title: Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions.
abstract: Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs.
  
  
  BACKGROUND
  Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation.
  
  
  METHODS
  cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, β-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies.
  
  
  RESULTS
  In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition.
  
  
  CONCLUSIONS
  The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts.
authors: Group Applied Stem Cell Technology, Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.
authors: S
authors: S
authors: Trettner
authors: A
authors: A
authors: Findeisen
authors: S
authors: S
authors: Taube
authors: P A
authors: PA
authors: Horn
authors: E
authors: E
authors: Sasaki
authors: N I
authors: NI
authors: zur Nieden
mesh_terms: D000818:Animals; D002144:Callithrix; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology; D059785:Karyotype; D051379:Mice; D023421:Models, Animal; D010006:Osteoblasts / Q000166:cytology; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D003470:Culture Media; D014212:Tretinoin