In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells.

Lineage-restricted cells can be reprogrammed to a pluripotent state known as induced pluripotent stem (iPS) cells through overexpression of 4 transcription factors. iPS cells are similar to human embryonic stem (hES) cells and have the same ability to generate all the cells of the human body, including blood cells. However, this process is extremely inefficient and to date has been unsuccessful at differentiating iPS into hematopoietic stem cells (HSCs). We hypothesized that iPS cells, injected into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ immunocompromised (NSG) mice could give rise to hematopoietic stem/progenitor cells (HSPCs) during teratoma formation. Here, we report a novel in vivo system in which human iPS cells differentiate within teratomas to derive functional myeloid and lymphoid cells. Similarly, HSPCs can be isolated from teratoma parenchyma and reconstitute a human immune system when transplanted into immunodeficient mice. Our data provide evidence that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation, and drug screening applications.

Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons.

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.

Repeats, longevity and the sources of mtDNA deletions: evidence from 'deletional spectra'.

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 overall number of perfect repeats in mammalian mitochondrial genomes negatively correlates with species' longevity. However, detailed examination of the distribution of mtDNA deletions challenges a special role of the 13 bp repeat 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.

Clonally expanded mitochondrial DNA deletions within the choroid plexus in multiple sclerosis.

Mitochondrial DNA deletions (∆-mtDNA) have been implicated in the pathogenesis of Alzheimer's disease (AD), multiple sclerosis (MS) and Parkinson's disease (PD), as well as ageing. Clonal expansion of ∆-mtDNA is the process by which a mutant mtDNA molecule increases to high levels within a single cell containing both wild-type and mutant mtDNA. Unlike in AD and PD, the diffuse inflammatory process in MS involves the choroid plexus, and mitochondria are exposed to reactive oxygen and nitrogen species over a prolonged period. We determined the extent of respiratory enzyme deficiency and ∆-mtDNA at a single cell level within choroid plexus epithelial cells in MS as well as in AD, PD and controls. The respiratory enzyme-deficient (lacking complex IV and with intact complex II activity) cells were more prevalent within the choroid plexus in AD, MS and PD compared with controls. The main catalytic subunit of complex IV (subunit-I of cytochrome c oxidase) was lacking in significantly more respiratory enzyme-deficient cells in MS compared with AD, PD and controls. The single cell analysis showed a fourfold increase in the percentage of respiratory enzyme-deficient choroid plexus epithelial cells harbouring clonally expanded ∆-mtDNA in MS. Our findings establish clonal expansion of ∆-mtDNA as a feature relatively more prominent within the choroid plexus epithelium in MS than AD, PD or controls. We propose clonal expansion of ∆-mtDNA as a molecular link between inflammation and part of a delayed cellular energy failure in MS.

Mitochondrial Pseudogenes Suggest Repeated Inter-Species Hybridization among Direct Human Ancestors.

The hypothesis that the evolution of humans involves hybridization between diverged species has been actively debated in recent years. We present the following novel evidence in support of this hypothesis: the analysis of nuclear pseudogenes of mtDNA ("NUMTs"). NUMTs are considered "mtDNA fossils" as they preserve sequences of ancient mtDNA and thus carry unique information about ancestral populations. Our comparison of a NUMT sequence shared by humans, chimpanzees, and gorillas with their mtDNAs implies that, around the time of divergence between humans and chimpanzees, our evolutionary history involved the interbreeding of individuals whose mtDNA had diverged as much as ~4.5 Myr prior. This large divergence suggests a distant interspecies hybridization. Additionally, analysis of two other NUMTs suggests that such events occur repeatedly. Our findings suggest a complex pattern of speciation in primate/human ancestors and provide one potential explanation for the mosaic nature of fossil morphology found at the emergence of the hominin lineage. A preliminary version of this manuscript was uploaded to the preprint server BioRxiv in 2017 (10.1101/134502).

Mitochondrial DNA deletions in mice in men: substantia nigra is much less affected in the mouse.

Mitochondrial DNA (mtDNA) deletions have been reported to accumulate to high levels in substantia nigra of older humans, and these mutations are suspected of causing age-related degeneration in this area. We have compared levels of mtDNA deletions in humans and mice and report here that levels of deletions in the mouse are very significantly lower than in humans. While human mtDNA from substantia nigra contained more than 5% of deleted molecules, mouse substantia nigra contained less than 0.5%. These results imply that mtDNA deletions are unlikely to play any significant role in of murine substantia nigra aging and further call for caution in using mouse models in studies of the role of mtDNA deletions in aging and neurodegeneration. On a more general note, these results support the view that critical targets of the various aging processes may differ significantly between distant species.

Collection of isolated cells for studying mitochondrial DNA mutations within individual cells.

Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. This chapter outlines the methods for collecting individual cells appropriate for analysis of mtDNA mutations by single-molecule PCR. In addition, we describe the protocols for respiratory complexes II and IV in these cells. Together, the identification of respiratory deficiency and mtDNA mutations within single cells provides a powerful means of evaluating the importance of these events in disease and aging.

Quantitative analysis of somatic mitochondrial DNA mutations by single-cell single-molecule PCR.

Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an ongoing mutation process. Single-molecule PCR (smPCR) has been shown to be an effective method that can be applied to all areas of quantitative studies. It has distinct advantages over conventional vector-based cloning techniques avoiding the well-known PCR-related artifacts such as the introduction of artificial mutations, preferential allelic amplifications, and "jumping" PCR. smPCR is a straightforward and robust method, which can be effectively used for molecule-by-molecule mutational analysis, even when mitochondrial whole genome (mtWG) analysis is involved. This chapter describes the key features of the smPCR method and provides three examples of its applications in single-cell analysis: di-plex smPCR for deletion quantification, smPCR cloning for clonal point mutation quantification, and smPCR cloning for whole genome sequencing (mtWGS).

Does premature aging of the mtDNA mutator mouse prove that mtDNA mutations are involved in natural aging?

Recent 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.

Amelioration of premature aging in mtDNA mutator mouse by exercise: the interplay of oxidative stress, PGC-1α, p53, and DNA damage. A hypothesis.

The mtDNA mutator mouse lacks the proofreading capacity of the sole mtDNA polymerase, leading to accumulation of somatic mtDNA mutations, and a profound premature aging phenotype including elevated oxidative stress and apoptosis, and reduced mitochondrial function. We have previously reported that endurance exercise alleviates the aging phenotype in the mutator mice, reduces oxidative stress, and enhances mitochondrial biogenesis. Here we summarize our findings, with the emphasis on the central role of p53 in these adaptations. We demonstrate that mtDNA in sedentary and exercised PolG mice carry similar amounts of mutations in muscle, but in addition to that sedentary mice have more non-mutational damage, which is mitigated by exercise. It follows therefore that the profound alleviation of the mtDNA mutator phenotype in muscle by exercise may not require a reduction in mtDNA mutational load, but rather a decrease of mtDNA damage and/or oxidative stress. We further hypothesize that the observed 'alleviation without a reduction of mutational load' implies that the oxidative stress in PolG muscle is maintained, at least in part, by the 'malicious cycle', a hypothetical positive feedback potentially driven by the 'transcriptional mutagenesis', that is the conversion of chemically modified nucleotides into mutant RNA bases by the mitochondrial RNA polymerase.

Quantitation of Mitochondrial DNA Deletions Via Restriction Digestion/Long-Range Single-Molecule PCR.

Quantification of deletions in mtDNA is a long-standing problem in mutational analysis. We describe here an approach that combines the power of single-molecule PCR of the entire mitochondrial genome with the enrichment of the deletions by restriction digestion. This approach is indispensable if information about wide range of deletion types in a sample is critical, such as in studies concerning distribution of deletion breakpoints (as opposed to approaches where fraction of a single deletion or a limited set of deletions is used as a proxy for total deletion load). Because deletions in a sample are quantified almost exhaustively, the other important application of this approach involves studies where only small amounts of tissue, such as biopsies, are available.

Therapeutic potential of targeting microRNA-10b in established intracranial glioblastoma: first steps toward the clinic.

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'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.

mtLOH (mitochondrial loss of heteroplasmy), aging, and 'surrogate self'.

In tribute to Dr Strehler, an attempt is made to use a style of reasoning found in some of his later papers as an outline of this article. First, general arguments in favor of the involvement of somatic mutations in mtDNA in the aging process are presented. Second, evidence is provided in support of a general tendency of mitochondrial genomes to reach homoplasmic state at the cellular level, for which we propose the term mitochondrial loss of heteroplasmy (mtLOH). This process is likely to facilitate the involvement of mtDNA mutations in the aging process by streamlining the phenotypic expression of the mutant genotype. Third, preliminary evidence of the very high incidence of clonal deletions in pigmented neurons of substantia nigra is reported. This observation highlights the possibility that accumulation of mtDNA mutations specific in certain cell types of a complex tissue may account for the involvement of mtDNA mutations in the aging process despite the relatively low average incidence of these mutations in the tissue as a whole. High incidence of mtDNA deletions in pigmented neurons evokes Strehler's idea that efforts to delay aging may not be the most cost-efficient way of preserving 'self awareness and a joyful sense of life', as he put it. A potential alternative suggested by Strehler, i.e. creation of a 'surrogate self' by computer simulation may deserve more attention than it currently enjoys.

Mutation and intracellular clonal expansion of mitochondrial genomes: two synergistic components of the aging process?

The foundations of the Mitochondrial mutational theory of aging include two assumptions: the high abundance of mitochondrial mutations and their ability to clonally expand within individual cells. The up-to-date data pertinent to these assumptions is reviewed and semi-quantitative estimates of the frequencies of mutants and intracellular expansions are offered. The incidence of mutations in various aged tissues may be on the order of one mutant per mitochondrial genome copy, and most of the cells are likely to be affected by intracellular clonal expansions of mitochondrial genomes. Thus aged tissue may be considered a mosaic of cells with different mutant mitochondrial genotypes. Interestingly, independent studies show that a wide range of aged tissues presents with a mosaic of cells with different mitochondrial phenotypes. The necessary methodologies are available to explore whether the two mosaics are causally related. The answer apparently is positive in muscle; other tissues, brain in particular, await exploration.

Where and when do somatic mtDNA mutations occur?

It is generally assumed that somatic mtDNA mutations are originally created in the cells where these mutations are currently found. Accumulating data indicate, however, that cells with a particular mtDNA mutation tend to "cluster," that is, occur repeatedly within a given sample, but not in the others. Clusters likely are clonal, which implies that mtDNA mutations do not originate in the cells that currently carry them, but rather in those cells' progenitors, such as stem or satellite cells, or even earlier in the development. Importantly, a majority of mtDNA mutations appear to belong to such clusters, and thus mutational events in progenitor cells may be one of the major sources of mtDNA mutations in healthy aging tissue. More research including the analysis of multiple samples per individual is needed to confirm the existence of clustering and to distinguish between the possible clustering mechanisms.

Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra.

It is often assumed mutant frequencies, as measured in a DNA sample, faithfully represent basic mutation rates associated with these mutations. This paradigm was extremely helpful for in vitro studies of the mechanisms of mutagenesis/repair and causes of mutations. However, in vivo, mutant fractions appear to vary dramatically and randomly from sample to sample. It's unlikely that basic mutational rates vary so much. Such variations are probably caused by clonal expansions of mutants within tissue. Whether a particular tissue sample includes an expansion or not, is a matter of chance, which explains the observed random fluctuations of mutant fractions. Well-known examples of clonal expansions involve pathological conditions such as cancer or mitochondrial disease. It is less appreciated that even in normal tissue, expansions of somatic mutants create local deviations from the "expected" mutant frequencies. The sizes of clonal expansions appear to span a wide range and thus, may affect samples of various sizes, from individual cells to individuals. In conclusion, human body appears to be a sort of a "gambling ground" for clonally expanding mutants. We speculate that expansion of early mutants rather than de novo mutation at old age may be the major source of at least some aging-specific mutants in our bodies.

When man got his mtDNA deletions?

Somatic mtDNA mutations and deletions in particular are known to clonally expand within cells, eventually reaching detrimental intracellular concentrations. The possibility that clonal expansion is a slow process taking a lifetime had prompted an idea that founder mutations of mutant clones that cause mitochondrial dysfunction in the aged tissue might have originated early in life. If, conversely, expansion was fast, founder mutations should predominantly originate later in life. This distinction is important: indeed, from which mutations should we protect ourselves - those of early development/childhood or those happening at old age? Recently, high-resolution data describing the distribution of mtDNA deletions have been obtained using a novel, highly efficient method (Taylor et al., ). These data have been interpreted as supporting predominantly early origin of founder mutations. Re-analysis of the data implies that the data actually better fit mostly late origin of founders, although more research is clearly needed to resolve the controversy.

On the timing and the extent of clonal expansion of mtDNA deletions: evidence from single-molecule PCR.

mtDNA deletions are pathogenic mutations that remove substantial portions of the mitochondrial genome. mtDNA deletions accumulate with age and have been implicated in various degenerative diseases. There are multiple mtDNA per cell and mtDNA mutations become toxic only if they accumulate to substantial intracellular levels, i.e. exceed so-called "phenotypic threshold". This is usually achieved via clonal expansion of a single initial mutated molecule. Intracellular mitochondrial genomes are analogous to a population of individuals in that mitochondria are born by division and die by degradation. Clonal expansion within cells is thus analogous to genetic drift within populations and is driven by a combination of random processes and selection. mtDNA mutations occurring early in development are expected to end up spread across tissues, while mutations of late origin are expected to be localized, i.e. limited a single post-mitotic cell or progeny of a single stem cell. We have explored the extent and timing of clonality of mtDNA deletion in human muscle using single-molecule PCR. We analyzed deletions from two nearby locations within the same tissue sample. Altogether we analyzed over 130 mutant molecules, but almost every deletion type detected was represented by several identical mutant molecules, so that altogether there were only 21 different kinds of deletions, implying that essentially all deletions were clonal. At the same time the sets of deletions in the two locations were completely different. This observation implies that all of the clonal expansions spanned very small areas and therefore that the corresponding mutations were likely events of older age. More studies are necessary to further validate these findings in muscle and to explore the other tissues.

Do mtDNA deletions drive premature aging in mtDNA mutator mice?

Deletions in mitochondrial DNA (mtDNA) have long been suspected to be involved in mammalian aging, but their role remains controversial. Recent research has demonstrated that relatively higher levels of mtDNA deletions correlate with premature aging in mtDNA mutator mice, which led to the conclusion that premature aging in these mice is driven by mtDNA deletions. However, it is reported here that the absolute level of deletions in mutator mice is quite low, especially when compared with the level of point mutations in these mice. It is thus argued that the available data are insufficient to conclude that mtDNA mutations drive premature aging in mtDNA mutator mice. It remains possible that clonal expansion of mtDNA deletions may result in sufficiently high levels to play a role in age-related dysfunction in some cells, but assessing this possibility will require studies of the distribution of these deletions among different cell types and in individual cells.