Epiphyseal chondro-progenitors provide a stable cell source for cartilage cell therapy

Articular cartilage regeneration poses particularly tough challenges for implementing cell-­‐based therapies. Many cell types have been investigated looking for a balanced combination of responsiveness and stability, yet techniques are still far from defining a gold standard. The work presented focuses on the reliable expansion and characterization of a clinical-­‐grade human epiphyseal chondro-­‐progenitor (ECP) cell bank from a single tissue donation. A parental human ECP cell bank was established which provides the seed material for master and working cell banks. ECPs were investigated at both low and high cumulative population doublings looking at morphology, monolayer expansion kinetics, resistance to cryogenic shock, colony forming efficiency and cell surface markers. Three dimensional micro-­‐pellet assays were used to determine spontaneous extracellular matrix deposition at varying population doublings and monolayer 2D differentiation studies were undertaken to assess the propensity for commitment into other lineages and their stability. ECPs exhibited remarkable homogeneity in expansion with a steady proliferative potential averaging 3 population doublings over eight days. Surface marker analysis revealed no detectable contaminating subpopulations or population enrichment during prolonged culture periods. Despite a slight reduction in Sox9 expression levels at higher population doublings in monolayer, nuclear localization was equivalent both in monolayer and in micro-­‐pellet format. Equally, ECPs were capable of depositing glycosaminoglycans, producing aggrecan, collagen I and collagen II in 3D pellets both at after low and high population doublings indicating a stable spontaneous chondrogenic potential. Osteogenic induction was differentially restricted in low and high population doublings as observed by Von Kossa staining of calcified matrix, with a notable collagen X, MMP13 and ADAMTS5 down-­‐regulation. Rare adipogenic induction was seen as evidenced by cytoplasmic lipid accumulation detectable by Oil Red O staining. These findings highlight the reliability, stability and responsiveness of ECPs over prolonged culture, making them ideal candidates in defining novel strategies for cartilage regeneration.