Adipose-derived stem cells (ASCs) for the treatment of osteochondral defects : comparison between human and porcine ASCs for a future pre-clinical model

Adipose-derived stem cells (ASCs) with their osteogenic and chondrogenic potential may represent a novel approach which may substitute or ameliorate the currently available therapies for the treatment of osteochondral defects due to trauma or osteo-degenerative diseases affecting joints. We characterized pASCs (pig Adipose-derived Stem Cells) and evaluated their osteogenic and chondrogenic potential comparing them to hASCs (human Adipose-derived Stem Cells). The number of hASCs per ml of raw adipose tissue (4.7x105±3.4x105, n=11) was higher compared to the number of pASCs (1.8x105±4.7x104, n=5). One week after isolation both cellular populations start to constantly proliferate obtaining about 107-108 hASCs and 108-109 pASCs in four weeks of culture. Both hASCs and pASCs show the typical fibroblast-like morphology of mesenchymal stem cells, maintained during their life span. ASCs from both human and pig are highly clonogenic with a percentage of colony forming units of about 15% until the IV passage in culture. The osteogenic differentiation of hASCs and pASCs in the absence or in the presence of clinical-grade hydroxyapatite granules (HA) was analyzed by ALP activity quantification. In the absence of scaffold, undifferentiated hASCs show about 10-fold higher ALP basal levels respect to pASCs, however both cellular populations possess a similar ability to respond to the differentiative osteogenic stimuli with an increased ALP activity of about 100% in hASCs and pASCs differentiated for 14 days. The presence of HA granules induces a significant increase in ALP level both for undifferentiated and differentiated hASCs and pASCs, in comparison to cells grown on monolayer, confirming our previous data about the in vitro HA osteo-inductive properties. Interestingly, we observed a synergistic effect produced by the combination of scaffold and osteogenic medium, supporting the future clinical applications of HA in association with in vitro differentiated ASCs. Moreover, both hASCs and pASCs, aggregated into micromasses where the physiological conditions of cartilage are partially recreated, have been differentiated towards cells of the chondrogenic lineage in the presence of 1% FBS and 10 ng/ml TGF-β1 for 21 days. Both cell types express an abundant amount of sulfated glycosaminoglycans (GAGs) showing a significant increase of about 115% and 95% in the GAGs content in comparison to undifferentiated cells. These data demonstrate that hASCs and pASCs share common features and possess a similar osteogenic and chondrogenic differentiative ability, supporting the idea that the pre-clinical autologous ASCs reimplantation model in pig might be predictive of the behaviour of ASCs in a future clinical model of cell therapy.