Using a novel single-molecule PCR approach to quantify the total burden of mitochondrial DNA (mtDNA) molecules with deletions, we show that a high proportion of individual pigmented neurons in the aged human substantia nigra contain very high levels of mtDNA deletions. Molecules with deletions are largely clonal within each neuron; that is, they originate from a single deleted mtDNA molecule that has expanded clonally. The fraction of mtDNA deletions is significantly higher in cytochrome c oxidase (COX)-deficient neurons than in COX-positive neurons, suggesting that mtDNA deletions may be directly responsible for impaired cellular respiration.

Key Points• Human hematopoietic cells develop within human iPSCderived teratomas in immunodeficient mice.• Co-transplantation of OP9 stromal cells along with human iPSCs increases hematopoietic specification within teratomas.

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MicroRNA-10b (miR-10b) is a unique oncogenic miRNA that is highly expressed in all GBM subtypes, while absent in normal neuroglial cells of the brain. miR-10b inhibition strongly impairs proliferation and survival of cultured glioma cells, including glioma-initiating stem-like cells (GSC). Although several miR-10b targets have been identified previously, the common mechanism conferring the miR-10b-sustained viability of GSC is unknown. Here, we demonstrate that in heterogeneous GSC, miR-10b regulates cell cycle and alternative splicing, often through the non-canonical targeting via 5 0 UTRs of its target genes, including MBNL1-3, SART3, and RSRC1. We have further assessed the inhibition of miR-10b in intracranial human GSC-derived xenograft and murine GL261 allograft models in athymic and immunocompetent mice. Three delivery routes for the miR-10b antisense oligonucleotide inhibitors (ASO), direct intratumoral injections, continuous osmotic delivery, and systemic intravenous injections, have been explored. In all cases, the treatment with miR-10b ASO led to targets' derepression, and attenuated growth and progression of established intracranial GBM. No significant systemic toxicity was observed upon ASO administration by local or systemic routes. Our results indicate that miR-10b is a promising candidate for the development of targeted therapies against all GBM subtypes.

Using single-cell sequence analysis, we discovered that a high proportion of cells in tissues as diverse as buccal epithelium and heart muscle contain high proportions of clonal mutant mtDNA expanded from single initial mutant mtDNA molecules. We demonstrate that intracellular clonal expansion of somatic point mutations is a common event in normal human tissues. This finding implies efficient homogenization of mitochondrial genomes within individual cells. Significant qualitative differences observed between the spectra of clonally expanded mutations in proliferating epithelial cells and postmitotic cardiomyocytes suggest, however, that either the processes generating these mutations or mechanisms driving them to homoplasmy are likely to be fundamentally different between the two tissues. Furthermore, the ability of somatic mtDNA mutations to expand (required for their phenotypic expression), as well as their apparently high incidence, reinforces the possibility that these mutations may be involved actively in various physiological processes such as aging and degenerative disease. The abundance of clonally expanded point mutations in individual cells of normal tissues also suggests that the recently discovered accumulation of mtDNA mutations in tumors may be explained by processes that are similar or identical to those operating in the normal tissue. somatic mutation ͉ clonal expansion ͉ single cell ͉ aging ͉ cancer

Perfect direct repeats and, in particular, the prominent 13-bp repeat, are thought to cause mitochondrial DNA (mtDNA) deletions, which have been associated with the aging process. Accordingly, individuals lacking the 13-bp repeat are highly prevalent among centenarians and the number of repeats negatively correlates with mammalian longevity. However, detailed examination of the distribution of mtDNA deletions challenges the role of the 13-bp repeat and other perfect repeats in generating mtDNA deletions. Instead, deletions appear to depend on long and stable, albeit imperfect, duplexes between distant mtDNA segments. Furthermore, significant dissimilarities in breakpoint distributions suggest that multiple mechanisms are involved in creating mtDNA deletions. Direct repeats in the mitochondrial genome and longevityThe premise that accumulation of mtDNA mutations [1] and, in particular, of large-scale deletions in mtDNA is one of the possible causes of aging has received substantial support from biochemical and longevity studies [2], [3], [4], [5]. mtDNA deletions are usually flanked by direct repeats, implying that these repeats are involved in the generation of deletions. Recombination [6], [7], slip-replication [8], and double-stranded break repair [9] have been suggested as potential alternative mechanisms involving direct repeats. In corroboration of the connection between mtDNA deletions and aging, the number of direct repeats in mtDNA of various mammal species is inversely correlated with longevity [10], [11]. Of particular interest is the so-called "common deletion" [6], the deletion most frequently detected in humans, which is flanked by a prominent 13-bp perfect direct repeat.Corresponding author: Konstantin Khrapko (kkhrapko@gmail.com). * XG and KP equally contributed to the study Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Interestingly, the carriers of the well studied D4a mitochondrial haplogroup, who are significantly enriched among Japanese centenarians [12], lack the 13-bp direct repeat in their mtDNA, and thus presumably lack the common deletion, which seems to support the premise that deletions are involved in the aging process [13]. It should be noted, however, that although the "common" deletion is the most abundant mtDNA deletion, it typically constitutes no more than 10% of all deletions in aging tissues [5]. Therefore D4a individuals would have at most 10% fewer deletions, which perhaps is too moderate a change to affect longevity. There is another possibility, though [13]. According to an elegant hypothesis by Samuels, Schon and Chinnery...

A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future.

SummaryRecent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice.