A Preliminary Study On The Mechanism Of Skeletal Abnormalities In Turner Syndrome Using Inducing Pluripotent Stem Cells (iPSCs)-based Disease Models

Background: Turner syndrome(TS)is caused by the complete or partial deletion of an X chromosome in all or part of a cell,and the incidence is 1/2500.Short stature and osteoporosis are prominent clinical manifestations in patients with Turner syndrome.The biological characteristics of induced pluripotent stem cells(iPSCs)are similar to those of embryonic stem cells,which can express pluripotent stem cell markers and have the ability to differentiate into three germ layers.Therefore,we can use diseased cells to establish disease models and explore the mechanism of disease occurrence.iPSCs have opened up new ideas and methods for the study of Turner syndrome.Objective: By inducing pluripotent stem cells from patients with Turner syndrome and induced pluripotent stem cells from normal humans into osteoblasts and osteoclasts,to analyze whether there are abnormalities in the phenotype of osteoblasts and osteoclasts in patients with Turner syndrome.Initially explore the causes of skeletal abnormalities in Turner syndrome.Methods: Two iPSCs are mechanically formed into embryoid bodies(EBs),inducing osteoblasts through the development of mesoderm,and we analyze genetic differences in the osteogenic marker genes ALP,RUNX2,OCN,COL1A1 using real-time quantitative polymerase chain reaction.The number of calcium nodules produced on the surface of osteoblasts induced by iPSCs was observed by alizarin red staining;Similarly,we induce osteoclast formation by forming EBs.We used RT-qPCR to analyze the markers of osteoclasts-TRAP,Mmp9,CA2,OSCAR,at the same time,TRAP staining was used to analyze abnormalities on the phenotype.Results: We successfully produced functional osteoblasts and osteoclasts from iPSCs through embryonic bodies(EBs)and mesoderm stages,showing obvious mineralized nodules and multinucleated giant cells with tartrate-resistant acid phosphatase(TRAP)staining.In the analysis of osteoblast marker genes,we found that the marker genes of osteoblasts in patients with Turner syndrome were not down-regulated or even slightly elevated compared with normal people,but there is no difference in the mineralized nodules produced by alizarin red staining;The marker gene of osteoclasts in patients with Turner syndrome has increased,and the results of TRAP staining were consistent with real-time quantitative results.Conclusion:(1)We applied techniques that induce pluripotent stem cells to induce osteoblasts and osteoclasts.The induced pluripotent stem cells of normal human and Turner syndrome patients were cultured into osteoblasts and osteoclasts,providing research materials for subsequent disease research.(2)By inducing iPSCs in TS patients as osteoblasts and osteoclasts,it was initially found that there was a difference in osteoclasts in patients with Turner syndrome,which needs further verification in the future.