Evaluation of the effect of human platelet lysate versus fetal bovine serum on human osteoarthritic cartilage derived chondroprogenitors

Abstract

Purpose: Articular chondroprogenitors are a sub-population of cells exhibiting clonal growth, propensity for enhanced chondrogenesis and reduced hypertrophy. Since these cells have been classified as mesenchymal stem cells (MSCs) as per International Society for Cellular Therapy criteria, they may be a suitable contender for cell-based therapy in cartilage repair following disease and degeneration, as seen in osteo-arthritis. Fetal bovine serum (FBS) is a widely used culture additive for expansion and differentiation of chondroprogenitors (CPs). However due to concerns related to potential transmission of zoonoses and possibility of transplant rejections following cell implantation, there is a need for a human derived alternative. Human platelet lysate (HPL) has been extensively used for expanding various cell lines due to existent pool of abundant growth factors, but data related to its use with chondroprogenitors has not been reported. We therefore aimed to culture human chondroprogenitors in HPL and compare their growth, senescence, phenotypic expression and differentiation capacity to that of FBS. Methods: CPs were isolated from three human osteo-arthritic knee joints (Age: 53.3 ± 10.21 years) following differential fibronectin adhesion assay. Passage 0 cells grown in a)10% FBS and b)10% HPL were considered for assessment of trilineage differentiation, growth kinetics (cell cycle analysis and galactosidase assay), surface marker expression (CD105, CD73, CD90, CD49e, CD29, CD146, CD166, CD34, CD45 and HLA-DR) and RT-PCR (markers of chondrogenesis: Collagen II, SOX9, Aggrecan; hypertrophic chondrocyte marker: Collagen I and that of hypertrophy: Collagen X, MMP13, RUNX2). Latent TGFß1 levels were also measured for each culture medium used. Data was compared using Student t-test and a P value of < 0.05 was considered significant. Results: Results showed that cellular proliferation was significantly higher in cells grown with HPL (P<0.05). Surface marker expression was comparable except in CD 146 where HPL group had significantly lower values (P<0.05). Comparison of mRNA expression revealed notably low values of Aggrecan, Collagen II, Collagen I and Collagen X (P<0.05). Trilineage differentiation was seen in both groups with higher uptake in the HPL group noted for alizarin red. There was also significantly higher level of latent TGFß1 in the medium containing HPL as compared to FBS (P<0.001). Conclusions: This is the first in-vitro xeno free study to affirm that HPL can serve as an optimal growth supplement for expansion of articular chondroprogenitors, although an in-depth assessment of resident growth factors and evaluation of different dilutions of HPL is required to assess suitability for use in translational research.