Abstract
BackgroundAchondroplasia (ACH) is the most common genetic form of dwarfism and belongs to dominant monogenic disorder caused by a gain-of-function point mutation in the transmembrane region of FGFR3. There are no effective treatments for ACH. Stem cells and gene-editing technology provide us with effective methods and ideas for ACH research and treatment.MethodsWe generated non-integrated iPSCs from an ACH girl’s skin and an ACH boy’s urine by Sendai virus. The mutation of ACH iPSCs was precisely corrected by CRISPR-Cas9.ResultsChondrogenic differentiation ability of ACH iPSCs was confined compared with that of healthy iPSCs. Chondrogenic differentiation ability of corrected ACH iPSCs could be restored. These corrected iPSCs displayed pluripotency, maintained normal karyotype, and demonstrated none of off-target indels.ConclusionsThis study may provide an important theoretical and experimental basis for the ACH research and treatment.
Highlights
Achondroplasia (ACH) is the most common genetic form of dwarfism and belongs to dominant monogenic disorder caused by a gain-of-function point mutation in the transmembrane region of fibroblast growth factor receptor 3 (FGFR3)
Recently Perlingeiro and his colleagues used limb girdle muscular dystrophy type 2A patient-derived iPSCs which were caused by mutations in the Calpain 3 (CAPN3) to perform gene correction by CRIPR-Cas9, and transplanted gene-corrected myogenic progenitors into a mouse model that combined immunodeficiency with a lack of CAPN3
We found that the chondrogenic differentiation ability of ACH iPSCs was confined compared with that of healthy iPSCs
Summary
Identification, isolation and culture of somatic cells from ACH patients We recruited three ACH patients, including an 8-yearold girl, a 7-year-old boy and a 37-year-old adult male. After single colonies were picked up and expanded, iPSC lines from the girl’s skin (GF) and the boy’s urine (BU) were established (Fig. 2c, d) These iPSCs expressed pluripotent proteins, including NANOG, OCT4, SOX2, SSEA-4 and TRA1-60, and did not express SSEA-1 (Fig. 2g). We found that corrected iPSCs expressed pluripotent proteins, including NANOG, OCT4, SOX2, SSEA-4 and TRA1-60, did not express SSEA-1 (Fig. 4d), and stained positive for the AP activity (Fig. 4e). By Safranin O staining, we discovered that the cartilage tissues derived from ACH iPSCs showed fewer positive areas and lower cartilage density than those from healthy iPSCs (Fig. 3c). RT-qPCR results revealed that, compared with cartilage tissues from uncorrected ACH iPSCs, those from corrected ACH iPSCs expressed higher chondrocyte-specific genes—SOX9, COL2A1, and ACAN (Fig. 5c).
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