title: Identification of differentiation-stage specific markers that define the ex vivo osteoblastic phenotype.
abstract: The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity. For the clinical use of human skeletal (stromal, mesenchymal) stem cells (hMSC) in therapy, there is also a need to identify a set of gene markers that predict in vivo bone forming capacity. Thus, we used RNA sequencing to examine changes in expression for a set of skeletally-related genes across 8 time points between 0 and 12days of ex vivo OB differentiation of hMSC. We identified 123 genes showing significant temporal expression change. Hierarchical clustering and Pearson's correlation generated 4 groups of genes: early stage differentiation genes (peak expression: 0-24h, n=28) which were enriched for extracellular matrix organisation, e.g. COL1A1, LOX, and SERPINH1; middle stage differentiating genes (peak expression days: 3 and 6, n=20) which were enriched for extracellular matrix/skeletal system development e.g. BMP4, CYP24A1, and TGFBR2; and late stage differentiation genes (peak expression days: 9 and 12, n=27) which were enriched for bone development/osteoblast differentiation, e.g. BMP2 and IGF2. In addition, we identified 13 genes with bimodal temporal expression (2 peaks of expression: days 0 and 12) including VEGFA, PDGFA and FGF2. We examined the specificity of the 123 genes' expression in skeletal tissues and thus propose a set of ex vivo differentiation-stage-specific markers (n=21). In an independent analysis, we identified a subset of genes (n=20, e.g. ELN, COL11A1, BMP4) to predict the bone forming capacity of hMSC and another set (n=20, e.g. IGF2, TGFB2, SMAD3) associated with the ex vivo phenotype of hMSC obtained from osteoporotic patients.
authors.affiliation: NSW Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Twine
authors.affiliation: Department of Endocrinology and Metabolism, Endocrine Research Laboratory (KMEB), Odense University Hospital, Odense, Denmark.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.affiliation: NSW Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Chi N
authors.initials: CN
authors.lastname: Pang
authors.affiliation: NSW Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Marc R
authors.initials: MR
authors.lastname: Wilkins
authors.affiliation: NSW Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia; Department of Endocrinology and Metabolism, Endocrine Research Laboratory (KMEB), Odense University Hospital, Odense, Denmark; The Danish Stem Cell Center (DanStem), University of Copenhagen, Copenhagen, Denmark. Electronic address: mkassem@health.sdu.dk.
authors.forename: Moustapha
authors.initials: M
authors.lastname: Kassem
mesh_terms: D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D020869:Gene Expression Profiling; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D010024:Osteoporosis / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000378:metabolism; D065668:Vitamin D3 24-Hydroxylase / Q000235:genetics / Q000378:metabolism; D000097522:Neuroserpin
chemical_list: D000943:Antigens, Differentiation; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D009479:Neuropeptides; D015843:Serpins; D065668:Vitamin D3 24-Hydroxylase; D000097522:Neuroserpin; C525718:BMP2 protein, human; C525809:BMP4 protein, human; C585549:CYP24A1 protein, human
keywords: Mesenchymal stem cells; Osteoblasts; Osteogenesis; Osteoporosis; RNA sequencing