Use Reprogramming Technology To Establish IPS Cell Disease Model Of Mitochondrial Genetic Disease MERRF

Stem cells are defined by self-renewal and pluripotency,and can be classified into groups including embryonic stem cell(ESCs),induced pluripotent stem cell(iPSCs)and adult stem cell,according to their resource.IPSCs can be generated from somatic cells by using a cocktail of four transcription factors(Oct4,Sox2,Klf4 and c-Myc)to another type of cells with a gene expression profile and developmental potential similar to ESCs.Different cytokines can make iPSCs differentiate into different types of somatic cells.In this way,iPSCs derived from patients could provide large quantities of disease-relevant cells and a variety of different cell types that were previously inaccessible,such as neurons and cardiomyocytes.The cells are used to reveal disease aetiology and to identify pathological mechanisms.Myoclonus epilepsy associated with ragged-red fibers(MERRF)is a genetic disorder caused by mitochondrial mutations.It is known that in most MERRF cases,m.A8344G mutation accounts for the genetic itology of the phenotypes.However,there is no effective treatment for these patients.Thus,a good disease model is required for studying the disease mechanism of this syndrome,which is currently not available.In the first part of this study,we established the platform for generating iPSCs from human PBMC and AFC.In the second part,we applied this technology to generate MERRF-iPSCs,and performed cardiac differentiation to generate an active disease model for MERRP.PART ? Generation of human induced pluripotent stem cellsfrom peripheral blood mononuclear cells and amniotic fluidcell using the non-integrating reprogramming techniqueObjective To construct the induced pluripotent stem cells(iPSCs)from human peripheral blood mononuclear cells(PBMCs)and amniotie fluid cell(AFCs)using the non-integrating reprogramming technique.Methods Peripheral blood samples from healthy persons were collected and the PBMCs were isolated.Then,the PBMCs and AFCs were infected with sendai virus containing four reprogramming factors-Oct3/4,Sox2,Klf4 and c-Myc,and the hPBMC-derived iPSCs and AFC-derived iPSCs were obtained by the culture system with feeder-free condition.The multipotency of the obtained hPBMC-derived iPSCs and AFC-derived iPSCs were verified by the observation of cell morphology,alkaline phosphatase(AKP)staining,immunofluorescence staining,and the embryoid body(EB)formation and differentiation assay,and its safety was evaluated by the karyotype assay and the detection of sendai virus genomic RNA.Results iPSCs-like colonies emerged at day 16 after the PBMCs and AFCs were infected with sendai virus,The obtained iPSCs were able to be cultured with a few passages,and were demonstrated to have the multipotency.More over,the hPBMC-derived iPSCs and AFC-derived iPSCs carried normal karyotype,and no any exogenous genomic RNA from sendai virus was detected in it.Conclusion A non-integrating reprogramming system for hPBMCs and AFCs was successfully constructed,and the iPSCs from hPBMCs and AFCs with multipotency,normal karyotype and the lack of exogenous genes were obtained.PART ? Generation of induced pluripotent stem cell linesfrom a MERRF patientObjective To generate induced pluripotent stem cell(iPSCs)from MERRF patient.Metheds Peripheral blood sample from one MERRF patient and amniotic fluid cell(AFCs)from proband were collected and peripheral blood mononuclear cells(PBMCs)were isolated.The PBMCs and the AFCs were infected with sendai virus containing four reprogramming factors-Oct4,Sox2,Klf4 and c-Myc.The pluripotency of the obtained patient-derived iPSCs was verified by cell morphology,immunofluorescence staining,the embryoid body(EB)formation and differentiation assay,and RT-PCR.Karyotypes of the iPSCs were revealed by GTG-banding assay.The ratios of mitochondrial carrying m.8344 point mutation in each cell lines were detected by pyrosequencing.The ATP synthesis level of different cell lines with different heterogeneity was detected by ATP detection kit.Finally,iPSCs were differentiated into cardiomyocytes.Myocardium-specific antibodies were used to identify differentiated cardiomyocytes by immunofluorescence staining.The beating frequency of the differentiated cardiac cells were counted to evaluate whether there is a difference in cardiac function between different myocardial cells carrying various mutant heteroplasmic level..Result MERRF patient-derived iPSCs and were demonstrated to have the pluripotency,and the karyotypes were proved to be normal.Among the 6 iPSCs lines of PBMC-derived,the ratios of mitochondrial carrying m.8344 mutant were 68.24%,60.51%,45.95%,24.06%,62.11%and 0%respectively.2 iPSCs lines of AFC-derived,the ratio of mitochondrial were 0%and 0%.The ATP synthesis level of the cell line with the level of heterogeneity of zero was 94 ?mol/gprot in the PBMC-iPSCs.The ATP synthesis level of the cell line with the highest level of heterogeneity was 66 ?mol/gprot.After successfully differentiating into cardiomyocytes,the level of heterogeneity of cardiomyocytes was detected by pyrosequencing.The heterplasmic level was not significantly altered comparing to the iPSCs respectively.The beating frequencies were found to be significantly different between myocardial cells derived from different MERRF-iPSCs,the trend of which is consistent with the one of heteroplasmic level of the mutant mitochondria.Conclusion We generated MERRF patient derived iPSCs lines by non-integrating reprogramming method.These iPSCs provide a great disease model for MERRF syndrome.