Generation and Dissemination of a Mesenchymal Stem Cell-Derived Functional Smooth Muscle Tissue

Grosha, Jonian

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Generation and Dissemination of a Mesenchymal Stem Cell-Derived Functional Smooth Muscle Tissue

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Pulmonary artery hypertension (PAH) is a cardiovascular disease that causes more than 200,000 hospitalizations every year. PAH results in a hyperproliferative, stiffer, and hypercontractile pulmonary artery. This poses great resistance to the heart resulting in death in 50% of cases 3 years after diagnosis. Tissue-engineered models of PAH could be used to test new drugs and treatments. We propose a self-assembly approach, that utilizes mesenchymal stem cells (MSCs) to address the limited proliferation rate of primary smooth muscle cells (SMCs). We investigated MSCs as a readily available patient-specific cell source, with high proliferation rates, and myogenic differentiation potential. We hypothesized that self-assembled MSC tissue rings would differentiate into SMC tissue when stimulated with TGF-β1 and BMP4. We concluded that the addition of BMP-4 did not cause a significant increase in tissue differentiation compared to TGF-β1 alone, which was enough to differentiate the tissues towards a contractile SMC phenotype. The differentiated tissues showed similar morphology to vascular SMCs in vivo, expressed early, mid, and late-stage protein and gene markers of smooth muscle differentiation, and ultimately responded to contractile agonists. These engineered SMC tissue rings could be used as screening tools for the development of new drug therapies. To establish a model of PAH we hypothesized that exposure to ET-1 of engineered MSC-derived tissue rings would cause expression of PAH characteristics, resulting in rings that are thicker and hypercontractile compared to tissues not exposed to ET-1. We determined that differentiating first into SMC and then exposing to ET-1 results in hypercontractile and thicker tissues. Since we created a tissue engineered product, the next step in the manufacturing pipeline would be to store or disseminate it. Tissue storage has currently been identified as a major bottleneck of the tissue engineering field. Cryopreservation is the mode of storage for cells and tissues. While there are currently cryopreserving agents (CPAs) for cell cryopreservation, creating CPAs for tissue cryopreservation has presented challenges. In addition, freezing parameters, such as freezing rate and freezing point are cell and tissue type specific. Studies have examined the preservation of engineered tissues, but have not used a standardized tissue, which hinders the translatability of these studies. Thus, there is a need for a high-throughput platform that uses a standard tissue where multiple cryopreservation parameters and CPAs can be tested. To achieve this, we used self-assembled engineered tissue rings as standard test units. To develop this platform, we re-designed the agarose wells where the tissues are cultured in order to fit in a 48-well plate, hence making this a high-throughput culture method. The molds containing the tissues also fit in standard cryovials. We validated this method by testing CPAs and identifying the optimal parameters for engineered SMC vascular tissues. In combination, this work generated a tissue engineered product and a platform to investigate storage parameters for its dissemination.

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