title: Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells.
abstract: BACKGROUND
  Notochordal cells (NCs) are influential in development of the intervertebral disc (IVD) and species that retain NCs do not degenerate. IVD repair using bone marrow derived mesenchymal stem cells (MSCs) is an attractive approach and the harsh microenvironment of the IVD suggests pre-differentiation is a necessary first step. The goal of this study was to use soluble factors from NCs in alginate and NCs in their native tissue to differentiate human MSCs to a young nucleus pulposus (NP) phenotype.
  
  
  METHODS
  Human MSCs (cultured under micromass conditions for 21 days in hypoxia) were differentiated with conditioned medium derived from porcine notochordal cells in native tissue (NCT) or in alginate beads (NCA), and compared with chondrogenic (TGFβ-3) or basal medium. A PCR array of 42 genes was utilized to screen a large number of genes known to be associated with the healthy NP phenotype and pellet cultures were also evaluated for glycosaminoglycan content, histology and viability. Proteomic analysis was used to assess candidate soluble factors in NCA and NCT.
  
  
  RESULTS
  Notochordal cell conditioned media had diverse effects on MSC phenotype. NCT resulted in the highest levels of glycosaminoglycan (GAG), as well as up-regulation of SOX9 and Collagen II gene expression. NCA demonstrated effects that were catabolic yet also anti-fibrotic and minimally hypertrophic with down-regulation of Collagens I and III and low levels of Collagen X, respectively. Micromass culture and hypoxic conditions were sufficient to promote chondrogenesis demonstrating that both basal and chondrogenic media produced similar phenotypes. Candidate matricellular proteins, clusterin and tenascin were identified by proteomics in the NCA group.
  
  
  CONCLUSIONS
  NCs secreted important soluble factors capable of differentiating MSCs to a NP phenotype synthesizing high levels of proteoglycan while also resisting collagen fiber expression and hypertrophy, yet results were sensitive to the conditions in which media was generated (cells in alginate versus cells in their native tissue) so that further mechanistic studies optimizing culture conditions and defining important NC secreted factors are required. Matricellular proteins, such as clusterin and tenascin, are likely to be important to optimize differentiation of MSCs for maximum GAG production and reduced collagen fiber expression.
authors.affiliation: Leni and Peter W, May Department of Orthopaedics, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1188, New York, NY 10029-6574, USA.
authors.forename: Devina
authors.initials: D
authors.lastname: Purmessur
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Schek
authors.forename: Rosalyn D
authors.initials: RD
authors.lastname: Abbott
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Ballif
authors.forename: Karolyn E
authors.initials: KE
authors.lastname: Godburn
authors.forename: James C
authors.initials: JC
authors.lastname: Iatridis
mesh_terms: D000328:Adult; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D060833:Cellular Microenvironment / Q000502:physiology; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D016207:Cytokines / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D007403:Intervertebral Disc / Q000166:cytology* / Q000196:embryology; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D009672:Notochord / Q000166:cytology* / Q000196:embryology; D010641:Phenotype; D011509:Proteoglycans / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013552:Swine; D055815:Young Adult
chemical_list: D017077:Culture Media, Conditioned; D016207:Cytokines; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D036341:Intercellular Signaling Peptides and Proteins; D011509:Proteoglycans