Poster Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Analyses of osteogenic differentiation in iPS cells derived from cleidocranial dysplasia patients (#337)

Akiko Saito 1 , Akio Oki 2 , Takashi Nakamura 3 , Shoko Onodera 1 , Hiromi Shino 1 , Daigo Hasegawa 4 , Kenjiro Kosaki 5 , Takeshi Onda 4 , Akira Watanabe 4 , Takahiko Shibahara 4 , Kenji Sueishi 2 , Toshifumi Azuma 1
  1. Department of Biochemistry, Tokyo Dental College, Tokyo
  2. Department of Orthodontist, Tokyo Dental College, Tokyo
  3. Department of Biochemistry&Integrative Medical Biology,, School of Medicine, Keio University, Tokyo
  4. Department of Oral and Maxillofacial Surgery, Tokyo Dental College, Tokyo
  5. Center for Medical Genetics, School of Medicine, Keio University, Tokyo

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.