Patient-Derived Skeletal Dysplasia Induced Pluripotent Stem Cells Display Abnormal Chondrogenic Marker Expression and Regulation byBMP2andTGFβ1

Abstract

Skeletal dysplasias (SDs) are caused by abnormal chondrogenesis during cartilage growth plate differentiation. To study early stages of aberrant cartilage formation in vitro, we generated the first induced pluripotent stem cells (iPSCs) from fibroblasts of an SD patient with a lethal form of metatropic dysplasia, caused by a dominant mutation (I604M) in the calcium channel gene TRPV4. When micromasses were grown in chondrogenic differentiation conditions and compared with control iPSCs, mutant TRPV4-iPSCs showed significantly (P < 0.05) decreased expression by quantitative real-time polymerase chain reaction of COL2A1 (IIA and IIB forms), SOX9, Aggrecan, COL10A1, and RUNX2, all of which are cartilage growth plate markers. We found that stimulation with BMP2, but not TGFb1, up-regulated COL2A1 (IIA and IIB) and SOX9 gene expression, only in control iPSCs. COL2A1 (Collagen II) expression data were confirmed at the protein level by western blot and immunofluorescence microscopy. TRPV4-iPSCs showed only focal areas of Alcian blue stain for proteoglycans, while in control iPSCs the stain was seen throughout the micromass sample. Similar staining patterns were found in neonatal cartilage from control and patient samples. We also found that COL1A1 (Collagen I), a marker of osteogenic differentiation, was significantly (P < 0.05) up-regulated at the mRNA level in TRPV4-iPSCs when compared with the control, and confirmed at the protein level. Collagen I expression in the TRPV4 model also may correlate with abnormal staining patterns seen in patient tissues. This study demonstrates that an iPSC model can recapitulate normal chondrogenesis and that mutant TRPV4-iPSCs reflect molecular evidence of aberrant chondrogenic developmental processes, which could be used to design therapeutic approaches for disorders of cartilage.

TRPV4 as a therapeutic target for joint diseases

“…A number of recent studies have shed light on the underlying mechanisms by which TRPV4 mutations lead to skeletal dysplasias (Leddy et al, 2014a, Leddy et al, 2014b, Saitta et al, 2014, Weinstein et al, 2014). The dysplasias result from improper endochondral ossification (Weinstein et al, 2014) due to increased Ca 2+ influx into chondrocytes through TRPV4 mutant channels, which in turn upregulates follistatin, a potent inhibitor of the bone morphogenetic protein (BMP) family of growth factors that are required for proper endochondral bone formation (Fig.…”

“…1) (Leddy et al, 2014b). Cells expressing these mutations are also less sensitive to BMP signaling, which prevents the normal chondrocyte hypertrophy cascade and leads to improper bone formation (Saitta et al, 2014). …”

“…In particular, iPSCs can be generated from patients with TRPV4 mutations that cause the spectrum of skeletal dysplasias (Saitta et al, 2014) and the arthopathies, or from patients with osteoporosis, rheumatoid arthritis, or osteoarthritis. Cartilage derived from iPSCs has been shown to replicate various aspects of human disease in vitro (Willard et al, 2014) and can be used to test therapeutic approaches for the spectrum of joint diseases.…”

Stem cells and bone diseases: New tools, new perspective

“…The use of iPS cells for disease modeling encompasses investigative as well as directly applicative avenues: the generation of patient-specific diseased and differentiated cell types, in which to seek disease mechanisms, but also a tool for high-throughput drug screening. iPS cells have been used to model rare diseases such as Fibrodysplasia Ossificans Progressiva [16] and metatropic dysplasia [17], revealing altered patterns of cartilaginous differentiation through the use, notably, of assays in fact developed for the study of postnatal stem cells. However, the notion that skeletal diseases could be modeled through stem cells precedes the development of the iPS cell technology.…”

Protein informatics combined with multiple data sources enriches the clinical characterization of novel TRPV4 variant causing an intermediate skeletal dysplasia

“…In wild‐type mammalian fetuses, chondrocytes produce a cartilage matrix that is slowly replaced and lengthened by bone, a process that is continued after birth (Mackie, Ahmed, Tatarczuch, Chen, & Mirams, ). TRPV4 pathogenic variants have been experimentally proven to cause incomplete endochondral ossification by interfering with proper chondrocyte function (Leddy et al., ; Saitta et al., ; Weinstein, Tompson, Chen, Lee, & Cohn, ). It is proposed that Ca 2+ entry through mutant TRPV4 triggers overproduction of follistatin, an inhibitor of bone morphogenetic protein 2 (BMP2).…”