title: Immortalization of human adipose-derived stromal cells: production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors.
abstract: BACKGROUND
  Human adipose-derived stromal cells (hASCs), due to their relative feasibility of isolation and ability to secrete large amounts of angiogenic factors, are being evaluated for regenerative medicine. However, their limited culture life span may represent an obstacle for both preclinical investigation and therapeutic use. To overcome this problem, hASCs immortalization was performed in order to obtain cells with in vitro prolonged life span but still maintain their mesenchymal marker expression and ability to secrete angiogenic factors.
  
  
  METHODS
  hASCs were transduced with the human telomerase reverse transcriptase (hTERT) gene alone or in combination with either SV-40 or HPV E6/E7 genes. Mesenchymal marker expression on immortalized hASCs lines was confirmed by flow cytometry (FC), differentiation potential was evaluated by immunocytochemistry and ELISA kits were used for evaluation of angiogenic factors. Green fluorescent protein (GFP) gene transduction was used to obtain fluorescent cells.
  
  
  RESULTS
  We found that hTERT alone failed to immortalize hASCs (hASCs-T), while hTERT/SV40 (hASCs-TS) or hTERT/HPV E6/E7 (hASCs-TE) co-transductions successfully immortalized cells. Both hASCs-TS and hASCs-TE were cultured for up to one year with a population doubling level (PDL) up to 100. Comparative studies between parental not transduced (hASCs-M) and immortalized cell lines showed that both hASCs-TS and hASCs-TE maintained a mesenchymal phenotypic profile, whereas differentiation properties were reduced particularly in hASCs-TS. Interestingly, hASCs-TS and hASCs-TE showed a capability to secrete significant amount of HGF and VEGF. Furthermore, hASCs-TS and hASCs-TE did not show tumorigenic properties in vitro although some chromosomal aberrations were detected. Finally, hASCs-TS and hASCs-TE lines were stably fluorescent upon transduction with the GFP gene.
  
  
  CONCLUSIONS
  Here we demonstrated, for the first time, that hASCs, upon immortalization, maintain a strong capacity to secrete potent angiogenic molecules. By combining hASCs immortalization and their paracrine characteristics, we have developed a "hybridoma-like model" of hASCs that could have potential applications for discovering and producing molecules to use in regenerative medicine (process scale-up).
authors.forename: Luigi
authors.initials: L
authors.lastname: Balducci
authors.forename: Antonella
authors.initials: A
authors.lastname: Blasi
authors.forename: Marilisa
authors.initials: M
authors.lastname: Saldarelli
authors.forename: Antonio
authors.initials: A
authors.lastname: Soleti
authors.forename: Augusto
authors.initials: A
authors.lastname: Pessina
authors.forename: Arianna
authors.initials: A
authors.lastname: Bonomi
authors.forename: Valentina
authors.initials: V
authors.lastname: Coccè
authors.forename: Marta
authors.initials: M
authors.lastname: Dossena
authors.forename: Valentina
authors.initials: V
authors.lastname: Tosetti
authors.forename: Valentina
authors.initials: V
authors.lastname: Ceserani
authors.forename: Stefania Elena
authors.initials: SE
authors.lastname: Navone
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Falchetti
authors.forename: Eugenio Agostino
authors.initials: EA
authors.lastname: Parati
authors.forename: Giulio
authors.initials: G
authors.lastname: Alessandri
mesh_terms: D000273:Adipose Tissue / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line / Q000166:cytology* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D017228:Hepatocyte Growth Factor / Q000096:biosynthesis; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D019098:Telomerase / Q000235:genetics; D014161:Transduction, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis
chemical_list: C066855:HGF protein, human; D042461:Vascular Endothelial Growth Factor A; D017228:Hepatocyte Growth Factor; C509186:TERT protein, human; D019098:Telomerase