Multiple epiphyseal dysplasia (MED) is a chondrodysplasia characterised by delayed epiphyseal endochondral ossification, resulting in disproportionate short stature and early onset osteoarthritis. MED can be caused by heterozygous mutations in COMP, MATN3, COL9A1, COL9A2 and COL9A3, or bi-allelic mutations in SLC26A2. Human induced pluripotent stem cells (hiPSCs) are reprogrammed somatic cells which can differentiate to form all body tissues and have excellent potential for tissue regeneration as well as providing models of human disease. Our aim is to generate an in vitro hiPSC model of growth-plate development in order to better understand MED. HiPSCs were generated from peripheral blood mononuclear cells (PBMCs) of 3 related MED individuals who are heterozygous for a MATN3 p.Val194Asp mutation (V194D) and 4 healthy controls, including one close relative. HiPSC were differentiated to growth-plate-like chondrocytes via an iPSC-MSC-like intermediate, followed by TGFβ3+ BMP2 induced chondrogenic pellet culture for 21 days. Healthy and V194D hiPSCs were able to differentiate to iPSC-MSCs which displayed typical MSC morphology, expressed MSC markers (CD90, CD105, CD44 and CD73) and were capable of generating cartilage and bone. After 21 days in TGFβ3+ BMP2-containing medium V194D chondrogenic pellets were significantly larger in size, stained more strongly for cartilage associated sulphated glycosaminoglycans (Alcian blue and Safranin O), and expressed significantly higher levels of transcript for the chondrogenic transcription factor SOX9, and the major cartilage matrix constituents COL2A1 and ACAN. These data suggest V194D mutant pellets respond differently during TGFβ3+ BMP2 induced chondrogenesis. RNA-Seq validated the high expression of chondrogenic associated transcripts in the V194D mutant pellets, recapitulating the delayed transition from cartilage to bone observed in the growth plate of MED patients. Immunohistochemistry and confocal co-localisation analysis of matrilin-3 (usually a matrix protein) with the endoplasmic reticulum (ER) marker GRP94, suggests matrilin-3 is retained within the ER of the V194D mutant pellets. As matrilin-3 interacts with TGFβ and BMP2, this may explain the differences in response to growth factors during pellet culture. This work provides novel insight into MED disease pathogenesis. Our in vitro growth-plate model will facilitate the identification of pathogenic pathways of growth-plate diseases that are suitable for pharmacological intervention, and will allow the screening of pharmaceutical products.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology