The Use of Pluripotent Stem Cells (PSCs) and CRISPR Genome Editing to Study the Roles of TRPV4 Ion Channels in Skeletal Malformation

© 2019 Yudha Nur PatriaTransient Receptor Potential Vanilloid 4 (TRPV4) is a non-selective calcium channel that plays an important role in the mechanotransduction system in chondrocytes. Heterozygous TRPV4 mutations cause skeletal disorders with varying severity. Heterologous cells such as fibroblasts and HEK-293 cells are commonly used to model TRPV4-inherited skeletal diseases in vitro. Studies using human chondrocytes are limited because cartilage is rarely available from patients and controls. Although heterologous cells cannot completely recapitulate the biological processes occurring in human chondrocytes, the studies show that two distinct disease phenotypes, TRPV4 skeletal dysplasia and arthropathy, might be caused by differences in the way the mutations change TRPV4 channel behaviour. TRPV4 skeletal dysplasia causing mutations show channel over-activity whereas arthropathy causing mutations show reduced channel activity upon channel stimulation. However, the downstream pathogenic mechanisms responsible for the distinct skeletal phenotypes remain undefined. Recognising the limitations of previous studies, human-induced pluripotent stem cells (hiPSCs) offer a new approach for inherited disease modeling. Their ability to differentiate into disease-relevant cells such as chondrocytes creates new opportunities for TRPV4-inherited skeletal disease modelling. Therefore, this PhD project aims to model the disorders caused by two TRPV4 mutations using hiPSCs and identify the pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal disease phenotypes. To obtain disease-relevant cells, establishing a robust and reproducible chondrocyte differentiation protocol is required. To do this, a reporter hiPSC line, SOX9-T2A-tdTom, was generated from MCRIi001-A (PB001.1) (1) using CRISPR/Cas9 genome editing. Thus in vitro chondrocyte differentiation could be monitored in real-time. The T2A linker and tdTomato fluorescent reporter gene were inserted downstream of the SOX9 coding sequence through homology-directed repair. The targeted allele was designed to produce SOX9 with the T2A sequence at the C-terminal end and a separate tdTom fluorescent protein. Genomic DNA sequencing of the SOX9-T2A-tdTom hiPSC line confirmed that the hiPSC line had one SOX9 allele with the T2A tdTom gene fusion and one wild type allele. Pluripotency was maintained as indicated by expression of pluripotency markers OCT4 and NANOG (immunostaining); CD9, CD326, and SSEA-4 (flowcytometry); and the ability to form tissues derived from three germ layers. SNP array showed there were no aneuploidies. The SOX9-T2A-tdTom hiPSC line had a similar capability to the parental line, MCRIi001-A, to form sclerotome. Western blotting showed that SOX9 protein expression was similar between SOX9-2A-tdTom and its parental line suggesting that adding tdTom gene sequence downstream of SOX9 gene did not disrupt the SOX9 expression and stability. The chondrocyte differentiation protocol was established using the SOX9-T2A-tdTom hiPSC line. Two stages of differentiation were performed. First, sclerotome induction was achieved by culturing hiPSCs in a 6-day multiple-step chemically defined culture mimicking embryonic development with pellet culture format was established on day 4. Secondly, chondrocyte differentiation was performed by transferring day-6 pellets into chondrogenic media in swirling culture format up to 10 weeks. A 4-week course of FGF2 treatment followed by an optional TGFB3 and GDF5 treatment until week 10 was performed during chondrocyte differentiation. RNA was collected every day during a 6-day sclerotome induction and at different time points during chondrocyte differentiation. The optimised protocol that involved a multiple-step chemically-defined 3-dimensional (3D) culture with swirling in an extended culture that included a 4-week FGF2 supplementation and optional subsequent TGFB3 and GDF5 treatment was able to generate cartilage that closely resembles fetal cartilage. CRISPR/Cas9 genome editing was also used to introduce two human TRPV4 mutations, a TRPV4 c.819C>G (p.F273L) mutation causing familial digital arthropathy with brachydactyly (FDAB) and a TRPV4 c.2396C>T (p.P799L) mutation causing metatropic dysplasia, into the SOX9-T2A-tdTom hiPSC line. For in vitro disease modelling, the mutant and their isogenic wild-type control (SOX9-T2A-tdTom) hiPSC lines were differentiated towards chondrocytes using optimised chondrocyte differentiation. The phenotypic differences between mutants and wild-type were assessed using various techniques including gene (RNA sequencing) and protein expression analysis. The two mutant cell lines and their isogenic wild-type control (SOX9-T2A-tdTom) were able to form cartilage. The pellet cartilage histology did not show any striking differences between the two mutants and their isogenic control. COL2A1 and TRPV4 protein expression was similar between mutants and control and this was consistent with the RNA sequencing data. RNA sequencing suggested that the pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal diseases were different. Compared to the isogenic control, F273L mutant cartilage had 263 differentially expressed genes. F273L cartilage showed a slight reduction in cartilage related gene expression including COL2A1, CSPG4, BGN, and CILP2. The F273L cartilage tissue was also less mature than the wild-type as indicated by increased SHH expression. On the other hand, P799L cartilage had more differentially expressed genes (655 genes) than F273L. MEF2C, the main regulator of chondrocyte hypertrophy, was upregulated in P799L. The hypertrophic chondrocyte markers such as RUNX2, SPP1 or osteopontin, and PTH1R, were also upregulated in P799L suggesting increased chondrocyte hypertrophy of P799L chondrocytes. The other characteristics of hypertrophic chondrocytes such as a reduction in cell proliferation and increased apoptosis were also observed in P799L cartilage. In conclusion, this study is the first study that conducts global gene expression analysis using RNA sequencing to characterise gene expression changes downstream of TRPV4 mutations in hiPSC-derived chondrocytes. The pathogenic mechanisms underlying the two distinct TRPV4-inherited skeletal diseases are different. The fewer differentially expressed genes in the F273L cartilage than in P799L suggests a milder disease phenotype. The slight reduction in cartilage marker expression in F273L cartilage might cause the cartilage tissues less resilient to physical forces thus leading to FDAB. In contrast, accelerated chondrocyte hypertrophic maturation can be the pathogenic mechanism underlying TRPV4 skeletal dysplasia phenotype. Accelerated chondrocyte hypertrophic maturation can disrupt growth plate development and cause systemic skeletal defects seen in patients. This thesis demonstrates that hiPSCs are a powerful tool to model inherited skeletal disease in vitro