title: Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture.
abstract: OBJECTIVE
  This study has aimed to repopulate 'primitive' cells from late-passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density.
  
  
  METHODS
  Effects of low density culture compared t high density culture on late-passage bone marrow (BM)-derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed.
  
  
  CONCLUSIONS
  We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2.
authors.affiliation: Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, South Korea.
authors.forename: D S
authors.initials: DS
authors.lastname: Yoon
authors.forename: Y H
authors.initials: YH
authors.lastname: Kim
authors.forename: H S
authors.initials: HS
authors.lastname: Jung
authors.forename: S
authors.initials: S
authors.lastname: Paik
authors.forename: J W
authors.initials: JW
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D002452:Cell Count; D018929:Cell Culture Techniques; D002453:Cell Cycle; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D005819:Genetic Markers; D018398:Homeodomain Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D006801:Humans; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000502:physiology*; D000071317:Nanog Homeobox Protein; D010012:Osteogenesis; D010641:Phenotype; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
chemical_list: D017931:DNA Primers; D005819:Genetic Markers; D018398:Homeodomain Proteins; C084119:MSX2 protein; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D012333:RNA, Messenger; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors