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Human iPSCs Harbor Homoplasmic and Heteroplasmic Mitochondrial DNA Mutations while Maintaining hESC-Like Metabolic Reprogramming

Two sets of human iPSCs derived from neonatal foreskin fibroblasts were utilised; previously generated HFF1- iPSCs (lines iPS2 and iPS4) (Prigione et al) and BJ-iPSCs (lines iB4 and iB5) which were generated for the study and shown to be fully pluripotent. All four cell lines displayed a decreased mtDNA copy number compared to the parental cells, in agreement with previous findings (Prigione et al and Armstrong et al). Massively parallel pyrosequencing of mtDNA was carried out employing Roche 454 sequencing, allowing complete coverage and thus comparative analyses of the mitochondrial target region for each of the iPSCs as well as their parental fibroblasts, using the Cambridge Reference Sequence (CRS) for human mitochondrial DNA as a reference. The mitochondrial genome exhibits a polyploid nature as it exists in several copies within every single cell. Homoplasmy occurs when all copies of mtDNA are identical whereas heteroplasmy occurs when a mixture of two mtDNA genotypes exist. The heteroplasmic level is of significance as pathogenic phenotypes are believed to occur only when the number of mutated molecules reaches a certain level (DiMauro and Schon). Also of importance is replicative segregation, the unequal distribution of mtDNA variants which can occur during proliferative division and which characterizes the mitochondrial genetic bottleneck, a phenomenon in which mothers harbouring a mixture of mutated and wild-type mtDNAs transmit varying proportions of mutated and wild-type mtDNA to their offspring (Cree et al and Wai et al ).
The mitochondrial genome of each of the four iPSC lines did not exhibit signs of large-scale rearrangement, but several point mutations were detected within iPSCs compared to their parental cells although no constant changes between the cells were observed. Homoplasmic variants included single base substitutions, single base insertions, and single base deletions and while base substitutions were highly frequent within HFF1-iPSCs, other types of homoplasmic or heteroplasmic variants occurred at approximately equal numbers across all iPSC lines. With respect to the rCRS control mitochondrial sequence, fibroblasts contained mtDNA variants which were either maintained or lost during reprogramming, while iPSCs also acquired homoplasmic mtDNA mutations that were not present in the parental somatic cells, suggesting that the reprogramming process introduces changes to the mitochondrial genome. Reprogrammed cells frequently gained consistent changes in the level of heteroplasmy of heteroplasmic variants with regards to their parental cells (e.g. a heteroplasmic variant whose level of heteroplasmy was 45% in fibroblasts increased to 100% in iB4 and decreased to 0% in iB5) and only remained unaltered in the in a small percentage of cases (+/-5%). Furthermore, iPSCs exhibited heteroplasmic mutations that were not contained in the original fibroblast samples (e.g. at the nucleotide at position 16126 HFF1 fibroblasts were homoplasmic for T, while HFF1-iPSCs acquired heteroplasmic T>C mutations [48% C and 50% C in iPS2 and iPS4, respectively]).
mtDNA mutations within iPSCs affected both non-coding genes, including components of the control region (CR) or of the translational machinery, such as tRNAs, 12sRNA, and 16sRNA, and genes encoding for proteins of the electron transfer chain. Synonymous (not functionally relevant) changes occurred in all iPSCs while non-synonymous mutations (giving rise to an amino acid change and therefore possibly a functional outcome) affected genes encoding for proteins of all the four mitochondrial-encoded electron transfer chain complexes. Further analysis of the mtDNA variants demonstrated that while some were associated with diseases or cancer, others were novel (mostly heteroplasmies), but the majority were ‘common variants’ in the general population.

Interestingly, regardless of variations within the mtDNA sequence, all iPSCs acquired a hESC-like transcriptional and metabolic signature indicative of a metabolic transition from respirative to glycolytic metabolism as previously demonstrated (Prigione et al). Q-PCR analysis demonstrated an up-regulation of genes regulating the first (SLC2A3, GCK and HK3) and last steps (PGAM2, ENO1, PKLR, and LDH genes) of the glycolytic cascade in all iPSCs and hESCs in comparison to fibroblasts. The expression of glucose 6-phosphate downstream glycolytic enzymes (GPI, PFK genes, and ALDO genes) were however reduced in pluripotent stem cells compared to fibroblasts, which in conjunction suggests a possible accumulation of glucose 6-phosphate which may be diverted into the pentose phosphate pathway (PPP). PPP is an alternative to glycolysis and generates NADPH during an oxidative phase, followed by the non-oxidative synthesis of pentoses (5-carbon sugars). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that glucose 6-phosphate was significantly increased in all hESCs and iPSCs compared to fibroblasts, while dihydroxyacetone phosphate (DHAP) was reduced in all pluripotent stem cells, possibly implying an increased flux towards pyruvate generation, suggesting an active glycolytic pathway. Most of the genes encoding for enzymes of the gluconeogenesis pathway (MDH1B, FBP1, G6PC and PCK1) were up-regulated in hESCs and iPSCs, while genes of the mitochondrial oxygen-utilising tricarboxylic acid cycle (TCA) cycle (ACO, IDH2, SUCLG2, SDHA, SDHD and FH) were mainly down-regulated in hESCs and iPSCs in comparison to fibroblasts. Among the regulators of bioenergetic metabolism, PRKAA1 was downregulated in hESCs and iPSCs, (PRKKAA1 is induced with decreased ATP content in mitochondrial-defective cells (Prigione and Cortopassi)), while pyruvate dehydrogenase kinase (PDK1, 3, and 4), was up-regulated in hESCs and all iPSCs irrespective of mtDNA sequence variants. PDK1 inhibits pyruvate dehydrogenase which limits pyruvate entry into the TCA cycle, therefore blocking oxidative phosphorylation and perhaps shunting this into the PPP. Moreover, PDK1 protein levels decreased during subsequent differentiation into fibroblasts obtained from hESCs (H1 line), from iPSCs with a low mtDNA mutational load (iB5), and from iPSCs with a high mtDNA mutational load (iPS2 line). Overall this suggests that iPSC become hESC-like with regards to their metabolic signature and strongly suggests a switch from oxidative phosphorylation to glycolysis.
Subsequent analysis of the bioenergetic profile of iPSCs suggests that they are highly similar to hESCs and drastically distinct from their parental somatic cells, with no relation of iPSC bioenergetic profiles to differences in mtDNA variations. Total cellular ATP levels in iPSCs were equal to that of hESCs and reduced compared to fibroblasts, while simultaneous quantification of mitochondrial respiration (oxygen consumption rate [OCR]) and glycolysis (extracellular acidification rate or ECAR) revealed that hESCs and iPSCs with either a relatively low or high mitochondrial mutational load demonstrated a similar switch in metabolism. Furthermore, ATP turnover, maximal respiration rate, magnitude of the proton leak, amount of non-mitochondrial oxygen consumption and level of ATP reserves were also all similar between iPSCs with differing amounts of mtDNA variants. One noted difference however was the ATP reserve level in iPSCs, which was more similar to fibroblasts than hESCs suggesting that hESCs and iPSCs may respond differently to increased energy demands, which may have important implications for the further differentiation of these cells.
The overall picture suggests that while changes in the mitochondrial genome occur during iPSC generation, this does not impact on iPSC functionality. A more expansive study using different methods of reprogramming and different target cells may allow for a more detailed analysis, but perhaps the most relevant prospect is to study this effect in cells of patients with mitochondrial mutation-associated diseases. Examination of the mechanisms of bioenergetic profile reprogramming during iPSC generation would also be of great interest and the influence of mitochondrial mutations in cells undergoing differentiation process also warrants investigation.

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