title: Brazilian minipig as a large-animal model for basic research and stem cell-based tissue engineering. Characterization and in vitro differentiation of bone marrow-derived mesenchymal stem cells.
abstract: Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources.
  
  
  OBJECTIVE
  This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering.
  
  
  METHODS
  Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR.
  
  
  RESULTS
  MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (p<0.001, p<0.001, p=0.031; respectively).
  
  
  CONCLUSIONS
  The isolation, cultivation, and differentiation of BM-MSCs from BR1 makes this animal eligible as a useful large-animal model for stem cell-based studies in Brazil.
authors.affiliation: Department of Oncology, Faculty of Medicine, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Roberta Targa
authors.initials: RT
authors.lastname: Stramandinoli-Zanicotti
authors.affiliation: Department of Oncology, Faculty of Medicine, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: André Lopes
authors.initials: AL
authors.lastname: Carvalho
authors.affiliation: Experimental Laboratory of Cell Culture, Pontifical Catholic University of Paraná, Curitiba, PR, Brazil.
authors.forename: Carmen Lúcia Kuniyoshi
authors.initials: CL
authors.lastname: Rebelatto
authors.affiliation: Service of Oral and Maxillofacial Surgery, Erasto Gaertner Hospital, Curitiba, PR, Brazil.
authors.forename: Laurindo Moacir
authors.initials: LM
authors.lastname: Sassi
authors.affiliation: Laboratory of Experimental Surgery, Positivo University/Department of Anatomy, Federal University of Paraná (UFPR), Curitiba, PR, Brazil., Curitiba, PR, Brazil.
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Torres
authors.affiliation: Experimental Laboratory of Cell Culture, Pontifical Catholic University of Paraná, Curitiba, PR, Brazil.
authors.forename: Alexandra Cristina
authors.initials: AC
authors.lastname: Senegaglia
authors.affiliation: Experimental Laboratory of Cell Culture, Pontifical Catholic University of Paraná, Curitiba, PR, Brazil.
authors.forename: Lidiane Maria
authors.initials: LM
authors.lastname: Boldrinileite
authors.affiliation: Laboratory of Basic Biology of Stem Cells, Carlos Chagas Institute, Curitiba, PR, Brazil.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Correa-Dominguez
authors.affiliation: Laboratory of Basic Biology of Stem Cells, Carlos Chagas Institute, Curitiba, PR, Brazil.
authors.forename: Crisciele
authors.initials: C
authors.lastname: Kuligovsky
authors.affiliation: Experimental Laboratory of Cell Culture, Pontifical Catholic University of Paraná, Curitiba, PR, Brazil.
authors.forename: Paulo Roberto Slud
authors.initials: PR
authors.lastname: Brofman
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology*; D001938:Brazil; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D005434:Flow Cytometry; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D023421:Models, Animal; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013556:Swine, Miniature; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D015703:Antigens, CD