Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal.

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells

A genetic interaction network model of a complex neurological disease.

Absence epilepsy (AE) is a complex, heritable disease characterized by a brief disruption of normal behavior and accompanying spike-wave discharges (SWD) on the electroencephalogram. Only a handful of genes has been definitively associated with AE in humans and rodent models. Most studies suggest that genetic interactions play a large role in the etiology and severity of AE, but mapping and understanding their architecture remains a challenge, requiring new computational approaches. Here we use combined analysis of pleiotropy and epistasis (CAPE) to detect and interpret genetic interactions in a meta-population derived from three C3H × B6J strain crosses, each of which is fixed for a different SWD-causing mutation. Although each mutation causes SWD through a different molecular mechanism, the phenotypes caused by each mutation are exacerbated on the C3H genetic background compared with B6J, suggesting common modifiers. By combining information across two phenotypic measures - SWD duration and frequency - CAPE showed a large, directed genetic network consisting of suppressive and enhancing interactions between loci on 10 chromosomes. These results illustrate the power of CAPE in identifying novel modifier loci and interactions in a complex neurological disease, toward a more comprehensive view of its underlying genetic architecture. Genes Brain Behav 2014 Nov; 13(8):831-40

Assessing Healthspan and Lifespan Measures in Aging Mice: Optimization of Testing Protocols, Replicability, and Rater Reliability.

The relationship between chronological age (lifespan) and biological age (healthspan) varies amongst individuals. Understanding the normal trajectory and characteristic traits of aging mice throughout their lifespan is important for selecting the most reliable and reproducible measures to test hypotheses. The protocols herein describe assays used for aging studies at The Jackson Laboratory\u27s Mouse Neurobehavioral Phenotyping Facility and include assessments of frailty, cognition, and sensory (hearing, vision, olfaction), motor, and fine motor function that can be used for assessing phenotypes in aged mice across their lifespan as well as provide guidance for setting up and validating these behavioral measures. Researchers aiming to study aging phenotypes require access to aged mice as a reference when initiating these types of studies in order to observe normal aging characteristics that cannot be observed in young adult mouse populations. © 2018 by John Wiley & Sons, Inc

Genotypes and features of inbred and congenic strains used in this study.

<p>Genotypes and features of inbred and congenic strains used in this study.</p

TALEN-induced frameshift alleles of <i>Pcnxl2</i> and suppression of <i>Gria4</i> SWD.

<p>A. Exon-intron structure of mouse <i>Pcnxl2</i> (from UCSC genome browser, version m38 mouse genome assembly), highlighting the position of the HeJ-specific IAP-1Δ1 insertion in intron 19, the two exons (exon 16 and exon 29, respectively) targeted for TALEN mutagenesis and the wildtype FeJ sequence of each together with the sequence of the respective frameshift alleles in TALEN mutations A+1 (FS2), A−11 (FS1), and B−2 (FS3). The asterisk indicates a premature translational stop codon in each mutant allele. B. Spike-wave discharge (SWD) incidence (top) and length (bottom) for parent FeJ-<i>Gria4</i>^IAP congenic and HeJ-<i>Gria4</i>^IAP inbred strain colonies, compared with littermate wildtype (wt), heterozygous or homozygous respective TALEN mutations created and maintained on the isogenic FeJ-<i>Gria4</i>^IAP congenic strain background. Asterisks indicate where homozygous mutant genotypes were significantly different (<i>p</i><0.01) from wildtype control. Sample sizes: FeJ-<i>Gria4</i>^IAP (9), HeJ- <i>Gria4</i>^IAP (18), wt (16), FS1 het (6), FS1 hom (6), FS2 het (9), FS2 hom (9), FS3 het (6), FS3 hom (6). Error bars are SEM. C. Spike-wave discharge (SWD) incidence (left) and length (right) for littermate TalA-11 genotypes (hom, het, wt) isogenic on the FeJ strain, in double mutant combination with <i>Gabrg2</i>^tm1Spet or <i>Scn8a</i>^8J/+ from a congenic FeJ strain background. None of the <i>Pcnxl2</i> mutant allele homozygotes were significantly different from the other <i>Pcnxl2</i> genotypes for either mutation or SWD measure. Sample sizes: <i>Gabrg2</i>^tm1Spet (3 hom, 12 het, 2 wt); <i>Scn8a</i>^8J/+ (2 hom, 4 het, 2 wt).</p

IAP-1Δ1 element diversity in C3H substrains.

<p>A. Strategy for detection of IAP-1Δ1 elements. For detection of host-IAP-1Δ1 5′ <i>Bgl</i>II-restricted junction fragments by dried gel hybridization, a 31-nt oligonucleotide hybridization probe (IAPd1oligo1-R) was designed to straddle the previously described 1.2 kb IAP-1Δ1 element common deletion <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004454#pgen.1004454-Ishihara1" target="_blank">[22]</a>. For cloning of IAP-1Δ1 elements by inverse PCR, a complementary oligonucleotide (IAPd1oligo2F) was designed to pair with an oligonucleotide specific for the IAP LTR (IAPLTR5′) for amplification of circularized genomic <i>Bgl</i>II fragments. B. Dried gel detection of IAP-1Δ1 5′ junction fragments from C3H substrains (HeJ, HeOuJ, HeSnJ, FeJ) plus the outlier B6J. Bands estimated as unique to HeJ are indicated with asterisks on the left. C. Example of an inverse PCR experiment from HeJ and FeJ substrains showing <i>Bgl</i>II restricted genomic DNA (two lanes each), unligated controls (one lane each), and location of apparent HeJ-specific bands prepared. for cloning and Sanger sequencing. In additional experiments not shown, higher molecular weight <i>Bgl</i>II restricted fragments were similarly processed. Also shown with connecting lines are locus identities for specific bands after sequencing, the sizes of which correspond to the junction fragment lengths determined from the sequence, and dashed lines indicate the corresponding region from the dried gel, for the purpose of highlighting the similar banding pattern (<i>N.b.</i> the absolute sizes of fragments in panel C are smaller because the portion of the IAP genome between primers is expectedly absent from the inverse PCR product). Sizes of <i>Hind</i>III-digested bacteriophage lambda DNA are shown on the right, of panel B, and <i>Hae</i>III-digested bacteriophage phi-X DNA markers are shown at the left of panel C.</p

Quantitative RT-PCR in <i>Pcnxl2</i> mutants.

a<p>Fold-difference for each pair of rows was calculated as 2^{(top row ΔCt – bottom row ΔCt)}, and is signed relative to the bottom row (e.g. <i>Pcnxl2</i> transcript is 2.0 fold higher in FeJ-<i>Gria4</i>^IAP compared to HeJ.</p

C3H/HeJ IAP-1Δ1 insertions absent from C3HeB/FeJ.

a<p>To detect both IAP insertion allele and the pre-insertion allele, the IAP5′LTR-F oligonucleotide is used in a 3′-primer assay with both the locus-specific forward and reverse primers. In cases where the product sizes of both alleles were identical, the IAP5′LTR-F+gene-F (or gene -R, depending upon IAP orientation) for the insertion allele and gene -F+gene -R primers are run separately for the pre-insertion allele.</p

SWD phenotypes conferred by <i>G4swdm1</i>, a Chr 15 strain modifier of <i>Gria4</i> absence seizures.

<p>A. LOD score plot, similar to those in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004454#pgen-1004454-g003" target="_blank">Figure 3</a>, except only for Chr 15 in 84 N₉F₁ or N₁₀F₁ intercross congenic mice. At the bottom are Mb (mouse genome assembly m38) positions in of Chr 15 markers used for this analysis and the genome-wide significance threshold is also shown, as determined by 5000 permutations. B. For the marker <i>D15Mit93</i> corresponding to the peak maximum likelihood location from panel A, quartile plots with data points for average SWD incidence or SWD length from individual mice, showing additivity for both traits. C. EEG from a <i>Gria4</i>^KO/KO, <i>G4swdm1^Fe/FeJ</i> double homozygote on the B6J congenic strain, showing a particularly long (15 second) SWD. D. EEG from a <i>Gria4</i>^KO/IAP compound heterozygous, <i>G4swdm1^Fe/FeJ</i> homozygous double mutant genotype on a (B6J.FeJ×FeJ)F₁ hybrid background, showing a particularly long (46 second) SWD. For panels C and D, two of the six recorded channels are shown (LF-RB – left front, right back; LB-RB – left back, right back, corresponding to the electrode placement relative to Bregma and midline). 1 s: 1 second.</p

Genome scans for SWD traits in <i>Gria4</i> deficient backcross mice.

<p>Shown are genome-wide interval mapping LOD score plots from 89 backcross <i>Gria4</i> mutant mice for two observed spike-wave discharge (SWD) traits - SWD incidence (panel A), SWD length (panel B) - and their first two principal components (panels C and D). The insets of panels A and B show the frequency distribution of the respective raw traits, although for interval mapping all four traits were first rank- and quartile-normalized prior to analysis. The chromosomes are shown at the bottom, with tick marks indicating positions of individual markers within each chromosome. Dotted lines show positions of genome-wide significance thresholds determined from 1000 permutations.</p