title: Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.
abstract: There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.
authors.affiliation: Oncoematologia Pediatrica, IRCCS Policlinico San Matteo, Università di Pavia, Pavia, Italy. mebernardo@gmail.com
authors.forename: M E
authors.initials: ME
authors.lastname: Bernardo
authors.forename: M A
authors.initials: MA
authors.lastname: Avanzini
authors.forename: C
authors.initials: C
authors.lastname: Perotti
authors.forename: A M
authors.initials: AM
authors.lastname: Cometa
authors.forename: A
authors.initials: A
authors.lastname: Moretta
authors.forename: E
authors.initials: E
authors.lastname: Lenta
authors.forename: C
authors.initials: C
authors.lastname: Del Fante
authors.forename: F
authors.initials: F
authors.lastname: Novara
authors.forename: A
authors.initials: A
authors.lastname: de Silvestri
authors.forename: G
authors.initials: G
authors.lastname: Amendola
authors.forename: O
authors.initials: O
authors.lastname: Zuffardi
authors.forename: R
authors.initials: R
authors.lastname: Maccario
authors.forename: F
authors.initials: F
authors.lastname: Locatelli
mesh_terms: D000293:Adolescent; D015704:CD4 Antigens / Q000276:immunology; D002452:Cell Count; D002454:Cell Differentiation; D049109:Cell Proliferation; D064987:Cell- and Tissue-Based Therapy; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology; D007621:Karyotyping; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology; D007700:Kinetics; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000276:immunology; D039902:Multipotent Stem Cells / Q000166:cytology*; D010641:Phenotype; D044967:Serum / Q000378:metabolism*; D017154:Stromal Cells / Q000166:cytology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
chemical_list: D015704:CD4 Antigens; D016207:Cytokines; D053645:Interleukin-2 Receptor alpha Subunit