Poster Presentation 6th Annual Meeting for Australasian Society for Stem Cell Research 2013

High Throughput Screening of Wnt Modulators for Osteogenesis (#159)

Harish Padmanabhan 1 , Jessica E Frith 1 , Drew M Titmarsh 1 , Justin J Cooper-White 1 2 3
  1. Tissue Engineering and Microfluidics, The Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia
  2. OCE Science Leader, Materials Science and Engineering Division, CSIRO, Clayton, Victoria, , Australia
  3. School of Chemical Engineering, The University of Queensland, Brisbane, Queensland, Australia

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine and have the potential to differentiate into osteo-, adipo- and chondrogenic lineages. However, many of the molecular pathways involved in the differentiation of MSCs to different lineages are still poorly understood. To explore the impact of Wnt signaling on the osteogenic differentiation of MSCs, we utilised a full factorial microbioreactor array (MBA)1  platform to  rapidly and combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects on osteogenic differentiation potential. The MBA screening system showed excellent consistency between the different MSC donors and experimental runs. Intriguingly, a Wnt agonist (CHIR99021) had a profoundly inhibitory effect on osteogenesis, which was contrary to our expectations and previous reports show that Wnt activation is required for osteogenesis. The Wnt antagonists (IWP-4 and IWR-1) also inhibited osteogenesis, which was certainly more in line with previous investigations. Importantly, we demonstrated that these results were translatable to standard, static culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we have shown that whilst CHIR did activate the Wnt pathway, it also exerted its effects on osteogenesis via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Previous reports using canonical Wnt’s (eg. Wnt3a) have shown that there was a decrease in ALP expression and mineralisation, but only when in the presence of Dexamethasone. The inhibitory effect of CHIR on ALP may thus be due to the presence of Dexamethasone, although this remains to be tested.   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 MSCs, with factors secreted by the MSCs 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 regulatory pathways, enabling the optimization of MSC growth and differentiation conditions for therapeutic applications. 

  1. Titmarsh DM, Hudson JE, Hidalgo A, Elefanty AG, Stanley EG, et al. (2012) Microbioreactor Arrays for Full Factorial Screening of Exogenous and Paracrine Factors in Human Embryonic Stem Cell Differentiation. PLoS ONE 7: e52405.