(252e) A Microfluidic Approach for the Isolation of a Highly Migratory Presenescent Stem Cell Subpopulation

Cellular senescence refers to the permanent cell cycle withdrawal (aka cell cycle arrest) as a result of prolonged cell expansion and it triggers a variety of phenotypic and structural alterations that diminishes stem cell potential. Ultimately, senescent cells contribute to disease progression and hinder tissue regeneration. The aim of this study is to devise a novel approach to eliminate senescent cells from adult stem cell populations in order to improve their therapeutic efficacy. To achieve this, we exploit the innate potential of stem cells to migrate toward damaged tissues in vivo. Since senescent stem cells display increased size and reduced motility and plasticity, we hypothesize that a Y-shaped microfluidic device that has been previously optimized to isolate highly migratory cells effectively distinguishes between presenescent and senescent stem cells. This device is composed of two parallel seeding and collection channels connected by a ladder-like arrangement of Y-shaped micron-sized channels, the dimensions of which recapitulate the size of pores and longitudinal channel-like tracks encountered by migrating cells in vivo.

To test our hypothesis, early, intermediate, or late passage human dermal fibroblasts or human adipose tissue-derived mesenchymal stem cells (hMSCs) were seeded close to the entrances of Y-shaped, polydimethylsiloxane (PDMS)-based microchannels and their migration was monitored for 13 hours using time-lapse microscopy. Highly migratory and non-migratory cells were isolated by the device and their morphological, molecular and functional properties were assessed. The results showed that highly migratory cells were significantly faster and smaller than non-migratory cells, especially at earlier passages. Irrespective of cell passage number, migratory cells also exhibited higher proliferative capacity (assessed via Ki-67 immunocytochemistry) and less DNA damage (assessed via γH2AX immunocytochemistry) compared to non-migratory cells. To determine the ability of our assay to detect presenescent cells within heterogeneous populations, we mixed early passage cells with RFP-labelled, senescent cells at a 1:1.5 ratio. The device identified ~70% of early passage cells and only ~30% of senescent cells, thereby enriching the % of control (presenescent) cells within the migratory cell population. In the future, we will assess whether the clonogenic, differentiation, immunosuppressive and proangiogenic potential of highly motile hMSCs is elevated compared to the parental stem cell population.

Taken together, our assay enables the physical isolation and characterization of a highly motile presenescent cell subpopulation in real-time and at the single-cell level. Such technology can improve the standardization of cell preparations, enhance stem cell potency, and facilitate the discovery of signature markers for highly motile, presenescent stem cells.