Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

SummaryReprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.PaperCli

Multi-ancestry GWAS reveals excitotoxicity associated with outcome after ischaemic stroke

During the first hours after stroke onset, neurological deficits can be highly unstable: some patients rapidly improve, while others deteriorate. This early neurological instability has a major impact on long-term outcome. Here, we aimed to determine the genetic architecture of early neurological instability measured by the difference between the National Institutes of Health Stroke Scale (NIHSS) within 6 h of stroke onset and NIHSS at 24 h. A total of 5876 individuals from seven countries (Spain, Finland, Poland, USA, Costa Rica, Mexico and Korea) were studied using a multi-ancestry meta-analyses. We found that 8.7% of NIHSS at 24 h of variance was explained by common genetic variations, and also that early neurological instability has a different genetic architecture from that of stroke risk. Eight loci (1p21.1, 1q42.2, 2p25.1, 2q31.2, 2q33.3, 5q33.2, 7p21.2 and 13q31.1) were genome-wide significant and explained 1.8% of the variability suggesting that additional variants influence early change in neurological deficits. We used functional genomics and bioinformatic annotation to identify the genes driving the association from each locus. Expression quantitative trait loci mapping and summary data-based Mendelian randomization indicate that ADAM23 (log Bayes factor = 5.41) was driving the association for 2q33.3. Gene-based analyses suggested that GRIA1 (log Bayes factor = 5.19), which is predominantly expressed in the brain, is the gene driving the association for the 5q33.2 locus. These analyses also nominated GNPAT (log Bayes factor = 7.64) ABCB5 (log Bayes factor = 5.97) for the 1p21.1 and 7p21.1 loci. Human brain single-nuclei RNA-sequencing indicates that the gene expression of ADAM23 and GRIA1 is enriched in neurons. ADAM23, a presynaptic protein and GRIA1, a protein subunit of the AMPA receptor, are part of a synaptic protein complex that modulates neuronal excitability. These data provide the first genetic evidence in humans that excitotoxicity may contribute to early neurological instability after acute ischaemic stroke. Ibanez et al. perform a multi-ancestry meta-analysis to investigate the genetic architecture of early stroke outcomes. Two of the eight genome-wide significant loci identified-ADAM23 and GRIA1-are involved in synaptic excitability, suggesting that excitotoxicity contributes to neurological instability after ischaemic stroke.Peer reviewe

Incompatibility between Nuclear and Mitochondrial Genomes Contributes to an Interspecies Reproductive Barrier.

Vertebrate cells carry two different genomes, nuclear (nDNA) and mitochondrial (mtDNA), both encoding proteins involved in oxidative phosphorylation. Because of the extensive interactions, adaptive coevolution of the two genomes must occur to ensure normal mitochondrial function. To investigate whether incompatibilities between these two genomes could contribute to interspecies reproductive barriers, we performed reciprocal mtDNA replacement (MR) in zygotes between widely divergent Mus m. domesticus (B6) and conplastic Mus m. musculus (PWD) mice. Transfer of MR1 cybrid embryos (B6nDNA-PWDmtDNA) supported normal development of F1 offspring with reduced male fertility but unaffected reproductive fitness in females. Furthermore, donor PWD mtDNA was faithfully transmitted through the germline into F2 and F3 generations. In contrast, reciprocal MR2 (PWDnDNA-B6mtDNA) produced high embryonic loss and stillborn rates, suggesting an association between mitochondrial function and infertility. These results strongly suggest that functional incompatibility between nuclear and mitochondrial genomes contributes to interspecies reproductive isolation in mammals

Incompatibility between Nuclear and Mitochondrial Genomes Contributes to an Interspecies Reproductive Barrier.

Vertebrate cells carry two different genomes, nuclear (nDNA) and mitochondrial (mtDNA), both encoding proteins involved in oxidative phosphorylation. Because of the extensive interactions, adaptive coevolution of the two genomes must occur to ensure normal mitochondrial function. To investigate whether incompatibilities between these two genomes could contribute to interspecies reproductive barriers, we performed reciprocal mtDNA replacement (MR) in zygotes between widely divergent Mus m. domesticus (B6) and conplastic Mus m. musculus (PWD) mice. Transfer of MR1 cybrid embryos (B6nDNA-PWDmtDNA) supported normal development of F1 offspring with reduced male fertility but unaffected reproductive fitness in females. Furthermore, donor PWD mtDNA was faithfully transmitted through the germline into F2 and F3 generations. In contrast, reciprocal MR2 (PWDnDNA-B6mtDNA) produced high embryonic loss and stillborn rates, suggesting an association between mitochondrial function and infertility. These results strongly suggest that functional incompatibility between nuclear and mitochondrial genomes contributes to interspecies reproductive isolation in mammals

Germline and somatic mtDNA mutations in mouse aging.

The accumulation of acquired mitochondrial genome (mtDNA) mutations with aging in somatic cells has been implicated in mitochondrial dysfunction and linked to age-onset diseases in humans. Here, we asked if somatic mtDNA mutations are also associated with aging in the mouse. MtDNA integrity in multiple organs and tissues in young and old (2-34 months) wild type (wt) mice was investigated by whole genome sequencing. Remarkably, no acquired somatic mutations were detected in tested tissues. However, we identified several non-synonymous germline mtDNA variants whose heteroplasmy levels (ratio of normal to mutant mtDNA) increased significantly with aging suggesting clonal expansion of inherited mtDNA mutations. Polg mutator mice, a model for premature aging, exhibited both germline and somatic mtDNA mutations whose numbers and heteroplasmy levels increased significantly with age implicating involvement in premature aging. Our results suggest that, in contrast to humans, acquired somatic mtDNA mutations do not accompany the aging process in wt mice

Characterization of mtDNA mutations in homozygous <i>Polg</i> mice with aging.

<p>(A). Comparison of mean number of germline mutations in wt and <i>Polg</i> mice at young and old age (n = 31 for young wt, n = 8 for young <i>Polg</i>, n = 44 for old wt and n = 14 for old <i>Polg</i>). Error bars, mean ± SEM. Asterisk indicates a significant increase in the number of mutations per tissue in old <i>Polg</i> compared to the old wt (P < 0.05, Student’s t-test). (B). Mean heteroplasmy levels of non-synonymous germline mutations with ≥2% heteroplasmy in <i>Polg</i> mice (mean ± SEM; asterisk, P < 0.05, Student’s t-test). (C) Pie charts showing gene distributions of non-synonymous germline mutations in young <i>Polg</i> mice (2 months, left) and old <i>Polg</i> mice (9 months, right). (D) Bar graphs representing the mean heteroplasmy levels of non-synonymous germline mutations in protein-coding and RNA-coding genes in <i>Polg</i> mice (asterisks, P < 0.05, Student’s t-test). (E) Pie charts showing the distribution of shared and unique mtDNA mutations detected in single skin fibroblast (SF) clones in young and old <i>Polg</i> mice. (F) Mean heteroplasmy changes for non-synonymous somatic mutations with ≥15% heteroplasmy in <i>Polg</i> mice. Error bars, mean ± SEM. Asterisk, P < 0.05, Student’s t-test. (G) Changes in number of non-synonymous somatic mutations with heteroplasmy levels ≥ 15% among different gene types with <i>Polg</i> mice aging. Error bars, mean ± SEM. Asterisks, P < 0.05, Student’s t-test.</p

Dynamics of mtDNA mutations during mouse aging.

<p>Dynamics of mtDNA mutations during mouse aging.</p

Early embryonic and NCR mtDNA mutations in <i>Polg</i> mice.

<p>(A) Bar graphs representing changes in mean heteroplasmy of early embryonic mutations with <i>Polg</i> mice aging. Error Bars, mean ± SEM. (B) Distribution of non-synonymous early embryonic mtDNA mutations among different genes. Error bars, mean ± SEM. (C) Quantification of mtDNA mutations in the non-coding region (NCR) in <i>Polg</i> mice (n = 12 for 2 months; n = 24 for 9 months). Error bars, mean ± SEM. (D) Bar graphs representing mean heteroplasmy levels of NCR mtDNA mutations in <i>Polg</i> mice with aging. Error bars, mean ± SEM. (E) Summary of mtDNA mutations found in the NCR region (mtDNA15423-16299) in <i>Polg</i> mice. Dots represent mtDNA mutations and numbers under dots represent the heteroplasmy levels. ETAS indicates the extended termination associated sequence and CSB indicates the conserved sequence block.</p