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The Effects of Nuclear Reprogramming on Mitochondrial DNA Replication

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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 nuclear-encoded 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 cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES 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 SC-ES 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, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES 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 re-established through the use of epigenetic modifiers.

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Acknowledgements

This 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 (NT-ES); and Ms Jacqui Johnson, Centre for Reproduction and Development, Monash Institute of Medical Research for expertise in stem cell culture.

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The authors indicate no potential conflicts of interest.

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  1. Joao Facucho-Oliveira

    Present address: Centre for Molecular and Structural Biomedicine, FCT Bld. 8, Campus of Gambelas, University of Algarve, Faro, Portugal

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  1. Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria, 3168, Australia

    Richard D. W. Kelly, Huseyin Sumer, Matthew McKenzie, Paul J. Verma & Justin C. St. John

  2. Mitochondrial and Reproductive Genetics Group, Clinical Sciences Research Institute, Warwick Medical School, The University of Warwick, Clifford Bridge Road, Coventry, CV2 2DX, UK

    Richard D. W. Kelly, Joao Facucho-Oliveira & Justin C. St. John

  3. Centre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia

    Ian A. Trounce

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Figure S1

The 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, SC-ES 1, and ES-ES 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 ± s.e.m; significant differences between cell types are indicated (* P < 0.05). (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 CI-IV from undifferentiated ESCs, SC-ES, iPS, ES-ES, NT-ES and MEF cells were resolved by Blue-Native Polyacrylamide gel electrophoresis (BN-PAGE) and probed with antibodies against: NADH dehydrogenase 1 alpha subcomplex subunit 9 (CI); succinate dehydrogenase flavoprotein subunit (CII); cytochrome b-c1 complex subunit 1 (CIII2); cytochrome c oxidase subunit 1 (CIV). (JPEG 231 kb)

High resolution image (TIFF 1013 kb)

Figure S2

The effects of somatic cell reprogramming on the expression of mtDNA-specific replication factors in undifferentiated cells. Expression of PolgA, Twinkle (Peo1) mtSsbp1, PolgB and Tfam was analysed by quantitative real-time PCR in undifferentiated mouse iPS cells (lines 1–3), SC-ES cells (lines 1–4), ES-ES cells, the parental MEF cell line (QS/Rosa26) and the parental ESC D3s. All samples were normalised to β-actin. Bars represent means ± s.e.m; significant differences between cell types are indicated (* P < 0.05, ** P < 0.01, *** P < 0.001). (JPEG 309 kb)

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Figure S3

Total ATP and steady state OXPHOS subunit levels in reprogrammed cells during in vitro differentiation. ATP levels were analysed in differentiating mouse ESC, iPS 2, SC-ES 1 and ES-ES cells on (A) Days 7 and (B) 14. The values are relative luminescent units (RLU) normalised to ESCs. Bars represent means ± s.e.m; significant differences between cell types are indicated (* P < 0.05; ** P < 0.01). (C) Total cell proteins were isolated and separated by BN-PAGE under non-denaturing conditions and probed with antibodies against: NADH dehydrogenase (ubiquitin) 1 alpha subcomplex subunit 9 (CI); succinate dehydrogenase flavoprotein subunit (CII); cytochrome b-c1 complex subunit 1 (CIII2); cytochrome c oxidase subunit 1 (CIV). (JPEG 259 kb)

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Kelly, R.D.W., Sumer, H., McKenzie, M. et al. The Effects of Nuclear Reprogramming on Mitochondrial DNA Replication. Stem Cell Rev and Rep 9, 1–15 (2013). https://doi.org/10.1007/s12015-011-9318-7

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