Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation

A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However, the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here, we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further, we investigated the transcriptional changes in mRNA and miRNA levels, using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs

Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines

Tissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (\u3e 200 reprogrammed iPSC lines) using this protocol

Assessing neural tuning for object perception in schizophrenia and bipolar disorder with multivariate pattern analysis of fMRI data.

IntroductionDeficits in visual perception are well-established in schizophrenia and are linked to abnormal activity in the lateral occipital complex (LOC). Related deficits may exist in bipolar disorder. LOC contains neurons tuned to object features. It is unknown whether neural tuning in LOC or other visual areas is abnormal in patients, contributing to abnormal perception during visual tasks. This study used multivariate pattern analysis (MVPA) to investigate perceptual tuning for objects in schizophrenia and bipolar disorder.MethodsFifty schizophrenia participants, 51 bipolar disorder participants, and 47 matched healthy controls completed five functional magnetic resonance imaging (fMRI) runs of a perceptual task in which they viewed pictures of four different objects and an outdoor scene. We performed classification analyses designed to assess the distinctiveness of activity corresponding to perception of each stimulus in LOC (a functionally localized region of interest). We also performed similar classification analyses throughout the brain using a searchlight technique. We compared classification accuracy and patterns of classification errors across groups.ResultsStimulus classification accuracy was significantly above chance in all groups in LOC and throughout visual cortex. Classification errors were mostly within-category confusions (e.g., misclassifying one chair as another chair). There were no group differences in classification accuracy or patterns of confusion.ConclusionsThe results show for the first time MVPA can be used successfully to classify individual perceptual stimuli in schizophrenia and bipolar disorder. However, the results do not provide evidence of abnormal neural tuning in schizophrenia and bipolar disorder

Fast and powerful heritability inference for family-based neuroimaging studies.

Heritability estimation has become an important tool for imaging genetics studies. The large number of voxel- and vertex-wise measurements in imaging genetics studies presents a challenge both in terms of computational intensity and the need to account for elevated false positive risk because of the multiple testing problem. There is a gap in existing tools, as standard neuroimaging software cannot estimate heritability, and yet standard quantitative genetics tools cannot provide essential neuroimaging inferences, like family-wise error corrected voxel-wise or cluster-wise P-values. Moreover, available heritability tools rely on P-values that can be inaccurate with usual parametric inference methods. In this work we develop fast estimation and inference procedures for voxel-wise heritability, drawing on recent methodological results that simplify heritability likelihood computations (Blangero et al., 2013). We review the family of score and Wald tests and propose novel inference methods based on explained sum of squares of an auxiliary linear model. To address problems with inaccuracies with the standard results used to find P-values, we propose four different permutation schemes to allow semi-parametric inference (parametric likelihood-based estimation, non-parametric sampling distribution). In total, we evaluate 5 different significance tests for heritability, with either asymptotic parametric or permutation-based P-value computations. We identify a number of tests that are both computationally efficient and powerful, making them ideal candidates for heritability studies in the massive data setting. We illustrate our method on fractional anisotropy measures in 859 subjects from the Genetics of Brain Structure study

Bridging the gap: improving correspondence between low-field and high-field magnetic resonance images in young people

BackgroundPortable low-field-strength magnetic resonance imaging (MRI) systems represent a promising alternative to traditional high-field-strength systems with the potential to make MR technology available at scale in low-resource settings. However, lower image quality and resolution may limit the research and clinical potential of these devices. We tested two super-resolution methods to enhance image quality in a low-field MR system and compared their correspondence with images acquired from a high-field system in a sample of young people.MethodsT1- and T2-weighted structural MR images were obtained from a low-field (64mT) Hyperfine and high-field (3T) Siemens system in N = 70 individuals (mean age = 20.39 years, range 9–26 years). We tested two super-resolution approaches to improve image correspondence between images acquired at high- and low-field: (1) processing via a convolutional neural network (‘SynthSR’), and (2) multi-orientation image averaging. We extracted brain region volumes, cortical thickness, and cortical surface area estimates. We used Pearson correlations to test the correspondence between these measures, and Steiger Z tests to compare the difference in correspondence between standard imaging and super-resolution approaches.ResultsSingle pairs of T1- and T2-weighted images acquired at low field showed high correspondence to high-field-strength images for estimates of total intracranial volume, surface area cortical volume, subcortical volume, and total brain volume (r range = 0.60–0.88). Correspondence was lower for cerebral white matter volume (r = 0.32, p = 0.007, q = 0.009) and non-significant for mean cortical thickness (r = −0.05, p = 0.664, q = 0.664). Processing images with SynthSR yielded significant improvements in correspondence for total brain volume, white matter volume, total surface area, subcortical volume, cortical volume, and total intracranial volume (r range = 0.85–0.97), with the exception of global mean cortical thickness (r = 0.14). An alternative multi-orientation image averaging approach improved correspondence for cerebral white matter and total brain volume. Processing with SynthSR also significantly improved correspondence across widespread regions for estimates of cortical volume, surface area and subcortical volume, as well as within isolated prefrontal and temporal regions for estimates of cortical thickness.ConclusionApplying super-resolution approaches to low-field imaging improves regional brain volume and surface area accuracy in young people. Finer-scale brain measurements, such as cortical thickness, remain challenging with the limited resolution of low-field systems

Progressive Bidirectional Age-Related Changes in Default Mode Network Effective Connectivity across Six Decades

The default mode network (DMN) is a set of regions that is tonically engaged during the resting state and exhibits task-related deactivation that is readily reproducible across a wide range of paradigms and modalities. The DMN has been implicated in numerous disorders of cognition and, in particular, in disorders exhibiting age-related cognitive decline. Despite these observations, investigations of the DMN in normal aging are scant. Here, we used blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) acquired during rest to investigate age-related changes in functional connectivity of the DMN in 120 healthy normal volunteers comprising six, 20-subject, decade cohorts (from 20–29 to 70–79). Structural equation modeling (SEM) was used to assess age-related changes in inter-regional connectivity within the DMN. SEM was applied both using a previously published, meta-analytically derived, node-and-edge model, and using exploratory modeling searching for connections that optimized model fit improvement. Although the two models were highly similar (only 3 of 13 paths differed), the sample demonstrated significantly better fit with the exploratory model. For this reason, the exploratory model was used to assess age-related changes across the decade cohorts. Progressive, highly significant changes in path weights were found in 8 (of 13) paths: four rising, and four falling (most changes were significant by the third or fourth decade). In all cases, rising paths and falling paths projected in pairs onto the same nodes, suggesting compensatory increases associated with age-related decreases. This study demonstrates that age-related changes in DMN physiology (inter-regional connectivity) are bidirectional, progressive, of early onset and part of normal aging

Progressive Bidirectional Age-Related Changes in Default Mode Network Effective Connectivity across Six Decades

The default mode network (DMN) is a set of regions that is tonically engaged during the resting state and exhibits task-related deactivation that is readily reproducible across a wide range of paradigms and modalities. The DMN has been implicated in numerous disorders of cognition and, in particular, in disorders exhibiting age-related cognitive decline. Despite these observations, investigations of the DMN in normal aging are scant. Here, we used blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) acquired during rest to investigate age-related changes in functional connectivity of the DMN in 120 healthy normal volunteers comprising six, 20-subject, decade cohorts (from 20–29 to 70–79). Structural equation modeling (SEM) was used to assess age-related changes in inter-regional connectivity within the DMN. SEM was applied both using a previously published, meta-analytically derived, node-and-edge model, and using exploratory modeling searching for connections that optimized model fit improvement. Although the two models were highly similar (only 3 of 13 paths differed), the sample demonstrated significantly better fit with the exploratory model. For this reason, the exploratory model was used to assess age-related changes across the decade cohorts. Progressive, highly significant changes in path weights were found in 8 (of 13) paths: four rising, and four falling (most changes were significant by the third or fourth decade). In all cases, rising paths and falling paths projected in pairs onto the same nodes, suggesting compensatory increases associated with age-related decreases. This study demonstrates that age-related changes in DMN physiology (inter-regional connectivity) are bidirectional, progressive, of early onset and part of normal aging

Deficits in visual working-memory capacity and general cognition in African Americans with psychosis

On average, patients with psychosis perform worse than controls on visual change-detection tasks, implying that psychosis is associated with reduced capacity of visual working memory (WM). In the present study, 79 patients diagnosed with various psychotic disorders and 166 controls, all African Americans, completed a change-detection task and several other neurocognitive measures. The aims of the study were to (1) determine whether we could observe a between-group difference in performance on the change-detection task in this sample; (2) establish whether such a difference could be specifically attributed to reduced WM capacity (k); and (3) estimate k in the context of the general cognitive deficit in psychosis. Consistent with previous studies, patients performed worse than controls on the change-detection task, on average. Bayesian hierarchical cognitive modeling of the data suggested that this between-group difference was driven by reduced k in patients, rather than differences in other psychologically meaningful model parameters (guessing behavior and lapse rate). Using the same modeling framework, we estimated the effect of psychosis on k while controlling for general intellectual ability (g, obtained from the other neurocognitive measures). The results suggested that reduced k in patients was stronger than predicted by the between-group difference in g. Moreover, a mediation analysis suggested that the relationship between psychosis and g (i.e., the general cognitive deficit) was mediated by k. The results were consistent with the idea that reduced k is a specific deficit in psychosis, which contributes to the general cognitive deficit

Role of miRNA-mRNA Interaction in Neural Stem Cell Differentiation of Induced Pluripotent Stem Cells

miRNA regulates the expression of protein coding genes and plays a regulatory role in human development and disease. The human iPSCs and their differentiated progenies provide a unique opportunity to identify these miRNA-mediated regulatory mechanisms. To identify miRNA–mRNA regulatory interactions in human nervous system development, well characterized NSCs were differentiated from six validated iPSC lines and analyzed for differentially expressed (DE) miRNome and transcriptome by RNA sequencing. Following the criteria, moderated t statistics, FDR-corrected p-value ≤ 0.05 and fold change—absolute (FC-abs) ≥2.0, 51 miRNAs and 4033 mRNAs were found to be significantly DE between iPSCs and NSCs. The miRNA target prediction analysis identified 513 interactions between 30 miRNA families (mapped to 51 DE miRNAs) and 456 DE mRNAs that were paradoxically oppositely expressed. These 513 interactions were highly enriched in nervous system development functions (154 mRNAs; FDR-adjusted p-value range: 8.06 × 10−15–1.44 × 10−4). Furthermore, we have shown that the upregulated miR-10a-5p, miR-30c-5p, miR23-3p, miR130a-3p and miR-17-5p miRNA families were predicted to down-regulate several genes associated with the differentiation of neurons, neurite outgrowth and synapse formation, suggesting their role in promoting the self-renewal of undifferentiated NSCs. This study also provides a comprehensive characterization of iPSC-generated NSCs as dorsal neuroepithelium, important for their potential use in in vitro modeling of human brain development and disease