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

Valentina Daprà; Daniela Alotto; Stefania Casarin; Stefano Gambarino; Carlotta Zavatto; Maddalena Dini; Ilaria Galliano; Carlotta Castagnoli; Massimiliano Bergallo

doi:10.1016/j.tice.2022.101923

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

Tissue Cell. 2022 Dec:79:101923. doi: 10.1016/j.tice.2022.101923. Epub 2022 Sep 13.

Authors

Valentina Daprà¹, Daniela Alotto², Stefania Casarin³, Stefano Gambarino⁴, Carlotta Zavatto⁵, Maddalena Dini⁶, Ilaria Galliano⁷, Carlotta Castagnoli⁸, Massimiliano Bergallo⁹

Affiliations

¹ Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy; BioMole srl, Spin-off University of Turin, 10135 Turin, Italy. Electronic address: valentina.dapr@yahoo.it.
² Skin Bank, Department of General and Specialized Surgery, University Hospital Città della Salute e della Scienza di Torino, 10126 Turin, Italy. Electronic address: dalotto@cittadellasaluto.to.it.
³ Skin Bank, Department of General and Specialized Surgery, University Hospital Città della Salute e della Scienza di Torino, 10126 Turin, Italy. Electronic address: stefania.casarin@gmail.com.
⁴ Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy; BioMole srl, Spin-off University of Turin, 10135 Turin, Italy. Electronic address: gambarino.stefano@gmail.com.
⁵ Skin Bank, Department of General and Specialized Surgery, University Hospital Città della Salute e della Scienza di Torino, 10126 Turin, Italy. Electronic address: carlotta.zavatto@gmail.com.
⁶ BioMole srl, Spin-off University of Turin, 10135 Turin, Italy. Electronic address: info@biomole.it.
⁷ Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy. Electronic address: ilaria.galliano@unito.it.
⁸ Skin Bank, Department of General and Specialized Surgery, University Hospital Città della Salute e della Scienza di Torino, 10126 Turin, Italy. Electronic address: ccastagnoli@cittadellasalute.to.it.
⁹ Department of Public Health and Pediatric Sciences, University of Turin, Medical School, Turin, Italy; BioMole srl, Spin-off University of Turin, 10135 Turin, Italy. Electronic address: massimiliano.bergallo@unito.it.

PMID: 36126415
DOI: 10.1016/j.tice.2022.101923

Abstract

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.

Keywords: ADSC (Adipose derived Stem Cells); Adipocytes; Chondroblasts; Mesenchymal stem cells (MSCs); Osteoblasts.

MeSH terms

Adipocytes
Adipose Tissue*
Cell Differentiation / genetics
Cells, Cultured
Humans
Osteogenesis* / genetics
Stem Cells