Desirability profiling to rank scalable mesenchymal stromal cell enhancement methods according to multimodal mechanisms

Abstract

Background & Aim Mesenchymal stromal cells (MSCs) are widely investigated due to their anti-inflammatory, angiogenic, and anti-fibrotic properties. However, insufficient potency, MSC donor variability, and deficient knowledge on therapeutic mechanisms can limit MSC treatment efficacy. The Viswanathan lab has pioneered a novel, non-genetic proprietary strategy to culture MSCs in 3D aggregates under serum-free conditions to augment anti-inflammatory functions. Hypoxic culture has also been shown to enhance MSC paracrine functions. Our objective is to systematically characterize the anti-inflammatory, angiogenic, and anti-fibrotic properties of 3D and hypoxic MSCs, and to apply desirability profiling to empirically rank the potency of the MSCs according to these different properties. Methods, Results & Conclusion METHODS: Human adipose tissue (AT) or bone marrow (BM) MSCs were expanded with serum-free medium. MSCs were then cultured for 24-h under 3D (static culture or in 0.1 L vertical-wheel bioreactors) or 2D hypoxic (5% O2) conditions, with 2D normoxic MSCs as controls. Gene expression was measured by RT-qPCR and using a NanoString 50-gene panel, while microRNAs (miR) were examined by miR-sequencing. In ongoing work, desirability profiling will be performed on these data and on additional functional read-outs in MSC co-cultures with human monocytes, endothelial cells, or fibroblasts. RESULTS 3D MSC culture under static and bioreactor conditions resulted in similar high viable cell yields (>80%). 3D MSCs had increased expression of immunomodulatory genes (e.g. IDO, TSG6 with 4- to 10-fold increased expression vs. 2D normoxic control) while hypoxic MSCs had greater expression of angiogenic genes (e.g. VEGF with 2-fold greater expression vs. control). Preliminary investigation into miR expression revealed that AT-MSCs displayed a distinct miR profile relative to BM-MSCs, and that 3D enhanced AT-MSCs had a differential miR profile vs. controls. The miRs and their target pathways are being investigated to identify mechanisms underlying the enhanced MSC functionality. CONCLUSION MSCs act through multimodal mechanisms that are enhanced through 3D or hypoxic cultures. Statistical modeling is being used to systematically rank the potency attributes of enhanced MSCs in terms of their anti-inflammatory, angiogenic, and anti-fibrotic functionality in vitro and in inflammatory mouse models. Ultimately, this work will generate methods for improved MSC products with therapeutic profiles that are “fit-for-purpose.”