Format

Send to

Choose Destination
Stem Cell Res Ther. 2018 Jan 22;9(1):12. doi: 10.1186/s13287-017-0754-4.

Targeted reversion of induced pluripotent stem cells from patients with human cleidocranial dysplasia improves bone regeneration in a rat calvarial bone defect model.

Author information

1
Department of Biochemistry, Tokyo Dental College, Tokyo, Japan. ayokoyama@tdc.ac.jp.
2
Department of Orthodontics, Tokyo Dental College, Tokyo, Japan.
3
Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
4
Department of Oral and Maxillofacial Surgery, Tokyo Dental College, Tokyo, Japan.
5
Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan.
6
Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.
7
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
8
Oral Health Science Center, Tokyo Dental College, Tokyo, Japan.

Abstract

BACKGROUND:

Runt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models.

METHODS:

Two cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-derived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis.

RESULTS:

Mutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with severe combined immunodeficiency showed poor regeneration. However, reverted iPSCs improved the abnormal osteoblast differentiation which resulted in much better engraftment into the rat calvarial bone defect.

CONCLUSIONS:

Taken together, these results demonstrate that patient-specific iPSC technology can not only provide a useful disease model to elucidate the role of RUNX2 in osteoblastic differentiation but also raises the tantalizing prospect that reverted iPSCs might provide a practical medical treatment for CCD.

KEYWORDS:

CRISPR/Cas; Cleidocranial dysplasia; Osteoblasts; Osteogenesis; RUNX2; iPSCs

PMID:
29357927
PMCID:
PMC5778688
DOI:
10.1186/s13287-017-0754-4
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for BioMed Central Icon for PubMed Central
Loading ...
Support Center