The importance of mesenchymal stem cell donor’s age for cartilage engineering

Abstract

In 1994, Brittber et coll. showed that cultured autologous chondrocytes could be used to repair deep cartilage defects of the knee joint. But, several problems are observed due to the differentiation of chondrocytes in culture leading to fibrocartilage formation. More recently, the use of bone marrow mesenchymal stem cells (BM-MSC) in cartilage engineering has been used. As hinted by the role of stem cell senescence and dysfunction in natural aging, donor or patient age will be a critical factor that must be accounted for in clinical and laboratory evaluations of bone marrow mesenchymal stem cell based technology.We evaluated in vitro chondrogenic differentiation of bone marrow mesenchymal stem cells in function of donor age and passage in culture. The objective of such a study design was to provide a controlled analysis of two variables (age and passage) and possible interaction between these crucial factors in developing adult stem cell based therapeutics and for which no consensus exists regarding their role in MSC differentiation. Materials and Methods: Mesenchymal stem cells were obtained from bone marrow from human donors of different ages (n = 18, 3 to 85 years old, mean age 49 ± 27). Cells were isolated by plastic adherence (5 104 mononuclear cells/cm2) and cultured in α-MEM with 10% SVF. After two weeks cells were detached from culture flasks and replated at 1000 cells/cm2. Then cells were passaged every two weeks. At each passage, cells were counted, clonogenicity was tested by a colony forming unit test and phenotype was verified by flow cytometry for the expression of several markers (CD90, CD73, CD105, CD44, CD45, CD34, HLA-DR). Chondrogenic potential was assayed after passage 1 and 4/5 using specific differentiation media containing TGF-β. 2.5 x 105 cells were used to form each chondrogenic pellet. After 28 days, Gene expression of chondrogenic markers was evaluated by qPCR and matrix synthesis by histology and alcian blue staining. Quantification of staining area was performed by using ImageJ. Results: Our study has revealed differences between proliferation capacities of BM-MSC in function of donor’s age. Cells from old donors proliferate slowly than cells from young donors and become senescent after 4 or 5 passages. Two groups of donors have been defined according to proliferation capacity (less than 40 years old and over 40 years old). Phenotype, clonogenicity and chondrogenic differentiation was studied in the two groups. While cells expressed mesenchymal markers (CD90, CD44, CD73, CD105) and did not expressed HLA-DR, CD45 or CD34, no differences were observed in the expression level of the markers according to donor’s age. Clonogenicity was inceased for cells coming from young donors, both at number or size levels. At the same time, young donors cells were characterized by high level expression of type 2 collagen, aggrecan and sox-9 genes after chondrogenic differentiation while cells from the > 40 years group showed a poor chondrogenic differentiation. Similar results were observed at the matrix synthesis levels after alcian blue staining. Cells from late passages showed decresed differentiation compared with cells from early passages. However, at early passages, cells from young donors presented a great differentiation potential than cells from aged donors. Conclusions: This study shows that differences on proliferation and chondrogenic differentiation exist between mesenchymal stem cells isolated from bone marrow obtained in young and old donors. These results could be considered in the choice of MSC for cartilage tissue engineering. The proliferation capacity of cells and their clonogenicity are good parameters to rapidly estimate cell quality.