title: In vitro differentiation of human calvarial suture derived cells with and without dexamethasone does not induce in vivo-like expression.
abstract: Osteogenic supplements are a requirement for osteoblastic cell differentiation during in vitro culture of human calvarial suture-derived cell populations. We investigated the ability of ascorbic acid and beta-glycerophosphate with and without the addition of dexamethasone to stimulate in vivo-like osteoblastic differentiation. Cells were isolated from unfused and prematurely fused suture tissue from patients with syndromic and non-syndromic craniosynostosis and cultured in each osteogenic medium for varying lengths of time. The effect of media supplementation was investigated with respect to the ability of cells to form mineralised bone nodules and the expression of five osteodifferentiation marker genes (COL1A1, ALP, BSP, OC and RUNX2), and five genes that are differentially expressed during human premature suture fusion (GPC3, RBP4, C1QTNF3, WIF1 and FGF2). Cells from unfused sutures responded more slowly to osteogenic media but formed comparable bone nodules to fused suture-derived cells after 16 days of culture in either osteogenic media. However, gene expression differed between unfused and fused suture-derived cells, as did expression in each osteogenic medium. When compared to expression in the explant tissue of origin, neither medium induced a level or profile of gene expression similar to that seen in vivo. Overall, our results demonstrate that cells from the same suture that are isolated during different stages of morphogenesis in vivo, despite being de-differentiated to a similar level in vitro, respond uniquely and differently to each osteogenic medium. Further, we suggest that neither cell culture medium recapitulates differentiation via activation of the same genetic cascades as occurs in vivo.
authors.affiliation: Cooperative Research Centre for Diagnostics, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Coussens
authors.forename: Ian P
authors.initials: IP
authors.lastname: Hughes
authors.forename: C Phillip
authors.initials: CP
authors.lastname: Morris
authors.forename: Barry C
authors.initials: BC
authors.lastname: Powell
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Anderson
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003393:Cranial Sutures / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D003398:Craniosynostoses / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D003470:Culture Media; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D005994:Glycerophosphates / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007223:Infant; D008297:Male; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D010641:Phenotype; D048069:Tumor Necrosis Factors / Q000235:genetics
chemical_list: C506069:C1QTNF3 protein, human; D003470:Culture Media; D017931:DNA Primers; D005994:Glycerophosphates; D048069:Tumor Necrosis Factors; D016222:Fibroblast Growth Factor 2; D003907:Dexamethasone; D001205:Ascorbic Acid; C031463:beta-glycerophosphoric acid