Aneuploidy effects on human gene expression across three cell types.

Aneuploidy syndromes impact multiple organ systems but understanding of tissue-specific aneuploidy effects remains limited-especially for the comparison between peripheral tissues and relatively inaccessible tissues like brain. Here, we address this gap in knowledge by studying the transcriptomic effects of chromosome X, Y, and 21 aneuploidies in lymphoblastoid cell lines, fibroblasts and iPSC-derived neuronal cells (LCLs, FCL, and iNs, respectively). We root our analyses in sex chromosome aneuploidies, which offer a uniquely wide karyotype range for dosage effect analysis. We first harness a large LCL RNA-seq dataset from 197 individuals with one of 6 sex chromosome dosages (SCDs: XX, XXX, XY, XXY, XYY, and XXYY) to i) validate theoretical models of SCD sensitivity and ii) define an expanded set of 41 genes that show obligate dosage sensitivity to SCD and are all in cis (i.e., reside on the X or Y chromosome). We then use multiple complementary analyses to show that cis effects of SCD in LCLs are preserved in both FCLs (n = 32) and iNs (n = 24), whereas trans effects (i.e., those on autosomal gene expression) are mostly not preserved. Analysis of additional datasets confirms that the greater cross-cell type reproducibility of cis vs. trans effects is also seen in trisomy 21 cell lines. These findings i) expand our understanding of X, Y, and 21 chromosome dosage effects on human gene expression and ii) suggest that LCLs may provide a good model system for understanding cis effects of aneuploidy in harder-to-access cell types.

High-Throughput Protein Crystallization via Microdialysis.

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

Density dependent regulation of inflammatory responses in macrophages.

Macrophage distribution density is tightly regulated within the body, yet the importance of macrophage crowding during in vitro culture is largely unstudied. Using a human induced pluripotent stem cell (iPSC)-derived macrophage model of tissue resident macrophages, we characterize how increasing macrophage culture density changes their morphology and phenotype before and after inflammatory stimulation. In particular, density drives changes in macrophage inflammatory cytokine and chemokine secretion in both resting and activated states. This density regulated inflammatory state is also evident in blood monocyte derived-macrophages, the human monocytic THP-1 immortalized cell line, and iPSC-derived microglia. Density-dependent changes appear to be driven by a transferable soluble factor, yet the precise mechanism remains unknown. Our findings highlight cell plating density as an important but frequently overlooked consideration of in vitro macrophage research relevant to a variety of fields ranging from basic macrophage cell biology to disease studies.

Differentiation of human induced pluripotent stem cells to authentic macrophages using a defined, serum-free, open-source medium.

Human induced pluripotent stem cells (iPSCs) and macrophages derived from them are increasingly popular tools for research into both infectious and degenerative diseases. However, as the field strives for greater modeling accuracy, it is becoming ever more challenging to justify the use of undefined and proprietary media for the culture of these cells. Here, we describe a defined, serum-free, open-source medium for the differentiation of iPSC-derived macrophages. This medium is equally capable of maintaining these cells compared with commercial alternatives. The macrophages differentiated in this medium display improved terminally differentiated cell characteristics, reduced basal expression of induced antiviral response genes, and improved polarization capacity. We conclude that cells cultured in this medium are an appropriate and malleable model for tissue-resident macrophages, on which future differentiation techniques can be built.

Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System.

Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

Sex-biased trajectories of amygdalo-hippocampal morphology change over human development.

The amygdala and hippocampus are two adjacent allocortical structures implicated in sex-biased and developmentally-emergent psychopathology. However, the spatiotemporal dynamics of amygdalo-hippocampal development remain poorly understood in healthy humans. The current study defined trajectories of volume and shape change for the amygdala and hippocampus by applying a multi-atlas segmentation pipeline (MAGeT-Brain) and semi-parametric mixed-effects spline modeling to 1,529 longitudinally-acquired structural MRI brain scans from a large, single-center cohort of 792 youth (403 males, 389 females) between the ages of 5 and 25 years old. We found that amygdala and hippocampus volumes both follow curvilinear and sexually dimorphic growth trajectories. These sex-biases were particularly striking in the amygdala: males showed a significantly later and slower adolescent deceleration in volume expansion (at age 20 years) than females (age 13 years). Shape analysis localized significant hot-spots of sex-biased anatomical development in sub-regional territories overlying rostral and caudal extremes of the CA1/2 in the hippocampus, and the centromedial nuclear group of the amygdala. In both sexes, principal components analysis revealed close integration of amygdala and hippocampus shape change along two main topographically-organized axes - low vs. high areal expansion, and early vs. late growth deceleration. These results (i) bring greater resolution to our spatiotemporal understanding of amygdalo-hippocampal development in healthy males and females, and (ii) uncover focal sex-differences in the structural maturation of the brain components that may contribute to differences in behavior and psychopathology that emerge during adolescence.

Longitudinally Mapping Childhood Socioeconomic Status Associations with Cortical and Subcortical Morphology.

Childhood socioeconomic status (SES) impacts cognitive development and mental health, but its association with human structural brain development is not yet well characterized. Here, we analyzed 1243 longitudinally acquired structural MRI scans from 623 youth (299 female/324 male) to investigate the relation between SES and cortical and subcortical morphology between ages 5 and 25 years. We found positive associations between SES and total volumes of the brain, cortical sheet, and four separate subcortical structures. These associations were stable between ages 5 and 25. Surface-based shape analysis revealed that higher SES is associated with areal expansion of lateral prefrontal, anterior cingulate, lateral temporal, and superior parietal cortices and ventrolateral thalamic, and medial amygdalo-hippocampal subregions. Meta-analyses of functional imaging data indicate that cortical correlates of SES are centered on brain systems subserving sensorimotor functions, language, memory, and emotional processing. We further show that anatomical variation within a subset of these cortical regions partially mediates the positive association between SES and IQ. Finally, we identify neuroanatomical correlates of SES that exist above and beyond accompanying variation in IQ. Although SES is clearly a complex construct that likely relates to development through diverse, nondeterministic processes, our findings elucidate potential neuroanatomical mediators of the association between SES and cognitive outcomes.SIGNIFICANCE STATEMENT Childhood socioeconomic status (SES) has been associated with developmental disparities in mental health, cognitive ability, and academic achievement, but efforts to understand underlying SES-brain relationships are ongoing. Here, we leverage a unique developmental neuroimaging dataset to longitudinally map the associations between SES and regional brain anatomy at high spatiotemporal resolution. We find widespread associations between SES and global cortical and subcortical volumes and surface area and localize these correlations to a distributed set of cortical, thalamic, and amygdalo-hippocampal subregions. Anatomical variation within a subset of these regions partially mediates the positive relationship between SES and IQ. Our findings help to localize cortical and subcortical systems that represent candidate biological substrates for the known relationships between SES and cognition.

Carriage of Supernumerary Sex Chromosomes Decreases the Volume and Alters the Shape of Limbic Structures.

Sex chromosome aneuploidy (SCA) increases risk for several psychiatric disorders associated with the limbic system, including mood and autism spectrum disorders. Thus, SCA offers a genetics-first model for understanding the biological basis of psychopathology. Additionally, the sex-biased prevalence of many psychiatric disorders could potentially reflect sex chromosome dosage effects on brain development. To clarify how limbic anatomy varies across sex and sex chromosome complement, we characterized amygdala and hippocampus structure in a uniquely large sample of patients carrying supernumerary sex chromosomes (n = 132) and typically developing controls (n = 166). After adjustment for sex-differences in brain size, karyotypically normal males (XY) and females (XX) did not differ in volume or shape of either structure. In contrast, all SCAs were associated with lowered amygdala volume relative to gonadally-matched controls. This effect was robust to three different methods for total brain volume adjustment, including an allometric analysis that derived normative scaling rules for these structures in a separate, typically developing population (n = 79). Hippocampal volume was insensitive to SCA after adjustment for total brain volume. However, surface-based analysis revealed that SCA, regardless of specific karyotype, was consistently associated with a spatially specific pattern of shape change in both amygdala and hippocampus. In particular, SCA was accompanied by contraction around the basomedial nucleus of the amygdala and an area crossing the hippocampal tail. These results demonstrate the power of SCA as a model to understand how copy number variation can precipitate changes in brain systems relevant to psychiatric disease.

Morphometric Similarity Networks Detect Microscale Cortical Organization and Predict Inter-Individual Cognitive Variation.

Macroscopic cortical networks are important for cognitive function, but it remains challenging to construct anatomically plausible individual structural connectomes from human neuroimaging. We introduce a new technique for cortical network mapping based on inter-regional similarity of multiple morphometric parameters measured using multimodal MRI. In three cohorts (two human, one macaque), we find that the resulting morphometric similarity networks (MSNs) have a complex topological organization comprising modules and high-degree hubs. Human MSN modules recapitulate known cortical cytoarchitectonic divisions, and greater inter-regional morphometric similarity was associated with stronger inter-regional co-expression of genes enriched for neuronal terms. Comparing macaque MSNs with tract-tracing data confirmed that morphometric similarity was related to axonal connectivity. Finally, variation in the degree of human MSN nodes accounted for about 40% of between-subject variability in IQ. Morphometric similarity mapping provides a novel, robust, and biologically plausible approach to understanding how human cortical networks underpin individual differences in psychological functions.

An Allometric Analysis of Sex and Sex Chromosome Dosage Effects on Subcortical Anatomy in Humans.

Structural neuroimaging of humans with typical and atypical sex-chromosome complements has established the marked influence of both Yand X-/Y-chromosome dosage on total brain volume (TBV) and identified potential cortical substrates for the psychiatric phenotypes associated with sex-chromosome aneuploidy (SCA). Here, in a cohort of 354 humans with varying karyotypes (XX, XY, XXX, XXY, XYY, XXYY, XXXXY), we investigate sex and SCA effects on subcortical size and shape; focusing on the striatum, pallidum and thalamus. We find large effect-size differences in the volume and shape of all three structures as a function of sex and SCA. We correct for TBV effects with a novel allometric method harnessing normative scaling rules for subcortical size and shape in humans, which we derive here for the first time. We show that all three subcortical volumes scale sublinearly with TBV among healthy humans, mirroring known relationships between subcortical volume and TBV among species. Traditional TBV correction methods assume linear scaling and can therefore invert or exaggerate sex and SCA effects on subcortical anatomy. Allometric analysis restricts sex-differences to: (1) greater pallidal volume (PV) in males, and (2) relative caudate head expansion and ventral striatum contraction in females. Allometric analysis of SCA reveals that supernumerary X- and Y-chromosomes both cause disproportionate reductions in PV, and coordinated deformations of striatopallidal shape. Our study provides a novel understanding of sex and sex-chromosome dosage effects on subcortical organization, using an allometric approach that can be generalized to other basic and clinical structural neuroimaging settings.

Lessons from ten years of crystallization experiments at the SGC.

Although protein crystallization is generally considered more art than science and remains significantly trial-and-error, large-scale data sets hold the promise of providing general learning. Observations are presented here from retrospective analyses of the strategies actively deployed for the extensive crystallization experiments at the Oxford site of the Structural Genomics Consortium (SGC), where comprehensive annotations by SGC scientists were recorded on a customized database infrastructure. The results point to the importance of using redundancy in crystallizing conditions, specifically by varying the mixing ratios of protein sample and precipitant, as well as incubation temperatures. No meaningful difference in performance could be identified between the four most widely used sparse-matrix screens, judged by the yield of crystals leading to deposited structures; this suggests that in general any comparison of screens will be meaningless without extensive cross-testing. Where protein sample is limiting, exploring more conditions has a higher likelihood of being informative by yielding hits than does redundancy of either mixing ratio or temperature. Finally, on the logistical question of how long experiments should be stored, 98% of all crystals that led to deposited structures appeared within 30 days. Overall, these analyses serve as practical guidelines for the design of initial screening experiments for new crystallization targets.