Differentiation of Human Induced Pluripotent Stem Cells to Growth-Plate like Cartilage to Understand Pseudoachondroplasia

Abstract

Pseudoachondroplasia (PSACH) is a rare genetic skeletal dysplasia, associated with dwarfism and premature joint degeneration. PSACH is caused by mutations in the gene encoding cartilage oligomeric protein (COMP) that lead to miss-folding and retention of COMP in the endoplasmic reticulum (ER) of growth-plate chondrocytes, during development. The aim of this PhD thesis was to use human induced pluripotent stem cells (hiPSCs) from individuals with two different PSACH associated COMP mutations (N489K and G465S) to generate chondrogenic pellets as a model system to explore the effects of these mutations in growth-plate cartilage development. HiPSCs showed expression of pluripotency markers and when differentiated into human induced mesenchymal stem cells (hiMSCs), they expressed human mesenchymal markers. CD73 was slightly elevated in both the mutant cell lines compared to control hiMSCs. At the chondrogenic pellet stage there were striking differences between the mutant and control pellets, which suggested diminished production of glycosaminoglycans as well as decreased expression and altered localisation/organisation of cartilage matrix proteins. For example, collagen fibres in mutant pellets were distorted. Addition of TGF-B3 and BMP2 in combination during chondrogenic pellet formation resulted in slightly increased expression of cartilage matrix proteins and associated transcripts, when compared to pellets formed in the absence of growth factors or with either TGF-B3 or BMP2 alone. However, these were small and didn't reverse the aberrant chondrocyte phenotype or the altered cartilage structure/composition associated with the N489K and G465S mutations. These results in hiPSC derived chondrogenic pellet models are consistent with the phenotype observed in PSACH patients as well as in mouse models studied previously. Exploration of the PSACH disease process in the hiPSCs model was further extended using RNA-sequencing to compare the transcriptomes of COMP mutant and control cells and identify signalling pathways that are disrupted during chondrogenesis in PSACH. Pathway analysis identified down regulation of TGF-B signalling at the hiMSCs stage and increased matrix degradation, inhibition of canonical WNT signalling and upregulation of oxidative stress at the pellets stage in the mutant cell lines compared to controls. The model system developed here complements previous studies and identifies targets for further research towards therapeutics for PSACH either using drugs or gene editing techniques to correct COMP mutations.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Susan Kimber (Supervisor) & Caroline Milner (Supervisor)