Several hundred microRNAs (miRNAs) have recently been cloned from a wide range of organisms across phylogeny. Despite the high degree of conservation of miRNAs, their functions in general, and in mammals particularly, are just beginning to be defined. Here we show that an oligonucleotide DNA array can be successfully used for the simultaneous analysis of miRNA expression profiles from tissues or cells. From a subset of miRNAs expressed in the brain we designed an oligonucleotide array spotted with probes specific for 44 mature miRNAs. These arrays demonstrated precise regulation of miRNA expression at mammalian brain developmental epochs. About 20% of the probed miRNAs changed significantly in their expression during normal brain development, and two of them, miR-9 and miR-131, were dysregulated in presenilin-1 null mice exhibiting severe brain developmental defects. Transcripts with regulated expression patterns on the arrays were validated by Northern blots. Additionally, a bioinformatic analysis of developmentally regulated miRNAs suggested potential mRNA targets. The arrays also revealed miRNAs distributed to translating polyribosomes in primary neurons where they are likely to modulate translation. Therefore, oligonucleotide arrays provide a new tool for studying miRNA expression in a variety of biological and pathobiological settings. Creating clusters of coexpressed miRNAs will contribute to understanding their regulation, functions, and discovery of mRNA targets.

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.

Researchers in several laboratories have reported a high frequency of homoplasmic mitochondrial DNA (mtDNA) mutations in human tumors. This observation has been interpreted to reflect a replicative advantage for mutated mtDNA copies, a growth advantage for a cell containing certain mtDNA mutations, and/or tumorigenic properties of mtDNA mutations. We consider another possibility-that the observed homoplasmy arose entirely by chance in tumor progenitor cells, without any physiological advantage or tumorigenic requirement. Through extensive computer modeling, we demonstrate that there is sufficient opportunity for a tumor progenitor cell to achieve homoplasmy through unbiased mtDNA replication and sorting during cell division. To test our model in vivo, we analyzed mtDNA homoplasmy in healthy human epithelial tissues and discovered that the model correctly predicts the considerable observed frequency of homoplasmic cells. Based on the available data on mitochondrial mutant fractions and cell division kinetics, we show that the predicted frequency of homoplasmy in tumor progenitor cells in the absence of selection is similar to the reported frequency of homoplasmic mutations in tumors. Although a role for other mechanisms is not excluded, random processes are sufficient to explain the incidence of homoplasmic mtDNA mutations in human tumors.

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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Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10(4) molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection at present is 3 x 10(-4), i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.

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

Quantitative information on the cell-to-cell distribution of all possible mitochondrial DNA (mtDNA) mutations in young and aged tissues is needed to assess the relevance of these mutations to the aging process. In the present study, we used PCR amplification of full-length mitochondrial genomes from single cells to scan human cardiomyocytes for all possible large deletions in mtDNA. Analysis of more than 350 individual cells that were derived from three middle-aged and four centenarian donors demonstrates that while most of the cells contain no deletions, in certain cardiomyocytes a significant portion of the mtDNA molecules carried one particular deletion. Different affected cells contained different deletions. Although similar numbers of cells were screened for each donor, these deletion-rich cells were found only in the hearts of old donors, where they occurred at a frequency of up to one in seven cells. These initial observations demonstrate the efficiency of the method and indicate that mitochondrial mutations have the potential to play an important role in human myocardial aging.