A new protocol for validation of Chondro, Adipo and Osteo differentiation kit of Cultured Adipose-Derived Stem Cells (ADSC) by real-time rt-QPCR.

Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose derived Stem Cells) show a great potential for degenerative diseases treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validated a commercially available kit able to explore changes in gene expression under osteogenic, adipogenic and chondrogenic differentiation of human ADSC. Initially, we selected a better indicators of trilineage differentiation by using third passages of cultured ADSC from stromal vascular fraction (SVF) isolated from fresh adipose tissue by enzymatic digestion. On the basis of statistically significant results ACAN, FABP4A and Col11a1 were chosen as indicators of chondrogenic, adipogenic and osteogenic differentiation respectively. An in-vitro aging analysis was then performed to evaluate the ADSC passage with the highest differentiation potential. Total RNA extraction from induced differentiation and controls ADSC from passage 2-6 and relative quantifications of mRNA expression of selected genes were performed according to rt-PCR kits tested. The chondrogenic differentiation test showed equivalent ∆∆Ct values for ACAN detection for cell passages ranging from P3 to P6, proving that they can be considered as equivalent samples for differentiation assays evaluation. For what concerns adipogenic differentiation and FABP4 detection, similar results were observed in all the cell passages tested; on the contrary only passage P6 showed suitable ∆∆Ct values for Col11a1 detection for osteogenic differentiation evaluation. In conclusion, we have validated a suitable real-time rt-QPCR protocol for osteogenic, chondrogenic and adipogenic ADSC differentiation ability evaluation in-vitro.