The Effects of Nuclear Reprogramming on Mitochondr

Authors: Richard D W Kelly Huseyin Sumer Matthew McKenzie Joao FacuchoOliveira Ian A Trounce Paul J Verma Justin C St John

Publish Date: 2011/10/13

Volume: 9, Issue: 1, Pages: 1-15

PDF Link

Abstract

Undifferentiated mouse embryonic stem cells ESCs possess low numbers of mitochondrial DNA mtDNA which encodes key subunits associated with the generation of ATP through oxidative phosphorylation OXPHOS As ESCs differentiate mtDNA copy number is regulated by the nuclearencoded mtDNA replication factors which initiate a major replication event on Day 6 of differentiation Here we examined mtDNA replication events in somatic cells reprogrammed to pluripotency namely somatic cellES SCES somatic cell nuclear transfer ES NTES and induced pluripotent stem iPS cells all at lowpassage MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SCES and NTES cells had significantly increased levels which correlated positively and negatively with Nanog and Sox2 expression respectively During pluripotency and differentiation the expression of the mtDNAspecific replication factors PolgA and Peo1 were differentially expressed in iPS and SCES cells when compared to ESCs Throughout differentiation reprogrammed somatic cells were unable to accumulate mtDNA copy number characteristic of ESCs especially on Day 6 In addition iPS and SCES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14 The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation 5Azacytidine prior to differentiation enabled iPS cells but not SCES and NTES cells to accumulate mtDNA copies per cell in a manner similar to ESCs These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be reestablished through the use of epigenetic modifiersThis work is supported by funding from the British Heart Foundation PG/04/117 and the Medical Research Council UK grant number G0600273 and the Victorian Government’s Operational Inftrastructure Support Program We are grateful to Dr Megan Munsie Australian Stem Cell Centre for the somatic cell nuclear transfer embryonic stem cell line NTES and Ms Jacqui Johnson Centre for Reproduction and Development Monash Institute of Medical Research for expertise in stem cell cultureThe effects of somatic cell reprogramming on ATP content and steady state levels of OXPHOS complexes in undifferentiated pluripotent cells A ATP levels were analysed in MEF ESC iPS 2 SCES 1 and ESES pluripotent stem cells in normal ESC media or media supplemented with 1 μg/ul R6G for 72 hrs The values are relative luminescent units RLU normalised to ESCs from three independent experiments Bars represent means ± sem significant differences between cell types are indicated P  005 B Treatment of pluripotent cells with R6G for 72 hrs to decrease mtDNA copy number Graph shows mtDNA copy number as a percentage of untreated cells C Steady state levels of CIIV from undifferentiated ESCs SCES iPS ESES NTES and MEF cells were resolved by BlueNative Polyacrylamide gel electrophoresis BNPAGE and probed with antibodies against NADH dehydrogenase 1 alpha subcomplex subunit 9 CI succinate dehydrogenase flavoprotein subunit CII cytochrome bc1 complex subunit 1 CIII2 cytochrome c oxidase subunit 1 CIV JPEG 231 kbThe effects of somatic cell reprogramming on the expression of mtDNAspecific replication factors in undifferentiated cells Expression of PolgA Twinkle Peo1 mtSsbp1 PolgB and Tfam was analysed by quantitative realtime PCR in undifferentiated mouse iPS cells lines 1–3 SCES cells lines 1–4 ESES cells the parental MEF cell line QS/Rosa26 and the parental ESC D3s All samples were normalised to βactin Bars represent means ± sem significant differences between cell types are indicated P  005 P  001 P  0001 JPEG 309 kbTotal ATP and steady state OXPHOS subunit levels in reprogrammed cells during in vitro differentiation ATP levels were analysed in differentiating mouse ESC iPS 2 SCES 1 and ESES cells on A Days 7 and B 14 The values are relative luminescent units RLU normalised to ESCs Bars represent means ± sem significant differences between cell types are indicated P  005 P  001 C Total cell proteins were isolated and separated by BNPAGE under nondenaturing conditions and probed with antibodies against NADH dehydrogenase ubiquitin 1 alpha subcomplex subunit 9 CI succinate dehydrogenase flavoprotein subunit CII cytochrome bc1 complex subunit 1 CIII2 cytochrome c oxidase subunit 1 CIV JPEG 259 kb

Keywords: