31 research outputs found
Art vs Design: Saving Power vs Enframing, or A Thing of the Past vs World-Making
No full texta debate with the co-author Cameron Tonkinwise of Carnegie Mellon University, USA, as to the ontological acumen of art versus design
Microbioreactor array screening of Wnt modulators and microenvironmental factors in osteogenic differentiation of Mesenchymal progenitor cells
No full textCellular 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
Significance of factor effects from factorial analysis.
No full text<p>Significance of factor effects from factorial analysis.</p
Analysis of selected inhibitor concentrations on osteogenesis under standard conditions.
No full text<p>A ELF97 (green) and PI (red) staining of MPCs treated with CHIR, IWP-4 and IWR-1 for 7 days. Scale bar, 100 µm. B Alizarin red staining of MPCs treated with combinations of CHIR, IWP-4 and IWR-1 for 21 days. Scale bar, 100 µm. C) RT-qPCR determination of expression of osteogenic marker genes after 7 days D) qPCR determination of expression of osteogenic markers genes after 21 days. RT-qPCR data is shown as mean±SEM. N = 3, p<0.05 (*), p<0.01 (**), p<0.001 (***).</p
qPCR determination of the expression of Wnt related factors.
No full text<p>qPCR determination of expression of Wnt pathway genes in MPCs after 7 and 21 days treatment. Data is shown as mean±SEM. N = 3, p<0.05 (*), p<0.01 (**), p<0.001 (***).</p
Screening MPC growth- and differentiation-conditioned medium in MBAs.
No full text<p>A Traces of ELF97/DNA expression index against row, from column 1 of all microbioreactor runs from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082931#pone-0082931-g002" target="_blank">Figure 2</a> (pooled arrays), and the average value. B Panel of conditions formed in conditioned medium screening experiment. C Heatmaps of total expression intensities (arbitrary units) for DNA, ELF97, and ELF97/DNA ratio. The average response of 3 technical replicates from one experimental run is shown. D Main effects plot showing effect of ROW, Growth-conditioned medium and Osteo-conditioned medium on expression index for ELF97/DNA ratio. E Interaction effects plot showing effects of 2 combined factors on ELF97/DNA ratio. F Traces of ELF97/DNA expression index against row, from columns 1, 8 and 9 (average of 3 technical replicates).</p
MBA screening of Wnt modulators in MPC osteogenesis.
No full text<p>A Panel of screening conditions in MBAs. Numbers denote concentrations of the various molecules, in µM. B Confocal microscopy images of endpoint PI (DNA) and ELF97 (alkaline phosphatase activity) staining from a representative experiment. Direction of fluid flow was from top to bottom. C Heatmaps of expression indices (see Methods) for DNA, ELF97, and ELF97/DNA ratio. The average expression index of 2 runs from each of 2 MPC donors (4 in total) is shown, and units represent global standard deviations of difference relative to the global mean. For data from individual runs, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082931#pone.0082931.s002" target="_blank">Figs. S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082931#pone.0082931.s005" target="_blank">S5</a>. D Higher magnification fluorescence images of representative MPCs in MBA displaying alkaline phosphatase activity (ELF97) and DNA staining (PI). Scale bar: 200 µm. E Main effects plot showing effect of DONOR, CHIR99021 (CHIR), IWP-4, IWR-1 and POSITION on expression index for ELF97/DNA ratio. F Interaction effects plot showing effects of 2 combined factors on ELF97/DNA ratio.</p
Validation of MBA culture parameters and MPC seeding.
No full text<p>A Comparison of cell morphology in 100 µm (top) versus 250 µm-high (bottom) devices. Scale bar, 200 µm. B Comparison of medium exchange regimes varied from conditions in top panel of A –10 µL/h flowrate (top) and periodic flow-stop (bottom). Scale bar, 200 µm. C Comparison of surface coating regimes varied from conditions in top panel of A – FBS-coated substrate (top) and collagen-I-coated substrate (bottom). Scale bar, 200 µm. D Phase contrast micrographs of MPCs in static plate controls and microbioreactor arrays in suspension directly after seeding, and attached after 4–5 h, just prior to the start of fluid flow. Scale bar, 200 µm. E Heatmap showing distribution of MPCs seeded into a MBA at representative experimental densities. F Graph showing average cells per chamber as a function of row. G Graph showing average cells per chamber as a function of column. H Live/dead staining of MPCs after 7 days. Scale bar, 100 µm.</p
Arrayed cellular environments for stem cells and regenerative medicine
No full textThe behavior and composition of both multipotent and pluripotent stem cell populations are exquisitely controlled by a complex, spatiotemporally variable interplay of physico-chemical, extracellular matrix, cell-cell interaction, and soluble factor cues that collectively define the stem cell niche. The push for stem cell-based regenerative medicine models and therapies has fuelled demands for increasingly accurate cellular environmental control and enhanced experimental throughput, driving an evolution of cell culture platforms away from conventional culture formats toward integrated systems. Arrayed cellular environments typically provide a set of discrete experimental elements with variation of one or several classes of stimuli across elements of the array. These are based on high-content/high-throughput detection, small sample volumes, and multiplexing of environments to increase experimental parameter space, and can be used to address a range of biological processes at the cell population, single-cell, or subcellular level. Arrayed cellular environments have the capability to provide an unprecedented understanding of the molecular and cellular events that underlie expansion and specification of stem cell and therapeutic cell populations, and thus generate successful regenerative medicine outcomes. This review focuses on recent key developments of arrayed cellular environments and their contribution and potential in stem cells and regenerative medicine
