| Background: Wilson's disease(WD)is a rare autosomal recessive disorder of copper metabolism caused by an ATP7 B gene mutation.Except for hepatic,neurological symptoms,lower bone mineral density is another most frequent clinical features of WD,but the underlying mechanisms have not been fully understood.Induced pluripotent stem cells(iPSCs)has the advantages of self-renewal,disease specific,and potential to differentiate into various diseases-associated cell types.And it has no restrictions on ethical? religious and laws compared with embryonic stem cells.Based on these advantages,iPSCs incorporated into gene therapy can establish a cell model of some diseases such as WD by in vitro culture,and then provide new ideas and methods for the treatment of the corresponding diseases.Objective: Exploring the causes of low bone density in WD;Preliminary study on the mechanism of low osteogenic activity in WD;Screening small molecules to promote osteogenic differentiation of WD.Methods: Osteogenesis induction from both normal and WD iPSCs was performed by the embryoid bodies(EBs)formation method and addition of osteogenic medium.And we compared whether there is a difference in osteogenesis between the normal controls and WD from both genotype and phenotype by real-time quantification and Alzizarin Red staining.The gene expression profile of WD and normal iPSCs-induced osteoblasts were analyzed using RNA-Seq technology.And we compared the differential genes between normal controls and WD,and then analyzed the sequencing results to obtain that the low osteogenic activity in the WD may be associated with the ?-catenin pathway.Further,it is confirmed whether the ?-catenin pathway is involved in regulating the low osteogenic activity in the WD by western blot and immunofluorescence.Finally,we found that SKL2001,a small molecule compound that can stabilize intracellular ?-catenin by disrupting its interaction with axin.Further,we verified whether SKL2001 can promote osteogenic differentiation in the WD by real-time quantification,western blot and immunofluorescence.Results: Osteogenesis induction from both normal and WD iPSCs was performed by the embryoid bodies(EBs)formation method and addition of osteogenic medium.In all stages of osteogenic differentiation,the expression of the osteogenic marker gene in all stages of osteogenesis induction were significantly lower in the WD osteoblasts compared to the normal controls,and the mineralization level was significantly lower in the WD osteoblasts than the normal controls.Transcriptome sequencing suggested that there was a significant difference in ?-catenin pathway between osteoblasts from WD and healthy control.Then we found that the total protein expression of ?-catenin is lower in the WD osteoblasts compared to the normal controls by western blot,and immunofluorescence assays and western blot also revealed that the ?-catenin nuclear protein level of the WD on days 7 and 21 during osteogenic differentiation was significantly lower compared to the normal controls.Finally,we found that SKL2001,a small molecule compound,stabilizes intracellular ?-catenin by disrupting the interaction between ?-catenin and axin.The result showed that the expression of the osteogenic marker gene in the WD osteoblasts treated with SKL2001 was significantly higher than that WD controls by real-time quantification.And the nucleus of ?-catenin in the WD osteoblasts treated with SKL2001 was significantly higher than WD controls by western blot and immunofluorescence.But these did not show a dose-dependent.Conclusion: In this study,osteogenesis induction from both normal and WD iPSCs was performed by the embryoid bodies(EBs)formation method and addition of osteogenic medium,and for the first time,we found that WD-iPSCs derived osteoblasts demonstrated a decreased osteogenesis activity than their normal controls.Through gene expression profiling,we identified and validated that there was a significant difference in ?-catenin pathway between osteoblasts from WD and healthy control.Most meaningfully,when treating with SKL2001,a small molecule compound that can stabilize intracellular ?-catenin by disrupting its interaction with axin.,can reverse decreased osteogenesis of WD.Therefore,our above findings indicated that low bone density and osteoporosis of WD patients might be due to the decreased osteogenesis caused by aberrant ?-catenin signaling pathway,and these patients might benefit from ?-catenin targeting strategies to improve the imbalance on bone remodeling. |
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