TY - JOUR
T1 - Targeted reversion of induced pluripotent stem cells from patients with human cleidocranial dysplasia improves bone regeneration in a rat calvarial bone defect model
AU - Saito, Akiko
AU - Ooki, Akio
AU - Nakamura, Takashi
AU - Onodera, Shoko
AU - Hayashi, Kamichika
AU - Hasegawa, Daigo
AU - Okudaira, Takahito
AU - Watanabe, Katsuhito
AU - Kato, Hiroshi
AU - Onda, Takeshi
AU - Watanabe, Akira
AU - Kosaki, Kenjiro
AU - Nishimura, Ken
AU - Ohtaka, Manami
AU - Nakanishi, Mahito
AU - Sakamoto, Teruo
AU - Yamaguchi, Akira
AU - Sueishi, Kenji
AU - Azuma, Toshifumi
N1 - Funding Information:
This work was supported by JSPS KAKENHI (C) (grant no. 25462900) and by JSPS KAKENHI for young scientists (B) (grant nos. 26861686, 26861560, 16 K20427, and 16 K20428).
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/1/22
Y1 - 2018/1/22
N2 - 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.
AB - 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.
KW - CRISPR/Cas
KW - Cleidocranial dysplasia
KW - Osteoblasts
KW - Osteogenesis
KW - RUNX2
KW - iPSCs
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U2 - 10.1186/s13287-017-0754-4
DO - 10.1186/s13287-017-0754-4
M3 - Article
C2 - 29357927
AN - SCOPUS:85040919122
VL - 9
JO - Stem Cell Research and Therapy
JF - Stem Cell Research and Therapy
SN - 1757-6512
IS - 1
M1 - 12
ER -