title: Microbioreactor array screening of Wnt modulators and microenvironmental factors in osteogenic differentiation of mesenchymal progenitor cells.
abstract: Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better understanding signaling mechanisms, as well as optimizing MPC growth and differentiation conditions for therapeutic applications.
authors.affiliation: Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, St. Lucia, Queensland, Australia.
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Frith
authors.affiliation: Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, St. Lucia, Queensland, Australia.
authors.forename: Drew M
authors.initials: DM
authors.lastname: Titmarsh
authors.affiliation: Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, St. Lucia, Queensland, Australia.
authors.forename: Harish
authors.initials: H
authors.lastname: Padmanabhan
authors.affiliation: Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, St. Lucia, Queensland, Australia ; School of Chemical Engineering, The University of Queensland, St. Lucia, Queensland, Australia ; Materials Science and Engineering Division, CSIRO, Clayton, Victoria, Australia.
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Cooper-White
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D019149:Bioreactors; D002454:Cell Differentiation; D002478:Cells, Cultured; D060833:Cellular Microenvironment; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007094:Imides / Q000494:pharmacology; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D046228:Microarray Analysis / Q000295:instrumentation*; D010012:Osteogenesis / Q000235:genetics*; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000494:pharmacology*; D051153:Wnt Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics*; D060449:Wnt Signaling Pathway / Q000187:drug effects
chemical_list: D015415:Biomarkers; C473711:Chir 99021; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D050659:Core Binding Factor Alpha 1 Subunit; D018398:Homeodomain Proteins; C585534:IWR-1 compound; D007094:Imides; D011725:Pyridines; D011743:Pyrimidines; D011804:Quinolines; C493709:RUNX2 protein, human; C506895:SPP1 protein, human; D054852:Small Molecule Libraries; D051153:Wnt Proteins; D053495:Osteopontin; D000469:Alkaline Phosphatase