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Introduction:Stem cell therapies have immense potential for the treatment of cardiovascular disease. However, their full potential has not yet been realized in clinical trials due to limited efficacy.Methods:We created a high throughput system that allows for the simultaneous application of mechanical load to 576 individual culture wells. Using this mechanobiological screening system, we examined the use of mechanical conditioning on human mesenchymal stem cells (hMSCs) to enhance their expression vascular cell markers. We combined mechanical forces with a library of kinase inhibitors to find synergy between pharmacological and biomechanical conditioning for enhancing the vascular phenotype of hMSCs.Results:We investigated various dynamic strain waveforms for their ability to activate the Hippo and TGF-? signaling pathways and found the optimal activation when induced with 7.5% strain waveform at a frequency of 0.1 Hz. In addition, we screened a library of small molecule kinase inhibitors and found a set of compounds that synergistically increased nuclear Yap/Taz localization and Smad2/3 phosphorylation. Specific combinations of these pharmacological inhibitors and a complex dynamic mechanical waveform dramatically increased the expression of endothelial cell and pericyte markers in hMSCs. When cultured on Matrigel, these MSCs had increased tube formation in comparison to untreated cells. The conditioned media from these cells was also able to induce greater tube formation in endothelial cells than that from control MSCs. Lastly, the optimally conditioned hMSCs increased angiogenesis and blood perfusion in nu/nu mice with hindlimb ischemia and after subcutaneous implantation (Fig. 1).Conclusions:Our results demonstrate that combined mechanical and pharmacological conditioning of hMSCs can dramatically enhance their regenerative capabilities.

Introduction:Induced pluripotent stem cells (iPSC) have great promise for providing new models of disease and generating novel cell types from patient derived samples. However, studies on iPSC derivation often involve viral genetic manipulation, which causes concerns in terms of using the derived cells for cellular therapeutics.Hypothesis:In this study, we hypothesized that mouse embryonic fibroblasts (MEFs) can be primed toward pluripotency through the use of mechanical conditioning.Methods:We have developed a high-throughput biaxial stretching device that is capable of inducing dynamic mechanical strain to 576 cell culture wells simultaneously. We applied varying levels of mechanical strain to MEFs with and without drug treatments and tracked the expression of Oct-4 using a promoter reporter construct. Following loading, we further confirmed expression of pluripotency related factors using RT-PCR and immunostaining.Results:Oct-4 expression increased 2.5-fold in MEFs stretched at 17.5% strain at 0.1 Hz. Under these conditions we also found a five-fold increase in Sox2 and a three-fold increase in SSEA1 in MEFs. We used the high throughput mechanical loading system to perform drug screening in the presence of mechanical loads and found several compounds that synergistically increased Oct-4 expression with mechanical load (Fig 1A). Combined treatment with mechanical load and the candidate compounds led to further increases in Sox2, SSEA1, Nanog, and some pluripotency related genes (Fig 1B, C).Conclusions:Taken together, our work suggests optimized mechanical conditions can prime MEFs for developing pluripotency and this effect can be increased with co-treatment with small molecule inhibitors.