Label-free separation of mesenchymal stem cell subpopulations with distinct differentiation potencies and paracrine effects

Mesenchymal stem cells (MSCs) have the capability to differentiate into multiple cell lineages, and produce trophic factors to facilitate tissue repair and regeneration, and disease regression. However, the heterogeneity of MSCs, whether inherent or developed during culture expansion, has a significant impact on their therapeutic efficacy. Therefore, the ability to identify and select an efficacious subpopulation of MSCs targeting specific tissue damage or disease holds great clinical significance. In this study, we separated three subpopulations from culture expanded human bone marrow derived MSCs according to cell size, using a high-throughput label-free microfluidic cell sorting technology. The size-sorted MSC subpopulations varied in tri-lineage differentiation potencies. The large MSCs showed the strongest osteogenesis, medium-size MSCs were advantageous in chondrogenesis and adipogenesis, and the small MSCs showed the weakest tri-lineage differentiation. The size-sorted MSC subpopulations also exhibited different secretome profiles. The large MSC secretome possessed highest levels of osteogenic promotor proteins and senescence-associated factors, but lower levels of osteogenic inhibitor proteins compared to the medium-size MSC secretome. The medium-size MSC secretome had high levels of chondrogenic promotor proteins, and contained lower levels of chondrogenic inhibitor proteins compared to the large MSC secretome. The secretome of size-sorted MSC subpopulations showed differences in paracrine effects. We found that the secretome of large MSCs enhanced osteogenic and adipogenic potencies during MSC culture expansion, but also induced cell senescence; and the secretome of medium-size MSCs promoted chondrogenesis. This study demonstrates size-dependent differentiation potency and secretome profile of MSC subpopulations, and provides an effective and practical technology to isolate the respective subpopulations, which may be used for more targeted tissue repair and regeneration.