Chondroprogenitor cells characterization in familial osteochondritis dissecans; identification of cellular pathologies

Purpose: Familial osteochondritis dissecans (FOCD) is an inherited cartilage defect characterized by multiple joint involvement, short stature and early osteoarthritis. We have studied a family from Northern Sweden (NS) with FOCD over five generations with all affected family members having a heterozygous missense mutation on exon 17 of the aggrecan gene. Privious study showed that this mutation results in a Val-Met amino acid replacement (V2303) in the G3 aggrecan C-type lectin domain (CLD). The aims of the study were to investigate the pathogenesis of FOCD-NS by examining chondrogenesis of patient-derived bone marrow mesenchymal stem cells (BM-MSCs) and induced pluripotent stem cells (iPSCs). This approach has given us a fascinating insight into the cellular pathology of the condition. Methods: BM-MSCs were obtained after institutional ethical approval and informed consent from a 33 year-old male healthy donor and a 49 year-old male patient. The cells were characterized by flow cytometry and tri-lineage differentiation. Chondrogenic pellets, derived from control and patient BM-MSCs, were harvested to study the effects of the mutation on early and late chondrogenesis. Toluidine blue staining was carried out to detect glycosaminoglycan (GAG) and immunohistochemistry (IHC) used to detect proteins of interest. The ultrastructure of cell organelles was examined by transmission electron microscopy (TEM). Mass spectrometry was employed to analyze the composition of extracellular matrix (ECM). Because of the difficulties in obtaining marrow samples from patients and the low yield of MSCs from this source, we generated patient-specific iPSCs from skin fibroblasts of two FOCD-NS patients, a 25 year-old son and 49 year-old mother. To assess the pluripotency of iPSCs generated, RT-PCR for pluripotency genes, teratoma assay and karyotype analysis were performed. One non-patient iPSC line was used as a positive control in all experiments. To validate the disease phenotype, cartilage formed in patient iPSC-derived teratoma was also analyzed. Results: All BM-MSCs expressed typical surface markers and were capable of tri-lineage differentiation. Chondrogenic differentiation in vitro revealed an irregular discoid morphology of patient pellets. An intracellular distribution of aggrecan in FOCD-NS pellets was observed at early stages of differentiation. Multiple immuno-staining of aggrecan and Bip, a marker of rough endoplasmic reticulum (rER), further confirmed that the mutated aggrecan was retained in the rER during chondrogenic processing. Membrane expansion of the rER was also detected in chondrocytes derived from patient BM-MSCs at day 56, indicating a prolonged folding defect within the organelle. Although collagen type II was produced by patient chondrocytes, TEM showed that they failure to assemble a normal ECM. GAGs distribution appeared normal in patient pellets during early chondrogenesis, which suggested that the non-mutated aggrecan allele was expressed. We cultured these pellets for up to 100 days to assess the impact during tissue maturation. The results of mass spectrometry on protein isolated from these pellets showed while the aggrecan G1 domain was presented in equal amounts in both groups, the G2 and G3 domains were reduced by approximately 50% in the patient pellets. Comparing the amount of other ECM proteins indicated that the composition of matrix was markedly different between control and patient. Four patient-derived iPS cell lines were morphologically, genetically and functionally similar to human embryonic stem cells. Detection of aggrecan in the cartilage differentiated from these showed the same disease phenotype found in chondrogenic pellets derived from the BM-MSCs. Conclusion: Our findings concluded that the V2303 mutation results in folding defect aggrecan G3 domain, which prevents processing of the protein core through rER. The accumulation of intracellular aggrecan results in an enlarged rER area and affects the function of FOCD-NS chondrocytes in assembling normal ECM. Although the non-mutated aggrecan was produced to a certain extent, it could not reverse the pathology of the disease during chondrogenesis. ECM with disrupted composition and reduced aggrecan levels could affect the mechanical properties of FOCD-NS cartilage. This study revels how insight into disease mechanism can be obtained by studying the regulation of differentiation of progenitor cells. It also provides, for the first time, a model of inherited osteoarthritic disease based on patient-derived iPS cells, and sheds further light on the critical role of the CLD in aggrecan folding and in normal matrix assembly.Figure 2Confocal microscopy images showing double Staining of aggrecan (red) and GRP78/Bip (green). Cell nuclei stained with DAPI (blue). The merged images indicated the relative location of aggrecan and Bip. Overlapping areas appeared yellow. Scale bar=20um. Samples were harvested at day 42.View Large Image Figure ViewerDownload Hi-res image Download (PPT)