Modulation of fibroblast-to-myofibroblast differentiation and fibroblast migration: in vitro assessment of the anti-fibrotic effects of human adipose derived multipotent mesenchymal stromal cells

Background \u0026 Aim Multipotent mesenchymal stromal cells (MSCs) are promising therapeutic options for a variety of diseases driven by inflammatory, vasculopathic and pro-fibrotic mechanisms. However, the anti-fibrotic effects of MSCs are not well characterized. To date, there are no recommendations on which in vitro assays should be used to test these effects. The development of anti-fibrotic readouts could inform the selection of MSC products to be used in studies of fibrotic diseases. Methods, Results \u0026 Conclusion Methods: Human adipose derived MSCs at passage 4 to 6 from six adult (mean age±SD: 70.2±8.7 years) and seven pediatric (16.9±2.4 years) donors were evaluated. Resting and cytokine primed (IFNg+TNFa) MSC conditioned media (MSC-CM) were assessed in immunopotency assays (MSC-CM modulation of PHA stimulated-CD4+ proliferation). The effect of MSC-CM on hTERT-immortalized human foreskin fibroblasts was tested in the following in vitro assays: (i) fibroblast to myofibroblast differentiation (immunostaining and Western blot for a-SMA and collagen 1); (ii) fibroblast migration (Incucyte scratch wound fibroblast cell migration); and (iii) fibroblast contraction (fibroblast-induced collagen gel contraction). Images were analyzed with ImageJ software. Results Primed MSC-CM inhibit CD4+ proliferation by 41.2% (resting vs primed, mean ± SD: 101.3±33.1 vs 58.8±23.1, n=10 per group, p Conclusion In vitro, human adipose derived MSC-CM inhibit the fibroblast-to-myofibroblast switch following TGF-β1 treatment and the non-directional migration of fibroblasts. MSC-CM promote the contraction of the extracellular matrix of fibroblasts and myofibroblasts. Whether these in vitro assays can be used as surrogates of the in vivo anti-fibrotic effects of MSCs remains to be defined.