Isolation and morphological characterization of IFP- derived stem-like cells: investigation on their potential role in osteoarthritis

Abstract

The extrasynovial adipose tissue Infrapatellar Fat Pad (IFP) is an emerging player in knee osteoarthritis (OA) [1,2]. While the role of constitutive adipocytes in secreting cytokines is known, little awareness on origin/function of the stem cells component exists. This study aims to isolate/ characterize IFP-derived stem cells (IFP-dSCs) from OA patients investigating their role in disease development. IFP samples were processed to discard matrix and the adipocyte fraction. The result- ing pellet was resuspended in proliferative medium and cultured routinely. IFP-dSCs morphol- ogy and ultrastructure were observed by optical microscope and Transmission Electron Microscope (TEM); expansion potential of cells was assessed by a population doubling level assay. IFP-dSCs vitality was assessed using a Apoptotic/Necrotic/Healthy Cells Detection Kit, while the metabol- ic activity of cell cultures was analysed by MTT assay. Flow cytometry analysis was performed to identify the presence of specific markers; in particular, IFP-dSCs were stained with antibodies against CD73/105/90/44/34/106, IL-6R/1R, VEGFR2. At last, IFP-dSCs plasticity was also assessed, evaluat- ing their commitment towards the adipogenic, chondrogenic and endothelial lineages. IFP-dSCs iso- lation required 14 h; cells were fibroblast-like and typically spindle shaped at low density, showing a greater polygonal nucleus at high density. Semithin-sections stained with toluidine blue revealed vesicles in the cytoplasm, as confirmed by TEM analysis. These rounded formations of electron- dense material were in proximity of empty round vesicles and in some contact areas a partial fusion between the external membranes was appreciable. An exponential growth during the entire long-term expansion period was observed, with a replication time of 42.7 ± 3.8 h. From passage (P)8 to P20, cells performed 11.9 ± 0.9 population doublings. IFP-dSCs immunophenotype was positive for the investigated markers, suggesting a role in modulation of inflammation; interestingly, cells were 100% CD73+bright both at low and high P in colture. Preliminary data about plasticity revealed the ability in differentiating towards adipogenic and endothelial lineages. Further analysis will be required to assess chondrogenic commitment. Experimental evidence on IFP-dSCs seem to correlate histopatho- logical features of IFP in OA (i.e. thickening of interlobular septa, increase in vascularization and innervation) with the stem cell component.