title: Human adipose tissue-derived stem cells cultured in xeno-free culture condition enhance c-MYC expression increasing proliferation but bypassing spontaneous cell transformation.
abstract: BACKGROUND
  Human adipose tissue-derived stem cells (hASCs) are attractive cells for therapeutic applications and are currently being evaluated in multiple clinical trials. Prior to their clinical application, hASCs must be expanded ex vivo to obtain the required number of cells for transplantation. Fetal bovine serum is the supplement most widely used for cell culture, but it has disadvantages and it is not safe for cell therapy due to the risks of pathogen transmission and immune reaction. Furthermore, the cell expansion poses a risk of accumulating genetic abnormalities that could lead to malignant cell transformation. In this study, our aim was to evaluate the proliferation pattern as well as the resistance to spontaneous transformation of hASCs during expansion in a xeno-free culture condition.
  
  
  METHODS
  hASCs were expanded in Dulbecco's modified Eagle's medium supplemented with pooled allogeneic human serum or fetal bovine serum to enable a side-by-side comparison. Cell viability and differentiation capacity toward the mesenchymal lineages were assessed, along with immunophenotype. Ki-67 expression and the proliferation kinetics were investigated. The expression of the transcription factors c-FOS and c-MYC was examined with Western blot, and MYC, CDKN2A, ERBB2 and TERT gene expression was assessed with quantitative PCR. Senescence was evaluated by β-gal staining. Karyotype analysis was performed and tumorigenesis assay in vivo was also evaluated.
  
  
  RESULTS
  The hASCs expanded in medium with pooled allogeneic human serum did not show remarkable differences in morphology, viability, differentiation capacity or immunophenotype. The main difference observed was a significantly higher proliferative effect on hASCs cultured in pooled allogeneic human serum. There was no significant difference in C-FOS expression; however, C-MYC protein expression was enhanced in pooled allogeneic human serum cultures compared to fetal bovine serum cultures. No difference was observed in MYC and TERT mRNA levels. Moreover, the hASCs presented normal karyotype undergoing senescence, and did not form in vivo tumors, eliminating the possibility that spontaneous immortalization of hASCs had occurred with pooled allogeneic human serum.
  
  
  CONCLUSIONS
  This complete characterization of hASCs cultivated in pooled allogeneic human serum, a suitable xeno-free approach, shows that pooled allogeneic human serum provides a high proliferation rate, which can be attributed for the first time to C-MYC protein expression, and showed cell stability for safe clinical applications in compliance with good manufacturing practice.
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-910, Brazil. ana.chagasdepaula@gmail.com.
authors.forename: Ana C C
authors.initials: AC
authors.lastname: Paula
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-910, Brazil. thaismmmartins@gmail.com.
authors.forename: Thaís M M
authors.initials: TM
authors.lastname: Martins
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-910, Brazil. alessandrazonari@gmail.com.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Zonari
authors.affiliation: Instituto Hermes Pardini, Av. das Nações, 2448, Vespasiano, Minas Gerais, 33200-000, Brazil. soraia.frade@labhpardini.com.br.
authors.forename: Soraia P P J
authors.initials: SP
authors.lastname: Frade
authors.affiliation: Instituto Hermes Pardini, Av. das Nações, 2448, Vespasiano, Minas Gerais, 33200-000, Brazil. patricia.angelo@labhpardini.com.br.
authors.forename: Patrícia C
authors.initials: PC
authors.lastname: Angelo
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-910, Brazil. dawidson.gomes@gmail.com.
authors.forename: Dawidson A
authors.initials: DA
authors.lastname: Gomes
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Department of Biochemistry and Immunology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, 31270-910, Brazil. goes@icb.ufmg.br.
authors.forename: Alfredo M
authors.initials: AM
authors.lastname: Goes
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology*; D006801:Humans; D007621:Karyotyping; D051379:Mice; D008819:Mice, Nude; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D044967:Serum / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019098:Telomerase / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D016760:Proto-Oncogene Proteins c-fos; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D019098:Telomerase