ovarian, small cell lung cancer as well as melanoma, glioblastoma and soft-tissue sarcoma. The studies showed the feasibility and efficacy of HDT with tolerable toxicity.High-dose chemotherapy will be of increasing importance in the treatment strategies of primary solid tumors, in the near future.
    Keywords: High-dose chemotherapy ; Peripheral blood stem cells ; Solid tumors
    ISSN: 0723-5003
    E-ISSN: 1615-6722
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  • 7
    Language: English
    In: Journal of Clinical Oncology, 05/1999, Vol.17(5), pp.1535-1535
    Description: PURPOSE: The expression of the carcinoma-associated mucin MUC-1 is thought to be restricted to epithelial cells and is used for micrometastatic tumor cell detection in patients with solid tumors, including those with breast cancer. Little is known, however, about the expression of MUC-1 epitopes in normal hematopoietic cells.MATERIALS AND METHODS: MUC-1 expression was analyzed by flow cytometry and immunocytology on bone marrow (BM) mononuclear cells and purified CD34+ cells from healthy volunteers, using different anti-MUC-1-specific monoclonal antibodies. In addition, Western blotting of MUC-1 proteins was performed.RESULTS: Surprisingly, 2% to 10% of normal human BM mononuclear cells expressed MUC-1, as defined by the anti-MUC-1 antibodies BM-2 (2E11), BM-7, 12H12, MAM-6, and HMFG-1. In contrast, two antibodies recognizing the BM-8 and the HMFG-2 epitopes of MUC-1 were not detected. MUC-1+ cells from normal BM consisted primarily of erythroblasts and normoblasts. In agreement with this, normal CD34+ cells cultured in vitro to differentiate into the erythroid lineage showed a strong MUC-1 expression on day 7 proerythroblasts. Western blotting of these cells confirmed that the reactive species is the known high molecular weight MUC-1 protein.CONCLUSION: Our data demonstrate that some MUC-1 epitopes are expressed on normal BM cells and particularly on cells of the erythroid lineage. Hence the application of anti-MUC-1 antibodies for disseminated tumor cell detection in BM or peripheral blood progenitor cells may provide false-positive results, and only carefully evaluated anti-MUC-1 antibodies (eg, HMFG-2) might be selected. Furthermore, MUC-1-targeted immunotherapy in cancer patients might be hampered by the suppression of erythropoiesis.
    Keywords: Medicine ; Pharmacy, Therapeutics, & Pharmacology;
    ISSN: 0732-183X
    E-ISSN: 1527-7755
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