Abstract
Summary Statement
Extended expansion of cells derived from equine articular cartilage reveal maintenance of chondrogenic potency and no evidence of senescence up to 100 population doublings. The data suggests the reclassification of these cells from progenitor cells to stem cells.
Introduction
One sign of ‘in vitro aging’ is the diminishing capacity for cell division. In contrast to embryonic stem cells that show no loss of proliferative potency, the maximal population doublings (PD) for mesenchymal stem cells (MSCs) in vitro is reported to be between 30 and 40 replications 1,2,3. We have isolated a population of chondroprogenitor cells from articular cartilage of several species, including equine4.
These cells have demonstrated functional equivalence in their differentiation capacity when compared with MSCs but have the advantage of retaining the highly desirable stable (permanent) chondrocyte phenotype. In this study, we examined the age-related capacity of these cells for extended division and retention of potency.
Methods
Chondroprogenitors were isolated from equine articular cartilage by adhesion onto fibronectin5. Cells were isolated from both skeletally immature (1 year-old) and mature animals (8 year-old). Clonal and polyclonal cell lines (at least 5 of each for each age) were cultured in the presence of 10% FCS, 1ng/ml TGFb-1 & 2.5 ng/ml FGF-2. Cells were seeded at low density and passaged weekly.
Results
Chondroprogenitors from both animals reached over 40 (mean) PD in 50 days with growth remaining linear. Little difference in growth rates was observed between clonal and polyclonal cell lines. For the mature animal, 96% of cells were BrDU positive at 22 PD whilst none of cells were (senescence associated) β-gal positive. At 44 PD, 88% of cells were BrDU positive and just 15% of cells were β-gal positive. Three clonal and three polyclonal cell lines from the mature animal were cultured beyond the 50-day time point. At 120 days, cells reached up to 90 PD with the same pattern of linear growth observed. When tested at 70 PD, 79% of these cells were still BrDU positive (range 55–97%) and just 11% of cells were β-gal positive (range 2–22%). Furthermore, little difference in cell morphology was observed throughout this extended expansion. At 70 PD, we found that both clonal and polyclonal cell lines in monolayer culture were still expressing the chondrogenic transcription factor; Sox-9. Expression of genes for aggrecan and collagen type II was also detected in cells that were chondrogenically induced for 72 hours.
Discussion & Conclusions
We have demonstrated for the first time the extended expansion of cells derived from articular cartilage that retain chondrogenic potency. These equine cells have since been cultured to over 100 PD without evidence of senescence. One hundred PD is equivalent to 1 × 1030 cells originating from a single cell. We have previously reported that the human equivalents of these cells surpass MSCs in doubling capacity but senesce at approximately 60 PD6. The properties of these equine chondroprogenitor cells make them ideal candidates for allogeneic cell therapy for articular cartilage repair. In addition, the data suggest the reclassification of these cells from progenitor cells to stem cells.
