title: Differential Regulation of SOX9 Protein During Chondrogenesis of Induced Pluripotent Stem Cells Versus Mesenchymal Stromal Cells: A Shortcoming for Cartilage Formation.
abstract: Induced pluripotent stem cells (iPSCs) are an attractive cell source for cartilage regeneration, but current in vitro chondrogenic differentiation protocols yield insufficient results. In search for shortcomings of iPSC chondrogenesis, this study investigated whether SOX9 protein was adequately regulated during multiphase chondrogenic differentiation of two human iPSC lines in a comparable manner like during mesenchymal stromal cell (MSC) chondrogenesis. Upon generation of intermediate mesenchymal progenitor cells (iMPCs), SOX9 was induced and reached variable protein levels compared to MSCs. Along with an altered condensation behavior, iMPC cartilage formation was less robust compared to MSCs and better in the iMPC line with higher SOX9 protein levels. Despite efficient Smad-2/3 phosphorylation, TGF-β-driven chondrogenic stimulation downregulated SOX9 protein in iMPCs rather than increasing levels like in MSCs. Chondrogenesis was further improved by cotreatment with TGF-β + BMP-4, which appeared to shorten the duration of the SOX9 protein decline. However, this was insufficient to overcome heterogenic outcome and came at the expense of undesired hypertrophy. In iMPCs, but not MSCs, high levels of the SOX9-antagonizing hsa-miR-145 correlated with low SOX9 protein quantity. Thus, considerable iMPC heterogeneity with variable SOX9 protein levels, an altered condensation pattern, and low early SOX9 inducibility appeared as critical shortcomings of iPSC chondrogenesis. We suggest consistent quality of intermediate cell populations with high SOX9 protein induction as important indicators to obtain robust cartilage formation from iPSCs. The impact of this study is the identification of a SOX9 protein regulation opposite to MSC chondrogenesis that will now enable a selective adaptation of the currently limited protocols to the specific needs of iPSCs.
authors.affiliation: 1 Research Center for Experimental Orthopedics, Orthopedic University Hospital Heidelberg , Heidelberg, Germany .
authors.forename: Solvig
authors.initials: S
authors.lastname: Diederichs
authors.affiliation: 1 Research Center for Experimental Orthopedics, Orthopedic University Hospital Heidelberg , Heidelberg, Germany .
authors.forename: Jessica
authors.initials: J
authors.lastname: Gabler
authors.affiliation: 1 Research Center for Experimental Orthopedics, Orthopedic University Hospital Heidelberg , Heidelberg, Germany .
authors.forename: Jennifer
authors.initials: J
authors.lastname: Autenrieth
authors.affiliation: 1 Research Center for Experimental Orthopedics, Orthopedic University Hospital Heidelberg , Heidelberg, Germany .
authors.forename: Katharina L
authors.initials: KL
authors.lastname: Kynast
authors.affiliation: 2 Clinic for Orthopedics and Trauma Surgery, Orthopedic University Hospital Heidelberg , Heidelberg, Germany .
authors.forename: Christian
authors.initials: C
authors.lastname: Merle
authors.affiliation: 3 Department Tissue Engineering and Regenerative Medicine (TERM), University Hospital Würzburg , Würzburg, Germany .|4 Fraunhofer Institute for Interfacial Engineering and Biotechnology, Translational Center Würzburg 'Regenerative Therapies in Oncology and Musculoskeletal Diseases'-Würzburg Branch , Würzburg, Germany .
authors.forename: Heike
authors.initials: H
authors.lastname: Walles
authors.affiliation: 5 Skin Cancer Unit, German Cancer Research Center (DKFZ) , Heidelberg, Germany .|6 Department of Dermatology, Venereology, and Allergology, University Medical Center Mannheim, Ruprecht-Karls University of Heidelberg , Mannheim, Germany .
authors.forename: Jochen
authors.initials: J
authors.lastname: Utikal
authors.affiliation: 1 Research Center for Experimental Orthopedics, Orthopedic University Hospital Heidelberg , Heidelberg, Germany .
authors.forename: Wiltrud
authors.initials: W
authors.lastname: Richter
mesh_terms: D055415:Bone Morphogenetic Protein 4 / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D020219:Chondrogenesis; D024043:Collagen Type II / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D035683:MicroRNAs / Q000235:genetics; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D034622:RNA Interference; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D055755:SOXD Transcription Factors / Q000235:genetics / Q000378:metabolism; D051785:Smad Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000502:physiology
chemical_list: C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D024043:Collagen Type II; C541010:MIRN145 microRNA, human; D035683:MicroRNAs; C490301:SOX5 protein, human; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D055755:SOXD Transcription Factors; D051785:Smad Proteins; D016212:Transforming Growth Factor beta