Date of Award
Doctor of Philosophy (PhD)
Cartilage is essential to joint development and function. However, there is a variety of cartilage diseases, ranging from developmental (e.g., skeletal dysplasias) to degenerative (e.g., arthritis), in which treatments and therapeutics are lacking. For example, specific point mutations in the ion channel transient receptor potential vanilloid 4 (TRPV4) prevent proper joint development, leading to mild brachyolmia and severe, neonatally lethal metatropic dysplasia. Tissue-engineered cartilage offers an opportunity to elucidate the underlying mechanisms of these cartilage diseases for the development of treatments. Human induced pluripotent stem cells (hiPSCs) are an improved cell source option for cartilage tissue engineering given their minimal donor site morbidity, absence of ethical concerns, and extensive proliferation, differentiation, and gene editing capacities. Unfortunately, previously published hiPSC chondrogenesis protocols were time consuming, difficult to reproduce, and resulted in off-target differentiation. Here, we used two methods to enhance hiPSC chondrogenesis using our previously published stepwise chondrogenic differentiation protocol. Next, we used the improved protocol to perform in vitro disease modeling of brachyolmia and metatropic dysplasia resulting from mutations in mechanosensor TRPV4.
To enhance chondrogenesis, we used a CRISPR-Cas9-edited hiPSC cell line with a GFP reporter to determine surface markers co-expressed with early chondrogenic marker and cartilage matrix protein COL2A1. We found that chondroprogenitors that were positive for PDGFRβ, CD146, and CD166 and negative for CD45 had enhanced chondrogenic potential. In fact, sorted chondroprogenitors from the reporter line and an unedited line had significantly improved homogeneity compared to unsorted as determined by single-cell RNA sequencing. Furthermore, the derived chondrocytes synthesized more homogenous and robust matrix proteins and had higher chondrogenic gene expression. In a continued effort to improve the chondrogenesis protocol, we used bulk and single-cell RNA sequencing to determine where the off-target differentiation occurred. We found that Wnt and melanocyte inducing transcription factor (MITF) signaling were driving the two primary off-target populations: neurogenic and melanogenic, respectively. Single-cell RNA sequencing, histology, and quantification of matrix production confirmed pan-Wnt and MITF inhibition during chondrogenesis improved homogeneity of the cells throughout differentiation and increased chondrogenic potential.
Using the findings from these studies, we created an hiPSC chondrogenesis protocol that follows the developmental mesodermal lineage and uses chemically defined medium. We also provide instructions for digesting the chondrogenic tissue to isolate hiPSC-derived chondrocytes at the single cell level. This protocol has applications for a variety of tissue engineering uses including regenerative therapies, gene editing, drug screening, and disease modeling.
In fact, we applied this protocol for disease modeling of TRPV4 mutations that result in skeletal dysplasias. Using CRISPR-Cas9 gene editing technology, we created two hiPSC lines harboring either the brachyolmia-causing V620I substitution or the metatropic dysplasia-causing T89I substitution. The hiPSCs were chondrogenically differentiated and then were treated with BMP4 to stimulate hypertrophic differentiation. We determined that TRPV4 mutations increased basal signaling but decreased sensitivity to chemical agonist GSK1016790A using electrophysiology techniques and confocal imaging. Furthermore, using bulk RNA sequencing, we found the mutations suppressed chondrocyte maturation and hypertrophy, likely preventing endochondral ossification and long bone formation leading to the disease phenotype.
We also used these cell lines to study the effects of the mutations on mechanotransduction. The hiPSC-derived chondrocytes were physiologically loaded in agarose constructs for 3 hours and then sequenced to elucidate the temporal response to loading. We found the mutant TRPV4 increased gene expression in response to loading compared to wildtype. Gene expression patterns indicated increased proliferation in mutant cells, which could prevent chondrocyte hypertrophic differentiation and endochondral ossification.
Overall, we have developed an improved chondrogenic hiPSC protocol. The resulting tissue-engineered cartilage has many uses including in vitro disease modeling of genetic, developmental conditions, as shown here. Our findings provide target genes for future drug development to treat brachyolmia and metatropic dysplasia. Furthermore, we have increased the understanding of TRPV4 function in chondrocytes, which can be applied to cartilage tissue engineering and other cartilage disease studies.
Nathaniel Huebsch, Spencer Lake, Ingrid Meulenbelt, Simon Tang,