Can cellular tracers attenuate regeneration potential and wound healing ability of adipose derived stem cells?

As the field of stem cell-based therapies continues to evolve, the ability to monitor the regenerative capacity of stem cell following delivery to target tissues has become increasingly relevant. While Bromodeoxyuridine (BrdU) and green-fluorescent protein (GFP) labeling techniques are often used, limited knowledge exists regarding their effects on the regeneration capacity of stem cells. In order to further understand the impact of these techniques in a clinical scenario, porcine adipose stem cells (ASCs) were derived, cultured and subsequently labeled with BrdU or GFP using transfection (GFP-t) or infection (GFP-i) by lentivirus, followed by investigation of downstream labeling effects on ASCs proliferation, differentiation, colony formation, secretion of regenerative cytokines, growth factors and healing ability. Neither BrdU nor GFP labeling led to gross morphological changes in adipose stem cells. Both groups of GFP labeled cells maintained a high percentage of signal intensity for at least 28 days, with a decrease in cell proliferation ability in the GFP-i group. In contrast, BrdU labelling began displaying weaker signal strength after 7-days of experiments. Both tracers negatively affected osteogenic and adipogenic differentiation of ASCs, causing decrease in the secretion of the regeneration markers VEGF and TNF-α. In contrast, in-vitro wound healing ability of labeled ASCs and secretion of IL-8, IL-10, and TGF-β was not affected by either method. Our results demonstrate that BrdU labelling may be more effective for short-term in-vitro studies due to the relative ease of preparation and shorter lifespan of the tracer signal. Contrarily, for studies with a longer duration, GFP labeling of cells by transfection provides more durable and stable signal. While further studies are needed, our results indicate that labelling mode can alter regeneration capacity of stem cells and should, therefore, be optimized prior to clinical translation experiments.