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A Role for Mitochondrial Genetics in ESCs Described

Original article from STEM CELLS

"Mitochondrial DNA Haplotypes Define Gene Expression Patterns in Pluripotent and Differentiating Embryonic Stem Cells"

The mitochondrial genome (mtDNA) encodes some of the key subunits of the electron transfer chain (ETC), essential for the generation of ATP through oxidative phosphorylation.    mtDNA haplotypes have evolved which lead to the existence of several different phenotypes (see paper for extended references), although what links mtDNA haplotypes and the mature phenotype is relatively unknown.   A great deal of research has however suggested that mtDNA is integral to cell differentiation and thus the cell phenotype (Crespo et al, Inoue et al, Mandal et al and Wang et al).   The effect of mtDNA haplotypes can be studied through the generation of cytoplasmic hybrids (cybrids) which can be made to have different mtDNA haplotypes through the use of divergent strains or species of animal (Kenyon and Moraes, McKenzie and Trounce and McKenzie et al).   Now, researchers from the laboratory of Justin C. St. John at Monash Institute of Medical Research, Monash University, Victoria, Australia, using mouse embryonic stem cell lines (mESCs) harbouring mitochondrial DNA haplotypes from different species, have found haplotype-specific expression of genes involved in pluripotency, differentiation, mitochondrial energy metabolism, and DNA methylation and their influence on differentiation is reported (Kelly et al).

Initial comparisons concentrated on pluripotency-associated gene expression differences between the mESC line from Mus musculus (CC9.3.1 cells; CC9mus) and ESC lines with same chromosomal genome but with mtDNA from Mus spretus (CC9spretus) or Mus terricolor (CC9dunni). This showed a general deregulation of pluripotency associated gene expression which was linked to DNA methylation and changes in expression of DNA methyltransferases (Dnmts). Differences in mtDNA copy number were noted, although alterations in ATP generation or oxygen consumption were not; all cells actively respired at their maximal capacity and also produced the same levels of lactate, an indicator of anaerobic metabolism. However, expression of nuclear- and mtDNA-encoded subunits of the ETC were significantly altered between the three lines.

Moving to analysis of early differentiation, embryoid bodies (EBs) were generated which demonstrated that their size, proliferation rate and ATP consumption were similar, while no differences were observed for resting O2 consumption rates, ETC coupling, or ETC capacity among the three cell lines. Lactate production fell, in line with changes to ATP generation mode, but no differences between cells were observed. Differentiation-associated gene expression patterns using a cell lineage identification qPCR array did however show mRNA expression differences between the three cell lines at day 7 of differentiation. Cardiomyocyte differentiation, as measured by number of beating EBs, was also altered with CC9spretus cells producing less beating foci than CC9mus and CC9dunni cells at day 7. The number of beating foci increased at day 12 but the differences between the cell types remained, with CC9dunni cells producing more beating EBs than CC9mus cells. Further long term analysis of differentiation (day 18 EBs) found that, compared to CC9mus cells, CC9spretus cells differentially expressed 31 lineage-specific genes while CC9dunni cells differentially expressed 18 genes. Markers of mesodermal and endodermal terminal differentiation were preferentially under-expressed in CC9spretus cells, while CC9dunni cells also under-expressed mesoderm and endoderm markers of terminal differentiation, as well as overexpressing immature progenitor and germ layer markers. Induced neural differentiation (ectoderm) demonstrated that levels of expression of the master regulators Sox2, Sox3, Musashi 1, and Pax6, and endpoint markers Tubb3 and Syp were significantly different between the three cell lines, with CC9dunni cells being the most divergent. The substantial changes in gene expression at day 18 were also accompanied by some changes to respiration, with lactate production increased in CC9dunni cells compared to CC9mus and CC9spretus cells suggesting a greater utilization of glycolysis.

Subsequent analysis focused on the role of a 3D environment on the differentiation potential of different mtDNA haplotypes through the culture of cells on nonwoven electrospun PCL scaffolds (Nisbet et al). Cells were differentiated as EBs and then seeded either onto polystyrene culture dishes (2D) or electrospun scaffolds (3D), and gene expression analysis after a total of 7 days of differentiation found Oct4, Sox2, and Nanog for each of the lines in the 3D environment to be at a higher level than when compared to the 2D environment. Under 2D conditions pluripotency gene expression was higher in the CC9pretus cells, while in 3D conditions, only Oct4 was at a higher level. Dnmt gene expression was also altered between 2D and 3D conditions with Dnmt1, 3a and 3b more highly expressed under 3D conditions. On day 18 of differentiation, in 2D conditions, mesodermal, ectodermal and endodermal gene expression was higher in CC9pretus cells and lowest in CC9dunni cells, while under 3D conditions, CC9dunni cells expressed higher levels of these genes with CC9spretus cells being the lowest expressers. Genes encoding nuclear- and mtDNA-encoded subunits of the ETC were also increased under 3D conditions but, again, different cell types expressed differing levels of these genes when comparing 2D vs. 3D growth conditions. Additionally Glut1 and Gapdh expression (associated with glycolysis) was increased under 3D conditions, especially in the CC9dunni cells. Overall gene expression results found that will on day 7 no differences were observed between CC9mus, CC9spretus, or CC9dunni cells in either the 2D or 3D environments, but by day 18, when cultured in 2D, the CC9spretus and CC9dunni cells displayed significantly reduced levels of gene expression. However, under 3D growth conditions, CC9spretus and CC9dunni cells had significantly altered gene expression, with CC9dunni cells having higher levels of expression.

Overall, these detailed findings suggest that mtDNA haplotype can greatly influence gene expression and DNA methylation in mESCs, which can influence cellular differentiation. Furthermore, gene expression changes are observed in both 2D and 3D culture systems although the effects in 3D are more pronounced and the 2D and 3D outcomes are dissimilar, suggesting an intricate interplay between mitochondrial genetics and environmental factors leading to altered differentiation propensities. Furthermore, this data proves that crosstalk between mtDNA and the nucleus is bidirectional with each genome affecting the other. The next step is to understand the mechanisms behind this crosstalk and how mitochondrial haplotypes affect specific signalling pathways leading to the observed alterations in gene expression and DNA methylation. Furthermore, systems like these may also be used to study how small changes in metabolism can influence ESC differentiation propensities.

References

  • Crespo FL et al. Mitochondrial reactive oxygen species mediate cardiomyocyte formation from embryonic stem cells in high glucose. Stem Cells 2010; 28: 1132–1142.
  • Inoue S K et al. Mitochondrial respiration defects modulate differentiation but not proliferation of hematopoietic stem and progenitor cells. FEBS Lett 2010; 584: 3402–3409.
  • Kenyon L, Moraes CT. Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids. Proc Natl Acad Sci USA 1997; 94: 9131–9135.
  • Mandal S et al. Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells. Stem Cells 2011; 29: 486–495.
  • McKenzie M et al. Production of homoplasmic xenomitochondrial mice. Proc Natl Acad Sci USA 2004; 101: 1685–1690.
  • McKenzie M, Trounce I. Expression of Rattus norvegicus mtDNA in Mus musculus cells results in multiple respiratory chain defects. J Biol Chem 2000; 275: 31514–31519.
  • Nisbet DR et al. Characterization of neural stem cells on electrospun poly(epsilon-caprolactone) submicron scaffolds: Evaluating their potential in neural tissue engineering. J Biomater Sci Polym Ed 2008; 19: 623–634.
  • Wang W et al. Mitochondrial DNA damage level determines neural stem cell differentiation fate. J Neurosci 2011; 31: 9746–9751.

STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.