Trilineage differentiation assays

To ensure that equine MSCs cultured in ePL were capable of trilineage differentiation, MSCs at P5 or P6 (n = 3), expanded with FBS or ePL culture media, were used for differentiation assays. Undifferentiated MSCs, cultured under standard cell culture conditions, were used as negative controls in all experiments. All experiments were performed in triplicate for each biological replicate.

Equine MSCs (n = 3) were plated at 100,000 cells/well in six-well plates in FBS or ePL culture media until reaching 90% confluency. Cell culture medium was replaced by HyClone AdvanceSTEM osteogenic medium supplemented with 50 μg/ml streptomycin and 50 U/ml penicillin, exchanged every 2–3 days for 28 days. Osteocytes were identified using Van Kossa staining. Specifically, cultures were fixed with 4% PFA on ice for 15 min and stained with 1% silver nitrate for 20 min under ultraviolet light. Plates were washed with distilled water, and unreacted silver was removed by the addition of 5% sodium thiosulfate for 5 min at room temperature. The plates were then washed again, and cultures were imaged using a Leica inverted microscope [32].

For the quantification of calcium deposition, equine MSCs (n = 3) were plated at 21,000 cells/cm² in a flat bottom 96-well plate and cultured with media supplemented with FBS or ePL. Upon reaching 90% confluency, cell culture medium was replaced by HyClone AdvanceSTEM osteogenic medium supplemented with 50 μg/ml streptomycin and 50 U/ml penicillin, exchanged every 2–3 days for 28 days. Differentiation of MSCs to osteocytes was determined using the Calcium Liquicolor® Test (StanBio) according the instructions of the manufacturer. Calcium was extracted by the addition of 0.6 N HCL, stored overnight at 4°C; supernatants were combined at a ratio of 1:20 with an equal portion mixture of the color and the base reagent and plates were read at 550 nm (SpectraMax).

Equine MSCs (n = 3) were plated at 100,000 cells/well in six-well plates and cultured with FBS or ePL culture media until reaching 90% confluency. Medium was then replaced by adipogenic media consisting of DMEM, 10% FBS, 5% rabbit serum, 0.5 μΜ dexamethasone, 60 μΜ indomethacin, 0.5 mM IBMX, 1 μM insulin, and 50 U/ml penicillin and 50 μg/ml streptomycin [31]. Medium was exchanged every 2–3 days for 21 days. Cultures were fixed with 4% PFA for 15 min over ice and rinsed with 60% isopropanol. For the identification of lipid droplets an Oil Red O (Sigma, St. Louis, MO) working solution in 60% isopropanol was added to the cultures for 20 min at room temperature. Cultures were imaged using a Leica inverted microscope.

One million equine MSCs (n = 3) cultured with FBS or ePL culture media were pelleted in sterile polypropylene 15-ml centrifuge tubes and incubated for 48 h with their respective media. The medium was then discharged and replaced with HyClone AdvanceSTEM chondrogenic medium supplemented with 50 μg/ml streptomycin and 50 U/ml penicillin with a medium change every 2–3 days for 28 days. Cultures were fixed with 4% PFA for 15 min over ice, rinsed with PBS, and submitted to histology for staining with Alcian Blue 8GX. Samples were visualized using an Olympus microscope.

For quantification of Alcian Blue staining, equine MSCs (n = 3) were plated at 100,000 cells/well in conical bottomed 96-well plates and centrifuged for 10 min; they remained in the presence of ePL or FBS media for 48 h. Cell culture medium was replenished with HyClone AdvanceSTEM chondrogenic medium every 2–3 days for 28 days. A 0.2% Alcian Blue 8GX in 0.1 M HCL solution was applied to the fixed chondrogenic pellets and incubated overnight at room temperature. Pellets were rinsed with PBS and Alcian Blue stain was extracted by the addition of 6 M guanidine/HCL for 24 h at 4 °C; absorbance was measured at 650 nm (Biotek Synergy).