title: Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.
abstract: BACKGROUND
  The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro.
  
  
  METHODS
  Nucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2- and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2).
  
  
  RESULTS
  After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2- NPC), respectively. Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2- cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2- cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001).
  
  
  CONCLUSIONS
  Our results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.
authors.affiliation: Tissue and Organ Mechanobiology, Institute for Surgical Technology & Biomechanics, Medical Faculty, University of Bern, Bern, Switzerland. adel.tekari@istb.unibe.ch.
authors.forename: Adel
authors.initials: A
authors.lastname: Tekari
authors.affiliation: Tissue and Organ Mechanobiology, Institute for Surgical Technology & Biomechanics, Medical Faculty, University of Bern, Bern, Switzerland.|Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, St Gallen, Switzerland.
authors.forename: Samantha C W
authors.initials: SCW
authors.lastname: Chan
authors.affiliation: Department for Orthopaedic Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan.|AO Spine Research Network, AO Spine International, Davos, Switzerland.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sakai
authors.affiliation: AO Research Institute Davos, Davos, Switzerland.|AO Spine Research Network, AO Spine International, Davos, Switzerland.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Grad
authors.affiliation: Tissue and Organ Mechanobiology, Institute for Surgical Technology & Biomechanics, Medical Faculty, University of Bern, Bern, Switzerland.|AO Spine Research Network, AO Spine International, Davos, Switzerland.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gantenbein
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D042683:Angiopoietin-1 / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002454:Cell Differentiation; D015687:Cell Hypoxia; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003050:Coccyx / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018920:Coculture Techniques; D015870:Gene Expression; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D000070614:Nucleus Pulposus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D042787:Receptor, TIE-2 / Q000235:genetics* / Q000378:metabolism; D044968:Regenerative Medicine; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; D036341:Intercellular Signaling Peptides and Proteins; D042787:Receptor, TIE-2
keywords: Fibroblast growth factor 2; Growth factors; Hypoxia; Intervertebral disc; Nucleus pulposus; Nucleus pulposus progenitor cells; Tie2