Immunophenotypic heterogeneity of bone marrow-derived mesenchymal stromal cells from patients with hematologic disorders: Correlation with bone marrow microenvironment

Section of Hematology, University Hospital, Ferrara, Italy.
Haematologica (Impact Factor: 5.81). 04/2006; 91(3):364-8.
Source: PubMed


The immunophenotypic analysis of ex vivo-expanded mesenchymal stromal cells (MSC) has so far been confined to single or dual staining analysis in normal subjects. In this study, using a four-color cytofluorimetric protocol, we demonstrated that cultured MSC derived from the bone marrow of patients with hematologic malignancies showed alterations in the expression of CD105, CD90, CD184, and HLA-DR molecules. The decrease in the percentage of CD105+ and CD90+ MSC correlated with an increased bone marrow angiogenesis. This paper provides evidence that multiparametric flow cytometry is essential for the establishment of a standardized protocol to identify various MSCs subsets and aberrant phenotypes.

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Available from: Francesco Lanza,
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    • "According to consensus, MSCs show no expression of HLA class II proteins [13]. An expression of HLA- DR has been reported for MSCs from BM in malignancy [17], but standard donor evaluation did not reveal any malignancy in donor 4. However, an expression of HLA-DR to a similar extent as seen in our donor was reported by Tarte et al. for the use of HPL-containing media. "
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    ABSTRACT: The immunomodulating capacity of multipotent mesenchymal stromal cells (MSCs) qualifies them as a therapeutic tool in several diseases. However, repeated transplantation with products of reproducible characteristics may be required. This could be achieved with cryopreserved aliquots of Good Manufacturing Practice (GMP)-grade MSCs. However, the impact of cryopreservation on the characteristics of GMP-MSCs is ill defined. We produced fresh and cryopreserved MSCs from human donors with a xenogen-free GMP protocol. Immunogenicity and immunomodulating capacity were tested in co-culture with putative recipient-specific peripheral blood mononuclear cells (PBMCs). Risk of malignant transformation was assessed in vitro and in vivo. Cryopreservation had no impact on viability and consensus criteria of MSCs. In co-culture with PBMCs, MSCs showed low immunogenicity and suppressed mitogen-stimulated proliferation of PBMC irrespective of cryopreservation. Cytogenetic aberrations were not observed consistently in fresh and cryopreserved products, and no signs of malignant transformation occurred in functional assays. MSC products from an elderly pretreated donor showed reduced functional quality, but imminent failure of functional criteria could be detected by an increased population doubling time in early passages. This study is the first systematic analysis on cryopreservation of xenogen-free human bone marrow-derived GMP-MSCs. The data support that cryopreservation does not alter the characteristics of the cells and thus may allow the generation of products for serial transplantation. In addition, the protocol allowed early detection of MSC products with low functional capacity. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
    Cytotherapy 02/2015; 17(2):186-98. DOI:10.1016/j.jcyt.2014.10.018 · 3.29 Impact Factor
    • "According to consensus, MSCs show no expression of HLA class II proteins [13]. An expression of HLA- DR has been reported for MSCs from BM in malignancy [17], but standard donor evaluation did not reveal any malignancy in donor 4. However, an expression of HLA-DR to a similar extent as seen in our donor was reported by Tarte et al. for the use of HPL-containing media. "
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    ABSTRACT: Background aims: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. Methods: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. Results: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. Conclusions: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.
    Cytotherapy 05/2014; 17(2). DOI:10.1016/j.jcyt.2014.04.002 · 3.29 Impact Factor
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    • "In general, no phenotypic changes have been reported in MSCs isolated from patients with haematological malignancies [33], [34]. Nevertheless, some studies reported decreased expression of some markers [35], [36]. Data in the literature are also contradictory with respect to differentiation capacities: some studies have reported normal osteogenic, chondrogenic and adipogenic differentiating potential in leukemic MSCs [34], whereas others have shown impaired chondrogenesis or adipogenesis [33], [37]. "
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    ABSTRACT: The relevance of tumor microenvironment for the development and progression of tumor cells in hematological malignancies has been extensively reported. Identification of factors involved in the information exchange between the malignant cells and the bone marrow mesenchymal stem cells (BM-MSCs) and the knowledge on their functioning may provide important information to eliminate leukemic cells from protective BM niches. We evaluated changes in BM-MSCs obtained from children with acute lymphoblastic leukemia (ALL) at different times in the course of disease. Whereas ALL-MSCs did not exhibit phenotypic changes compared to BM-derived MSCs isolated from healthy donors, they exhibited increased adipogenic capacity. In addition, the viability of healthy CD34+ hematopoietic progenitors was significantly reduced when co-cultured with ALL-MSCs. ALL-MSCs grow less efficiently, although gradually recover normal growth with treatment. Accordingly, proliferation is particularly low in MSCs obtained at diagnosis and in the first days of treatment (+15 days), recovering to control levels after 35 days of treatment. Correlating these results with bone morphogenetic protein 4 (BMP4) production, a molecule demonstrated to affect MSC biology, we found higher production of BMP4 in ALL-MSCs derived from patients over the course of disease but not in those free of leukemia. However, no significant differences in the expression of different members of the BMP4 signaling pathway were observed. Furthermore, an inverse correlation between high levels of BMP4 production in the cultures and MSC proliferation was found, as observed in MSCs derived from patients at diagnosis that produce high BMP4 levels. In addition, co-culturing ALL-MSC with the REH leukemia cell line, but not CD34+ hematopoietic progenitors, powerfully enhanced BMP4 production, suggesting an intimate crosstalk among ALL-MSCs isolated from BM colonized by ALL cells that presumably also occurs in situ conditions. Our data may support the participation of BMP4 in BM niche, but the mechanism remains to be elucidated.
    PLoS ONE 01/2014; 9(1):e84496. DOI:10.1371/journal.pone.0084496 · 3.23 Impact Factor
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