title: [Time differences of bone marrow mesenchymal stem cells and nucleus pulposus-like cells in a non contact co-culture system].
abstract: OBJECTIVE
  To investigate time differences in directional differentiation of bone marrow mesenchymal stem cells (BMSCs) into nucleus pulposus-like cells (NPCs) in a non contact co-culture system so as to search for the best time for transplantation in vivo.
  
  
  METHODS
  Six New Zealand white rabbits (aged 6 weeks, weighing 1.5-2.0 kg) were selected. BMSCs were collected and cultured for immunocytochemistry identification of CD34, CD44, CD45, and CD90; NPCs were isolated and identified immunocytochemically by RT-PCR. The 2nd passage BMSCs and the primary NPCs were co-cultured in a non contact co-culture system. The cell morphological changes were observed and the cell growth curves were made at 1, 3, and 5 passages after co-culture. The expressions of the aggrecan and collagen type II genes were detected by RT-PCR in BMSCs at 5, 10, and 15 days after co-culture; the expressions of the aggrecan and collagen type II proteins were detected by Western blot at 5, 10, 15, 20, 25, and 30 days after co-culture.
  
  
  RESULTS
  The expressions of CD44 and CD90 were positive, CD34 and CD45 were negative in BMSCs. The expressions of the collagen type II and aggrecan were positive in NPCs. At 2 weeks after co-culture, the morphology of BMSCs changed obviously, the cells were polygonal and irregular shape. The cell growth rate showed no difference within 3 passages, but decreased obviously after 3 passages. RT-PCR showed that the expressions of collagen type II and aggrecan genes at 10 and 15 days were significantly higher than those at 5 days (P < 0.05), no significant difference was found between at 10 days and at 15 days (P > 0.05). Western blot showed that the expressions of collagen type II and aggrecan proteins gradually increased with time, and there was significant difference within 5, 10, and 15 days (P < 0.05), but no significant difference was found after 15 days of co-culture (P > 0.05).
  
  
  CONCLUSIONS
  In a non contact co-culture system, BMSCs can differentiate into the NPCs. The expression of collagen type II and aggrecan can reach a stable level at 15 days after co-culture, and it is the suitable time for transplantation in vivo.
authors.affiliation: Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University, Fuzhou Fujian, 350004, P.R.China.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Xiunian
authors.initials: X
authors.lastname: Hu
authors.forename: Lengxi
authors.initials: L
authors.lastname: Fu
authors.forename: Junjian
authors.initials: J
authors.lastname: Ye
mesh_terms: D053674:Aggrecans / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D024043:Collagen Type II / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000628:therapy; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods
chemical_list: D053674:Aggrecans; D024043:Collagen Type II; D012333:RNA, Messenger