Generation Of Non-integration Induced Pluripotent Stem Cells From Chorionic Villi Cells Of A Duchenne Muscular Dystrophy Fetus

BackgroundDuchenne muscular dystrophy(DMD)is the most common X-linked recessive genetic disease.The incidence rate of DMD is approximately 1/3500 in males born.In males affected with DMD,symptoms such as progressive muscle weakness manifest around the age of 2-5 and finally resulting in the inability to walk around the age of 12.The patients finally die of cardiopulmonary failure caused by the weakness of heart and respiratory muscles.Clinically,regular glucocorticoid therapy is considered as the main therapy for DMD.However,the prognosis in patients with DMD has not been improved.Currently,gene therapy is the research hotspot for DMD therapy.Since Yamanaka discovered that four factors,namely Oct4?Sox2?Klf4 and c-Myc,can reprogram various somatic cells to induced pluripotent stem cells with multilineage differentiation potential.And patient-derived iPSCs have been considered as great cell models.At present,there are many non-integration induction systems such as Sendai virus,episome-adjuvant vector,microRNA,and recombinant protein,which are used in the reprogramming of human fibroblasts,urine cells,blood cells,and amniotic fluid cells.However,the induced pluripotent stem cells drived from fetal villi cells with non-integration induction systems are still not established.In this study,chronic vill cells with the nonsense mutation were electroporated with oriP/EBNA1-based vectors plasmids(pEP4-EO2S-ET2K),which express OCT4,SOX2,SV40 LT and KLF4,and miRNA cluster 302-367 vecotor plasmid(pCEP4-miR-302-367 cluster)which express miRNA 302-367 cluster to promote the reprogramming efficiency.Identified DMD-CV-iPSCs were the suitable cell model in the researches on DMD gene therapy and DMD pathogenesis.ObjectivesTo construct and identify the system of non-integration induced pluripotent stem cell line derived from fetal chorionic villi cells with a DMD gene mutation,which can be considered as great models in the followed DMD gene editing research.MethodsAfter the pregnant woman and her family agreed and signed the informed consent,chorionic villi cells were obtained from a fetus with a point nonsense mutation c.4583 del in the exon33.The chronic vill cells with the nonsense mutation at passage 7 were electroporated with oriP/EBNA1-based vectors plasmids(pEP4-EO2S-ET2K),which express OCT4,SOX2,SV40 LT and KLF4,and miRNA cluster 302-367 vecotor plasmid(pCEP4-miR-302-367 cluster)which express miRNA 302-367 cluster to promote the reprogramming efficiency.Chromosome karyotype analysis,STR linkage analysis and Sanger sequencing were used to determine whether the DMD-CV-iPSCs were drived from the origin of villi cells.Alkaline phosphatase staining,immunofluorescence staining and teratoma differentiation experiments in vivo were used to determine the multi-directional differentiation of the DMD-CV-iPSCs in this study.ResultsIn this study,DMD-CV-iPSCs cell lines derived from chorionic villi cells a nonsense mutation c.4583 delA in exon33 of the DMD gene were established.Karyotype analysis,STR linkage analysis,and Sanger sequencing results of DMD-CV-iPSCs were consistent with the fetal villus cells,which indicated that DMD-CV-iPSCs were derived from fetal villus cells.DMD-CV-iPSCs clones were positive for alkaline phosphatase staining.Immunofluorescence staining showed that the DMD-CV-iPSCs expressed pluripotency markers Oct4,SSEA4,TRA-1-60 and TRA-1-81.Teratoma formation in vivo found that the DMD-CV-iPSCs had multi-directional differentiation in vivo.ConclusionChorionic villi cells with DMD gene mustation can be reprogrammed into induced pluripotent stem cells with multi-directional differentiation potential by episomal vector plasmids.