title: Prolonged in vitro expansion partially affects phenotypic features and osteogenic potential of ovine amniotic fluid-derived mesenchymal stromal cells.
abstract: OBJECTIVE
  Ovine amniotic fluid mesenchymal stromal cells (oAFMSCs) are an emerging alternative source of stem cells to develop pre-clinical cell replacement protocols. For tissue engineering purposes, oAFMSCs can be used either immediately after isolation or after in vitro expansion. However, detailed studies are still required to investigate the advantages and drawbacks of their in vitro expansion.
  
  
  METHODS
  The phenotype and osteogenic differentiation potential of oAFMSCs were analyzed in relation to in vitro expansion that was carried out for 20 consecutive passages. Expanded oAFMSCs were analyzed for proliferation index, expression profiles of several surface, pluripotency-associated and HLA antigens, global DNA methylation, telomere length and karyotype. The osteogenic differentiation ability of expanded oAFMSCs was assessed by qualitative and quantitative methods.
  
  
  RESULTS
  Expanded oAFMSCs reduced their proliferative activity after 10 passages and partially modified the expression of surface antigens and the intracellular distribution of pluripotency-associated markers (NANOG, SOX2 and TERT) after 20 passages. The phenotypic alteration of cultured oAFMSCs was associated with a reduction of in vitro osteogenic plasticity. In detail, after 20 passages of cellular expansion, oAFMSCs lost the ability to increase osteocalcin and decreased collagen type I messenger RNA expression. Also, a lower percentage of cells displayed intracellular calcium release after stimulation with salmon calcitonin.
  
  
  CONCLUSIONS
  The results presented here suggest that long-term in vitro expansion may cause significant alterations in phenotypic features and plasticity of oAFMSCs, suggesting a careful re-evaluation of in vitro cultural and temporal conditions before employing expanded oAFMSCs for therapeutic purposes.
authors.affiliation: Department of Comparative Biomedical Sciences, University of Teramo, Teramo, Italy. acolosimo@unite.it
authors.forename: Alessia
authors.initials: A
authors.lastname: Colosimo
authors.forename: Valentina
authors.initials: V
authors.lastname: Russo
authors.forename: Annunziata
authors.initials: A
authors.lastname: Mauro
authors.forename: Valentina
authors.initials: V
authors.lastname: Curini
authors.forename: Marco
authors.initials: M
authors.lastname: Marchisio
authors.forename: Nicola
authors.initials: N
authors.lastname: Bernabò
authors.forename: Melissa
authors.initials: M
authors.lastname: Alfonsi
authors.forename: Mauro
authors.initials: M
authors.lastname: Mattioli
authors.forename: Barbara
authors.initials: B
authors.lastname: Barboni
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology*; D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis / Q000235:genetics; D019175:DNA Methylation; D005260:Female; D018398:Homeodomain Proteins / Q000378:metabolism; D059785:Karyotype; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D015675:Osteocalcin / Q000096:biosynthesis; D010012:Osteogenesis; D010641:Phenotype; D012333:RNA, Messenger / Q000096:biosynthesis; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D034561:Sheep, Domestic; D019098:Telomerase / Q000378:metabolism; D016615:Telomere / Q000502:physiology; D023822:Tissue Engineering
chemical_list: D000954:Antigens, Surface; D024042:Collagen Type I; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D055748:SOXB1 Transcription Factors; D015675:Osteocalcin; D019098:Telomerase
keywords: amniotic fluid mesenchymal stromal cells; in vitro expansion; osteogenic differentiation potential; sheep