Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3
Introduction: Achondroplasia (ACH) is a congenital disease which causes dwarfism and many symptoms resulting from skeletal dysplasia. Because present therapeutic strategies are mainly surgical procedures as symptomatic treatments, development of a radical treatment is desired. Clarification of the A...
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doaj-ad9172bd259f4776ac2d7e9e34bff5802020-11-25T00:37:05ZengElsevierRegenerative Therapy2352-32042017-06-016C152010.1016/j.reth.2016.11.002Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3Naohiro Horie0Atsuhiko Hikita1Satoru Nishizawa2Sakura Uto3Tsuyoshi Takato4Kazuto Hoshi5Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, JapanDepartment of Cartilage and Bone Regeneration (Fujisoft), Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, JapanTranslational Research Center, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, JapanDepartment of Cartilage and Bone Regeneration (Fujisoft), Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, JapanDepartment of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, JapanDepartment of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, JapanIntroduction: Achondroplasia (ACH) is a congenital disease which causes dwarfism and many symptoms resulting from skeletal dysplasia. Because present therapeutic strategies are mainly surgical procedures as symptomatic treatments, development of a radical treatment is desired. Clarification of the ACH pathology is essential for creating a new remedy. However, there are many questions about the disease mechanisms that have not been answered. Methods: As a single base substitution of the FGFR3 gene had been proved to be the ACH causing genome mutation, our group established disease specific iPS cells by introducing the causative mutation of achondroplasia into human iPS cells by CRISPR/Cas9 based genome editing. These cells were differentiated towards chondrocytes, then the gene and protein expressions were examined by real time RT-PCR and Western blotting, respectively. Results: Based on the western blotting analysis, the FGFR3 protein and phosphorylated ERK were increased in the FGFR3 mutated iPS cells compared to the control cells, while the FGFR3 gene expression was suppressed in the FGFR3 mutated iPS cells. According to chondrogenic differentiation experiments, the IHH expression level was increased in the control cells as the differentiation progressed. On the other hand, up-regulation of the IHH gene expression was suppressed in the FGFR3 mutated iPS cells. Conclusions: These results suggested that chondrocyte maturation was impaired between the proliferative stage and prehypertrophic stage in the chondrocytes of ACH. The development of chemical compounds which affect the specific maturation stage of chondrocytes is expected to contribute to the ACH treatment, and FGFR3 genome-edited hiPSCs will be a valuable tool in such research studies.http://www.sciencedirect.com/science/article/pii/S2352320416300608iPS cellAchondroplasiaGenome editingDifferentiationChondrocyte |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Naohiro Horie Atsuhiko Hikita Satoru Nishizawa Sakura Uto Tsuyoshi Takato Kazuto Hoshi |
spellingShingle |
Naohiro Horie Atsuhiko Hikita Satoru Nishizawa Sakura Uto Tsuyoshi Takato Kazuto Hoshi Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3 Regenerative Therapy iPS cell Achondroplasia Genome editing Differentiation Chondrocyte |
author_facet |
Naohiro Horie Atsuhiko Hikita Satoru Nishizawa Sakura Uto Tsuyoshi Takato Kazuto Hoshi |
author_sort |
Naohiro Horie |
title |
Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3 |
title_short |
Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3 |
title_full |
Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3 |
title_fullStr |
Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3 |
title_full_unstemmed |
Impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3 |
title_sort |
impairment of the transition from proliferative stage to prehypertrophic stage in chondrogenic differentiation of human induced pluripotent stem cells harboring the causative mutation of achondroplasia in fibroblast growth factor receptor 3 |
publisher |
Elsevier |
series |
Regenerative Therapy |
issn |
2352-3204 |
publishDate |
2017-06-01 |
description |
Introduction: Achondroplasia (ACH) is a congenital disease which causes dwarfism and many symptoms resulting from skeletal dysplasia. Because present therapeutic strategies are mainly surgical procedures as symptomatic treatments, development of a radical treatment is desired. Clarification of the ACH pathology is essential for creating a new remedy. However, there are many questions about the disease mechanisms that have not been answered.
Methods: As a single base substitution of the FGFR3 gene had been proved to be the ACH causing genome mutation, our group established disease specific iPS cells by introducing the causative mutation of achondroplasia into human iPS cells by CRISPR/Cas9 based genome editing. These cells were differentiated towards chondrocytes, then the gene and protein expressions were examined by real time RT-PCR and Western blotting, respectively.
Results: Based on the western blotting analysis, the FGFR3 protein and phosphorylated ERK were increased in the FGFR3 mutated iPS cells compared to the control cells, while the FGFR3 gene expression was suppressed in the FGFR3 mutated iPS cells. According to chondrogenic differentiation experiments, the IHH expression level was increased in the control cells as the differentiation progressed. On the other hand, up-regulation of the IHH gene expression was suppressed in the FGFR3 mutated iPS cells.
Conclusions: These results suggested that chondrocyte maturation was impaired between the proliferative stage and prehypertrophic stage in the chondrocytes of ACH. The development of chemical compounds which affect the specific maturation stage of chondrocytes is expected to contribute to the ACH treatment, and FGFR3 genome-edited hiPSCs will be a valuable tool in such research studies. |
topic |
iPS cell Achondroplasia Genome editing Differentiation Chondrocyte |
url |
http://www.sciencedirect.com/science/article/pii/S2352320416300608 |
work_keys_str_mv |
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