Fig. 4

Osteogenic, chondrogenic, and adipogenic differentiation of T-MPCs. a Alizarin red S staining demonstrates calcium deposits in T-MPCs treated with osteogenic differentiation media (ODM). b Undifferentiated T-MPCs grown in complete media (CM) are negative. Real-time qPCR analysis validates the differentiation of T-MPCs in osteogenic media and shows a significant increase in expression of c osteoblast markers and d osteocyte markers compared to control. e Oil red O is negative in the control cells incubated at the same time and for the same duration in complete medium, while f Oil droplets in T-MPCs differentiated in adipogenesis medium (ADM). g Real time RT-qPCR shows T-MPC-derived adipocytes express high levels of the adipogenic-specific genes PPRAG and Leptin compared to undifferentiated controls in CM. h Alcian blue staining is negative in T-MPCs grown in CM, and i T-MPC-derived chondrocytes grown in chondrogenesis media (CDM) stained in blue. j RT-qPCR confirms an increase in the chondrogenic-specific markers ACAN, COL10A1, and ATP2A2 following differentiation compared to undifferentiated T-MPC controls. All results are presented as mean ± SEM obtained in triplicate from donors in multiple independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ALP alkaline phosphatase, BMP2 bone morphogenetic protein 2, DMP1 dentin matrix protein 1, FGF23 fibroblast growth factor 23, MEPE matrix extracellular phospho-glycoprotein, OCN osteocalcin, OPN osteopontin, RUNX2 runt-related transcription factor 2, SOST sclerostin