title: Phenotypic and differentiation stability of human embryonic stem cell-derived osteoblasts.
abstract: OBJECTIVE
  To ensure the efficiency and safety of transplanted human embryonic stem cell (hESC)-derived osteoblast-like cells (hESC-OS) for bone regeneration, this study was designed to determine the effects of continuous cell expansion on the osteoblastic differentiation stability, pluripotency, and tumorigenic potential of long-term expanded hESC-OS.
  
  
  METHODS
  hESCs manually harvested as cell aggregates or enzymatically dissociated as single cells were directly incubated in osteogenic medium and serially passaged to passage 25. Expression of osteoblast-related genes, pluripotent regulator genes, and genes related to tumorigenesis were examined at the primary passage and every 5 passages thereafter. hESC-OS were subcutaneously transplanted into nude mice for 4-24 weeks to test for teratoma formation. hESC-OS were recultivated in hESC culture conditions to evaluate the extent to which reverse differentiation back to the undifferentiated stage may occur.
  
  
  RESULTS
  hESC-OS derived from hESC aggregates and dissociated cells exhibited comparable osteoblast differentiation patterns. Expression levels of osteoblast-related genes reached plateau levels at passages 5-10 before declining in higher passages. Expression of tumor-associated genes was not significantly increased. Only hESC-OS at primary and first passages formed teratomas after 4 weeks in vivo. The hESC-OS were not able to revert to hESCs.
  
  
  CONCLUSIONS
  Expanded hESC-OS demonstrated lineage-specific differentiation stability, did not maintain the pluripotency of hES cells, and were genetically stable. Thus, hESC-OS may be considered for large animal preclinical studies.
authors.affiliation: Department of Oral and Maxillofacial Surgery, School of Dentistry, Prince of Songkla University, Hat Yai, Thailand.
authors.forename: Premjit
authors.initials: P
authors.lastname: Arpornmaeklong
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pressler
authors.forename: Paul H
authors.initials: PH
authors.lastname: Krebsbach
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D005838:Genotype; D006801:Humans; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype