Pooled umbilical cord-derived MSCs retain key phenotypic and immunomodulatory properties - A path toward consistent...

Mesenchymal stromal stem cells (MSCs) represent a promising cell therapy tool, by merit of their regenerative and unique immunomodulatory properties. However, their clinical utility is often hindered by batch-to-batch variability, which is the result of several factors, particularly those of donor heterogeneity and differences in manufacturing. To address this issue, we developed a protocol for pooling umbilical cord-derived MSCs (UC-MSCs) from multiple donors that demonstrates comparable characteristics across pooled and single donor MSCs. We see this as a basis to generate a more homogenous and scalable cell products in the future, allowing for low passage manufacture, termed equipotent MSCs (EQ-MSCs). We characterized EQ-MSCs in comparison to UC-MSCs through extensive phenotypic analysis and by functional and immunological assessment. We demonstrate that EQ-MSCs retain the characteristic phenotypic features of single donor MSCs, with stable expression across multiple passages. Morphological evaluation of mixed donor cultures showed no significant alterations in cellular shape or growth patterns. The analysis of multilineage differentiation capacity demonstrated equivalent adipogenic, osteogenic, and chondrogenic potential, to single donor UC-MSCs. Strong colony forming ability of EQ-MSCs, indicative of preserved clonogenic potential, was also demonstrated. In terms of function, EQ-MSCs expressed key inhibitory molecules, including PD-L1, PD-L2, B7-H3, and adhesion molecule ICAM-1, with further upregulation upon IFN-γ priming, suggesting their capacity for enhanced immunomodulatory profile in response to an inflammatory environment. Functional co-culture assays with human PBMCs revealed a dose-dependent suppression of T cell proliferation, where EQ-MSCs showed comparable immunomodulatory activity to UC-MSCs. Our results support the pooling strategy of UC-MSCs as a viable strategy toward increasing product homogeneity, while at the same time maintaining key biological features.