To verify multipotency, BM-MSC, DFSC and adMSC were subjected to in vitro differentiation towards osteogenic, chondrogenic and adipogenic lineages using the Mesenchymal Stem Cell Functional Identification Kit (R & D). Differentiation was induced by maintaining cells under different culture conditions according to the manufacturer instructions for 20 days. Subsequently, cells were fluorescently labelled to detect fatty acid-binding protein 4 (FABP4), Aggrecan and Osteocalcin to visualize successful differentiation into adipocytes, chondrocytes, and osteocytes.
For labelling of cardiac markers, cells were seeded on coverslips and fixed with 4% PFA. Antibody staining was performed as described elsewhere [34 (link)]. Cells were labelled with anti sarcomeric α-actinin (abcam, ab9465), anti-NKX2.5 (Santa Cruz, sc-8697), anti-TBX5 (abcam, ab137833) and anti-MEF2C (Santa Cruz, sc-313).
To visualize intracellular calcium, cells were cultured on 8 well chamberslides (Ibidi). Three days after seeding, cells were incubated with the calcium sensitive dye Cal520 (AATBioquest) for one hour at 37 °C and subjected to fluorescence microscopy. All fluorescence images were acquired using Zeiss ELYRA LSM 780 (Zeiss, Oberkochen, Germany).
For labelling of cardiac markers, cells were seeded on coverslips and fixed with 4% PFA. Antibody staining was performed as described elsewhere [34 (link)]. Cells were labelled with anti sarcomeric α-actinin (abcam, ab9465), anti-NKX2.5 (Santa Cruz, sc-8697), anti-TBX5 (abcam, ab137833) and anti-MEF2C (Santa Cruz, sc-313).
To visualize intracellular calcium, cells were cultured on 8 well chamberslides (Ibidi). Three days after seeding, cells were incubated with the calcium sensitive dye Cal520 (AATBioquest) for one hour at 37 °C and subjected to fluorescence microscopy. All fluorescence images were acquired using Zeiss ELYRA LSM 780 (Zeiss, Oberkochen, Germany).