Purpose: Cleidocranial dysplasia (CCD) is a skeletal disorder with autosomal dominant inheritance and is caused by heterozygous mutation of RUNX2. In this study, we generated iPSCs derived from CCD patient, and performed a functional analysis to utilize for pathophysiological analysis and new therapeutic development.
Methods: All experimental procedures were approved by the Tokyo Dental College Ethics Committee. CCD-iPSCs were generated from the patient’ oral mucosa fibroblasts. Revertant (Rev-iPSCs) was generated from CCD-iPSCs corrected by gene editing using the CRISPR/Cas9 system, was confirmed pluripotency. After osteogenic differentiation start, sequentially collected samples were analyzed by ALP activity staining and RT-qPCR. Calvaria bone defect models experiments was performed using SCID-Rat to transplant CCD-osteoblasts (CCD-OBs). At 4 weeks after transplantation, the newly formed bone was evaluated by μCT and histological analysis. Lamin A/C expression of CCD-OBs under mechanical stress was analyzed by RT-qPCR.
Results: A sequencing analysis of the genomic DNA extracted from the patients’ oral fibroblasts (CCD-OF) revealed a heterozygous mutation (R391X as CCD1, Q67X as CCD2) in the RUNX2 gene. CCD-iPSCs and Rev-iPSCs were confirmed pluripotency. Expressions of RUNX2 target genes (ALP, OC and OSX) and transcription factors (MSX2, DLX5 and TWIST1) were sharply increased in Rev-iPSCs at 9 days after OBM induction. But their expressions in CCD-iPSCs were hardly rise. Calvarial bone defect models experiments showed the poor regeneration capability of CCD-OBs. Nuclear morphology of CCD-OBs was distorted and expression of Lamin A/C was significantly low. Moreover, Lamin A/C expression of Rev-OBs was increased by stress load, and the expression of osteogenic marker was increased. However, CCD-OBs were weak reactivity to mechanical stress.
Conclusion: CCD-iPSCs showed aberrant nuclear morphology with low osteogenic abilities, which could be partly due to poor response RUNX2 target molecule LaminA/C to mechanical stress.