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The beneficial effects of mesenchymal stem cell (MSC)-based cellular therapies are believed to be mediated primarily by the ability odansf MSCs to suppress inflammation associated with chronic or acute injury, infection, autoimmunity, and graft-versus-host disease. To specifically address the effects of frictional force caused by blood flow, or wall shear stress (WSS), on human MSC immunomodulatory function, we have utilized microfluidics to model WSS at the luminal wall of arteries. Anti-inflammatory potency of MSCs was subsequently quantified via measurement of TNF-α production by activated murine splenocytes in co-culture assays. The TNF-α suppression assay serves as a reproducible platform for functional assessment of MSC potency and demonstrates predictive value as a surrogate assay for MSC therapeutic efficacy.
Immunomodulatory activity of mesenchymal stem cells (MSCs) is mediated by direct cellular interactions and paracrine factors (Singer and Caplan, 2011; English, 2013). MSCs are believed to originate from pericytes that associate with endothelial cells of vasculature within the bone marrow and various tissues (Sacchetti et al., 2007; Crisan et al., 2008). This unique perivascular location positions them in close proximity to inflammatory and other soluble factors in the blood stream, poising them to monitor systemic signals. Indeed, recruitment of mural cells to the endothelium is a key event in vessel maturation, and pericytes play a critical role in vascular maintenance and integrity (Benjamin et al., 1998; Schrimpf et al., 2014). Pericytes likely monitor systemic signals by fluid outflow from arterioles and capillaries through interendothelial clefts or gaps in the basement membrane, which can expose the basolateral surface of endothelial cells outside the vessel to considerable fluid frictional force, or wall shear stress (WSS), that approximates intraluminal forces (Scallan et al., 2010). MSCs and other classes of pericytes might also view the intraluminal environment from openings between vascular endothelial cells by protrusion into the vascular lumen with cytoplasmic projections much like megakaryocytes, though more typically they ensheathe the blood vessel with branching processes (Shepro and Morel, 1993; Murphy et al., 2013). In instances of inflammation or injury, for example due to trauma to the central nervous system, pericytes have been shown to migrate away from microvessels concurrent with perivascular edema and toward injured tissue in association with blood vessel sprouting (Dore-Duffy et al., 2000; Göritz et al., 2011). Cells described as having features of MSCs have been detected circulating in human peripheral blood (Zvaifler et al., 2000), though there is some controversy surrounding evidence for MSCs in the circulation of healthy and even injured individuals (Hoogduijn et al., 2014). In those cases, disruption of endothelial-pericyte interactions could be expected to exacerbate vascular hyperpermeability which could impact migration or intravasation of MSCs (Mills et al., 2013). As MSCs are anchorage-dependent cells, a likely means of motility would include attachment to the vessel wall resulting in direct exposure to intraluminal WSS. In therapeutic applications wherein MSCs are administered intravenously, WSS would be an unavoidable stimulus during handling, infusion, and trafficking (Nitzsche et al., 2017).
We have shown that WSS typical of arterial blood flow promotes signaling through focal adhesion kinase (FAK), NF-κB, and COX2 (Diaz et al., 2017; Lee et al., 2017). Increased COX2 results in elevated prostaglandin E2 (PGE2) biosynthesis. PGE2 secreted by MSCs plays a central role in regulation of innate and adaptive immune cells. Thus, MSCs exposed to WSS more potently suppress immune cell activation in the presence of inflammatory cues (Diaz et al., 2017; Lee et al., 2017). To quantify MSC immunomodulatory activity in cells exposed to fluid flow, we co-cultured MSCs and lipopolysaccharide-activated murine splenocytes in an adaptation of the commonly used mixed lymphocyte reaction (Plumas et al., 2005). TNF-α was measured by species specific ELISA to determine cytokine production from activated murine splenocytes, thus restricting analysis to immune cell activity and enabling separate determinations of cytokine production by human MSCs. Employing this assay as a surrogate measure of MSC potency, we determined that transient exposure of MSCs to fluid shear stress improved their ability to limit activation of immune cells in the presence of inflammatory stimulus. Preconditioning of MSCs by as little as 3 h of WSS in culture was an effective means of enhancing therapeutic efficacy in treatment of a rat traumatic brain injury model. These data demonstrate that WSS enhances the immunomodulatory and neuroprotective function of MSCs. Together with complementary studies implicating PGE2 as a potency marker of MSC therapeutic efficacy (Kota et al., 2017), our studies suggest that mechanotransduction could be leveraged to improve cellular therapies available for patients with neurological injury. This co-culture assay could easily be adapted for analysis of anti-inflammatory potency of MSCs subjected to a variety of treatments, including genetically engineered MSCs.
This work was supported by grants from the State of Texas Emerging Technology Fund, American Society of Hematology Scholar Award, National Institutes of Health K01DK092365, and Mission Connect: a Program of the TIRR Foundation (014-121, 016-118) to P.L.W.