title: Foetal stem cell derivation & characterization for osteogenic lineage.
abstract: OBJECTIVE
  Mesencymal stem cells (MSCs) derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications.
  
  
  METHODS
  Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR) analysis and confirmed by sequencing using genetic analyzer.
  
  
  RESULTS
  Ovine foetal samples were processed to obtain mononuclear (MNC) cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45⁻/CD14⁻ was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP) activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells.
  
  
  CONCLUSIONS
  The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established. Criteria proposed for defining MSCs by this study includes the cell adherence to culture plates, specific surface protein profiles and differentiation to osteogenic lineage. The MSCs and osteogenic differentiated cells in this ovine animal model may serve as a large source for stem cell applications in regenerative medical therapies.
authors.affiliation: Department of Animal Biotechnology, Madras Veterinary College, Chennai, India. gowrivalavan@hotmail.com
authors.forename: A Mangala
authors.initials: AM
authors.lastname: Gowri
authors.forename: G
authors.initials: G
authors.lastname: Kavitha
authors.forename: M
authors.initials: M
authors.lastname: Rajasundari
authors.forename: S Mubeen
authors.initials: SM
authors.lastname: Fathima
authors.forename: T M A Senthil
authors.initials: TM
authors.lastname: Kumar
authors.forename: G Dhinakar
authors.initials: GD
authors.lastname: Raj
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D053686:Fetal Stem Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010012:Osteogenesis; D044968:Regenerative Medicine; D012756:Sheep; D023822:Tissue Engineering
chemical_list: D000469:Alkaline Phosphatase
references.citation: J Cell Sci. 2003 May 1;116(Pt 9):1827-35
references.pmid: 12665563
references.citation: J Cell Biochem Suppl. 2002;38:7-12
references.pmid: 12046852
references.citation: Blood. 1997 Nov 1;90(9):3471-81
references.pmid: 9345031
references.citation: J Orthop Res. 1991 Sep;9(5):641-50
references.pmid: 1870029
references.citation: Stem Cells. 2002;20(3):249-58
references.pmid: 12004083
references.citation: Calcif Tissue Int. 2003 May;72(5):537-47
references.pmid: 12724828
references.citation: Cytotherapy. 2006;8(3):194-5
references.pmid: 16793727
references.citation: Nat Med. 1999 Mar;5(3):262-4
references.pmid: 10086373
references.citation: Wound Repair Regen. 2007 Sep-Oct;15 Suppl 1:S18-26
references.pmid: 17727462
references.citation: J Cell Biochem. 2008 Aug 1;104(5):1793-802
references.pmid: 18348263
references.citation: Exp Hematol. 1976 Sep;4(5):267-74
references.pmid: 976387
references.citation: Int J Biol Macromol. 2011 Mar 1;48(2):276-85
references.pmid: 21126533
references.citation: Exp Biol Med (Maywood). 2001 Jun;226(6):507-20
references.pmid: 11395921
references.citation: Cytotherapy. 2006;8(4):315-7
references.pmid: 16923606
references.citation: J Cell Physiol. 2005 Jul;204(1):336-43
references.pmid: 15693018
references.citation: Science. 1999 Apr 2;284(5411):143-7
references.pmid: 10102814
references.citation: Blood. 2004 Mar 1;103(5):1669-75
references.pmid: 14576065
references.citation: Stem Cells. 2006 Jul;24(7):1707-18
references.pmid: 16574750
references.citation: J Cell Mol Med. 2004 Jul-Sep;8(3):301-16
references.pmid: 15491506
references.citation: Cell Tissue Kinet. 1970 Oct;3(4):393-403
references.pmid: 5523063