Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains.

Genetic background is known to play a role in the ability to derive pluripotent, embryonic stem cells (ESC), a trait referred to as permissiveness. Previously we demonstrated that induced pluripotent stem cells (iPSC) can be readily derived from non-permissive mouse strains by addition of serum-based media supplemented with GSK3B and MEK inhibitors, termed 2iS media, 3 days into reprogramming. Here, we describe the derivation of second type of iPSC colony from non-permissive mouse strains that can be stably maintained independently of 2iS media. The resulting cells display transcriptional heterogeneity similar to that observed in ESC from permissive genetic backgrounds derived in conventional serum containing media supplemented with leukemia inhibitor factor. However, unlike previous studies that report exclusive subpopulations, we observe both exclusive and simultaneous expression of naive and primed cell surface markers. Herein, we explore shifts in pluripotency in the presence of 2iS and characterize heterogenous subpopulations to determine their pluripotent state and role in heterogenous iPSCs derived from the non-permissive NOD/ShiLtJ strain. We conclude that heterogeneity is a naturally occurring, necessary quality of stem cells that allows for the maintenance of pluripotency. This study further demonstrates the efficacy of the 2iS reprogramming technique. It is also the first study to derive stable ESC-like stem cells from the non-permissive NOD/ShiLtJ and WSB/EiJ strains, enabling easier and broader research possibilities into pluripotency for these and similar non-permissive mouse strains and species.

Permissiveness to form pluripotent stem cells may be an evolutionarily derived characteristic in Mus musculus.

Mus musculus is the only known species from which embryonic stem cells (ESC) can be isolated under conditions requiring only leukemia inhibitory factor (LIF). Other species are non-permissive in LIF media, and form developmentally primed epiblast stem cells (EpiSC) similar to cells derived from post-implantation, egg cylinders. To evaluate whether non-permissiveness extends to induced pluripotent stem cells (iPSC), we derived iPSC from the eight founder strains of the mouse Collaborative Cross. Two strains, NOD/ShiLtJ and the WSB/EiJ, were non-permissive, consistent with the previous classification of NOD/ShiLtJ as non-permissive to ESC derivation. We determined non-permissiveness is recessive, and that non-permissive genomes do not compliment. We overcame iPSC non-permissiveness by using GSK3B and MEK inhibitors with serum, a technique we termed 2iS reprogramming. Although used for ESC derivation, GSK3B and MEK inhibitors have not been used during iPSC reprogramming because they inhibit survival of progenitor differentiated cells. iPSC derived in 2iS are more transcriptionally similar to ESC than EpiSC, indicating that 2iS reprogramming acts to overcome genetic background constraints. Finally, of species tested for ESC or iPSC derivation, only some M. musculus strains are permissive under LIF culture conditions suggesting that this is an evolutionarily derived characteristic in the M. musculus lineage.

Common variants in psychiatric risk genes predict brain structure at birth.

Studies in adolescents and adults have demonstrated that polymorphisms in putative psychiatric risk genes are associated with differences in brain structure, but cannot address when in development these relationships arise. To determine if common genetic variants in disrupted-in-schizophrenia-1 (DISC1; rs821616 and rs6675281), catechol-O-methyltransferase (COMT; rs4680), neuregulin 1 (NRG1; rs35753505 and rs6994992), apolipoprotein E (APOE; ε3ε4 vs. ε3ε3), estrogen receptor alpha (ESR1; rs9340799 and rs2234693), brain-derived neurotrophic factor (BDNF; rs6265), and glutamate decarboxylase 1 (GAD1; rs2270335) are associated with individual differences in brain tissue volumes in neonates, we applied both automated region-of-interest volumetry and tensor-based morphometry to a sample of 272 neonates who had received high-resolution magnetic resonance imaging scans. ESR1 (rs9340799) predicted intracranial volume. Local variation in gray matter (GM) volume was significantly associated with polymorphisms in DISC1 (rs821616), COMT, NRG1, APOE, ESR1 (rs9340799), and BDNF. No associations were identified for DISC1 (rs6675281), ESR1 (rs2234693), or GAD1. Of note, neonates homozygous for the DISC1 (rs821616) serine allele exhibited numerous large clusters of reduced GM in the frontal lobes, and neonates homozygous for the COMT valine allele exhibited reduced GM in the temporal cortex and hippocampus, mirroring findings in adults. The results highlight the importance of prenatal brain development in mediating psychiatric risk.

Impact of sex and gonadal steroids on neonatal brain structure.

There are numerous reports of sexual dimorphism in brain structure in children and adults, but data on sex differences in infancy are extremely limited. Our primary goal was to identify sex differences in neonatal brain structure. Our secondary goal was to explore whether brain structure was related to androgen exposure or sensitivity. Two hundred and ninety-three neonates (149 males) received high-resolution structural magnetic resonance imaging scans. Sensitivity to androgen was measured using the number of cytosine, adenine, guanine (CAG) triplets in the androgen receptor gene and the ratio of the second to fourth digit, provided a proxy measure of prenatal androgen exposure. There was a significant sex difference in intracranial volume of 5.87%, which was not related to CAG triplets or digit ratios. Tensor-based morphometry identified extensive areas of local sexual dimorphism. Males had larger volumes in medial temporal cortex and rolandic operculum, and females had larger volumes in dorsolateral prefrontal, motor, and visual cortices. Androgen exposure and sensitivity had minor sex-specific effects on local gray matter volume, but did not appear to be the primary determinant of sexual dimorphism at this age. Comparing our study with the existing literature suggests that sex differences in cortical structure vary in a complex and highly dynamic way across the human lifespan.

2D:4D ratios in the first 2 years of life: Stability and relation to testosterone exposure and sensitivity.

The relative lengths of the 2nd and 4th digits (2D:4D) may provide an easily measurable and stable anthropometric index of prenatal androgen exposure, but no study has examined the development of 2D:4D in infancy and the potential impact of neonatal testosterone levels. We collected 2D:4D ratios from 364 children between 0 and 2 years of age. Saliva samples were collected from 236 of these children 3 months after birth and analyzed for testosterone. In addition, 259 children provided DNA samples which were genotyped for the CAG repeat polymorphism in the androgen receptor. There was substantial variability across age in 2D:4D. Sex differences were small compared to adults and did not consistently reach statistical significance. This suggests that 2D:4D may not function well as a proxy measure of prenatal testosterone exposure in infancy. In addition, the interaction of salivary T and CAG repeats predicted right hand digit ratio at 12 months and left hand digit ratio at 12 months and 24 months in males. The interaction of salivary testosterone and CAG repeat length also predicted change in left hand 2D:4D from 2 weeks to 12 months in males. This suggests that 2D:4D in adults may reflect, in part, neonatal testosterone exposure. No significant relationships were observed within females. No significant relationships were observed when salivary testosterone and CAG repeats were examined independent of each other. Results have important implications for the design and interpretation of studies which use 2D:4D as a proxy measure of prenatal testosterone exposure.

No association of the serotonin transporter polymorphisms 5-HTTLPR and RS25531 with schizophrenia or neurocognition.

A promoter polymorphism in the serotonin transporter gene has been widely studied in neuropsychiatry. We genotyped the 5-HTTLPR/rs25531 triallelic polymorphism in 728 schizophrenia cases from the CATIE study and 724 control subjects. In a logistic regression with case/control status as dependent variable and 7 ancestry-informative principal components as covariates, the effect of 5-HTTLPR/rs25531 composite genotype was not significant (odds ratio = 1.008, 95% CI 0.868-1.172, P = 0.91). In cases only, 5-HTTLPR/rs25531 was not associated with neurocognition (summary neurocognitive index P = 0.21, working memory P = 0.32) or symptomatology (PANSS positive P = 0.67 and negative symptoms P = 0.46). We were unable to identify association of the triallelic 5-HTTLPR with schizophrenia, neurocognition, or core psychotic symptoms even at levels of significance unadjusted for multiple comparisons.

A searchable database of genetic evidence for psychiatric disorders.

This paper describes a new bioinformatic tool for use in psychiatric research, "SLEP" (Sullivan Lab Evidence Project). SLEP is a searchable archive of findings from psychiatric genetics that is freely available on the web for non-commercial use (http://slep.unc.edu). Via a simple interface, users can retrieve findings from genome-wide linkage, genome-wide association, and microarray studies for ADHD, autism, bipolar disorder, eating disorders, major depression, nicotine dependence, and schizophrenia. Findings can be save to disk or viewed via a genome browser.