title: A xeno-free culturing protocol for pluripotent stem cell-derived oligodendrocyte precursor cell production.
abstract: OBJECTIVE
  To show that human embryonic stem cells (hESCs) can be efficiently differentiated into oligodendrocyte precursor cells (OPCs) in a xeno-free medium with a specific medium supplement and specific human recombinant growth factors.
  
  
  METHODS
  The xeno-free OPC-differentiation medium for pluripotent stem cells was developed by using StemPro® neural stem cell xeno-free medium supplement together with human recombinant growth factors SHH, PDGF-AA, IGF-1, EGF, basic FGF and CNTF, in addition to RA, T3, human laminin and ascorbic acid. We analyzed the differentiated hESC-derived OPCs and oligodendrocytes with quantitative real-time (RT)-PCR, RT-PCR, flow cytometry and immunocytochemistry, and we performed NG2-positive selection for OPC cultures with fluorescence-activated cell sorting.
  
  
  RESULTS
  Based on quantitative RT-PCR analysis, OPCs after 9 weeks of differentiation in xeno-free medium expressed OLIG2, SOX10 and NKX2.2 at elevated levels compared with control conditions. According to the flow cytometric analysis, the cells expressed A2B5 (>70%) and NG2 (40-60%) at 5 weeks time point whereas maturing oligodendrocytes expressed O4 (60-80%) at 11 weeks time point. In addition, hESC-derived OPC populations were purified based on NG2-positive selection using fluorescence-activated cell sorting. NG2-positive OPC populations survived and differentiated further into O4 expressing oligodendrocytes in xeno-free medium, and the sorted cell populations were free from pluripotent Tra1-81 and Oct-4 -positive cells.
  
  
  CONCLUSIONS
  This study confirms that the xeno-free culturing method can support the differentiation and purification of hESC-derived OPC populations and provides an initial step toward safe cell graft production for the future clinical applications.
authors.affiliation: Regea - Institute for Regenerative Medicine, University of Tampere & Tampere University Hospital, Tampere, Finland.
authors.forename: Maria
authors.initials: M
authors.lastname: Sundberg
authors.forename: Anu
authors.initials: A
authors.lastname: Hyysalo
authors.forename: Heli
authors.initials: H
authors.lastname: Skottman
authors.forename: Soojung
authors.initials: S
authors.lastname: Shin
authors.forename: Mohan
authors.initials: M
authors.lastname: Vemuri
authors.forename: Riitta
authors.initials: R
authors.lastname: Suuronen
authors.forename: Susanna
authors.initials: S
authors.lastname: Narkilahti
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008297:Male; D009687:Nuclear Proteins; D009836:Oligodendroglia / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000715631:NKX2-2 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors