The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

Gene coregulation across a population is an important aspect of the considerable variability of the human immune response to virus infection. Methodology to investigate it must rely on a number of ingredients ranging from gene clustering to transcription factor enrichment analysis.

Developmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2 ± ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2 ± mice, but not in Atg7(CKO) neuronal autophagy-deficient mice or Tsc2 ± :Atg7(CKO) double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.

Molecular adaptations in the striatum mediated by dopamine (DA) denervation and/or levodopa (L-dopa) treatments have been implicated in the motor deficits found in Parkinson's disease (PD). Alterations in inflammatory response mechanisms and glutamatergic neurotransmission are reported to play important roles in mediating these changes. However, the mechanisms mediating the molecular adaptations in the striatum are not well understood. Small non-coding microRNAs (miRNAs) influence numerous biological processes including the development and maintenance of striatal neurons by regulating gene expression post-transcriptionally. To investigate miRNA function in human PD striatum, we examined the global expression of miRNAs in postmortem putamen (putamen along with caudate forms the striatum) tissues obtained from PD patients and neurologically normal controls using Nanostring miRNA assays. We found that 6 miRNAs were significantly (p≤0.05) upregulated and 7 miRNAs were downregulated in PD putamen when compared with control. The differential expression (DE) of the 4 highest scoring miRNAs was further confirmed by reverse transcription polymerase chain reaction. Ingenuity pathway analysis demonstrated that these miRNAs are enriched in the processes of inflammatory responses. We found that the expression of DE miRNAs in PD putamen negatively correlates with the expression of gene transcripts implicated in inflammatory response with p53 and NF-kB as central signaling molecules. Taken together, our results suggest that in PD striatum, the DE miRNAs are associated with the oxidative stress pathway. This mechanism may contribute to the molecular adaptations and related motor complications found in PD.

Autism spectrum disorder (ASD) consists of a group of complex developmental disabilities characterized by impaired social interactions, deficits in communication and repetitive behavior. Multiple lines of evidence implicate mitochondrial dysfunction in ASD. In postmortem BA21 temporal cortex, a region that exhibits synaptic pathology in ASD, we found that compared to controls, ASD patients exhibited altered protein levels of mitochondria respiratory chain protein complexes, decreased Complex I and IV activities, decreased mitochondrial antioxidant enzyme SOD2, and greater oxidative DNA damage. Mitochondrial membrane mass was higher in ASD brain, as indicated by higher protein levels of mitochondrial membrane proteins Tom20, Tim23 and porin. No differences were observed in either mitochondrial DNA or levels of the mitochondrial gene transcription factor TFAM or cofactor PGC1α, indicating that a mechanism other than alterations in mitochondrial genome or mitochondrial biogenesis underlies these mitochondrial abnormalities. We further identified higher levels of the mitochondrial fission proteins (Fis1 and Drp1) and decreased levels of the fusion proteins (Mfn1, Mfn2 and Opa1) in ASD patients, indicating altered mitochondrial dynamics in ASD brain. Many of these changes were evident in cortical pyramidal neurons, and were observed in ASD children but were less pronounced or absent in adult patients. Together, these findings provide evidence that mitochondrial function and intracellular redox status are compromised in pyramidal neurons in ASD brain and that mitochondrial dysfunction occurs during early childhood when ASD symptoms appear.

Heterozygous mutations in GBA, the gene encoding the lysosomal enzyme glucosylceramidase beta/β-glucocerebrosidase, comprise the most common genetic risk factor for Parkinson disease (PD), but the mechanisms underlying this association remain unclear. Here, we show that in GbaL444P/WT knockin mice, the L444P heterozygous Gba mutation triggers mitochondrial dysfunction by inhibiting autophagy and mitochondrial priming, two steps critical for the selective removal of dysfunctional mitochondria by autophagy, a process known as mitophagy. In SHSY-5Y neuroblastoma cells, the overexpression of L444P GBA impeded mitochondrial priming and autophagy induction when endogenous lysosomal GBA activity remained intact. By contrast, genetic depletion of GBA inhibited lysosomal clearance of autophagic cargo. The link between heterozygous GBA mutations and impaired mitophagy was corroborated in postmortem brain tissue from PD patients carrying heterozygous GBA mutations, where we found increased mitochondrial content, mitochondria oxidative stress and impaired autophagy. Our findings thus suggest a mechanistic basis for mitochondrial dysfunction associated with GBA heterozygous mutations. Abbreviations: AMBRA1: autophagy/beclin 1 regulator 1; BECN1: beclin 1, autophagy related; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chloroyphenylhydrazone; CYCS: cytochrome c, somatic; DNM1L/DRP1: dynamin 1-like; ER: endoplasmic reticulum; GBA: glucosylceramidase beta; GBA-PD: Parkinson disease with heterozygous GBA mutations; GD: Gaucher disease; GFP: green fluorescent protein; LC3B: microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated form of microtubule-associated protein 1 light chain 3 beta; MitoGreen: MitoTracker Green; MitoRed: MitoTracker Red; MMP: mitochondrial membrane potential; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH transcription factor; NBR1: NBR1, autophagy cargo receptor; Non-GBA-PD: Parkinson disease without GBA mutations; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; ROS: reactive oxygen species; SNCA: synuclein alpha; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1/Porin: voltage dependent anion channel 1; WT: wild type.

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.

Abnormalities of SNAP25 (synaptosome-associated protein 25) amount and protein-protein interactions occur in schizophrenia, and may contribute to abnormalities of neurotransmitter release in patients. However, presynaptic terminal function depends on multiple subcellular mechanisms, including energy provided by mitochondria. To explore the SNAP25 interactome in schizophrenia, we immunoprecipitated SNAP25 along with interacting proteins from the ventromedial caudate of 15 cases of schizophrenia and 13 controls. Proteins were identified with mass spectrometry-based proteomics. As well as 15 SNARE- (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) associated proteins, we identified 17 mitochondria-associated and four other proteins. The mitochondrial small GTPase ARF1 (ADP-ribosylation factor 1) was identified in eight schizophrenia SNAP25 immunoprecipitates and none from controls (P = 0.004). Although the ARF1-SNAP25 interaction may be increased, immunoblotting demonstrated 21% lower ARF1-21 (21 kiloDaltons) in schizophrenia samples (P = 0.04). In contrast, the mitochondrial protein UQCRC1 (ubiquinol-cytochrome c reductase core protein 1) did not differ. Lower ARF1-21 levels were associated with the previously reported increased SNAP25-syntaxin interaction in schizophrenia (r = -0.39, P = 0.04). Additional immunoprecipitation studies confirmed the ARF1-21-SNAP25 interaction, independent of UQCRC1. Both ARF1 and SNAP25 were localized to synaptosomes. Confocal microscopy demonstrated co-localization of ARF1 and SNAP25, and further suggested fivefold enrichment of ARF1 in synaptosomes containing an excitatory marker (vesicular glutamate transporter) compared with synaptosomes containing an inhibitory marker (vesicular GABA transporter). The present findings suggest an association between abnormalities of SNARE proteins involved with vesicular neurotransmission and the mitochondrial protein ARF1 that may contribute to the pathophysiology of schizophrenia.

Stress exposure is associated with the pathogenesis of psychiatric disorders, including post-traumatic stress disorder (PTSD) and major depressive disorder (MDD). Here, we show in rodents that chronic stress exposure rapidly and transiently elevates hippocampal expression of Kruppel-like factor 9 (Klf9). Inducible genetic silencing of Klf9 expression in excitatory forebrain neurons in adulthood prior to, but not after, onset of stressor prevented chronic restraint stress (CRS)-induced potentiation of contextual fear acquisition in female mice and chronic corticosterone (CORT) exposure-induced fear generalization in male mice. Klf9 silencing prevented chronic CORT and CRS induced enlargement of dendritic spines in the ventral hippocampus of male and female mice, respectively. KLF9 mRNA density was increased in the anterior dentate gyrus of women, but not men, with more severe recent stressful life events and increased mortality. Thus, Klf9 functions as a stress-responsive transcription factor that mediates circuit and behavioral resilience in a sex-specific manner.

Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cell interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection [MOI]) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread.IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.

A promising new approach, transcranial direct current stimulation (tDCS) has recently been used as a therapeutic modality for cerebellar ataxia. However, the strength of the conclusions drawn from individual studies in the current literature may be constrained by the small sample size of each trial. Following a systematic literature retrieval of studies, meta-analyses were conducted by pooling the standardized mean differences (SMDs) using random-effects models to assess the efficacy of tDCS on cerebellar ataxia, measured by standard clinical rating scales. Domain-specific effects of tDCS on gait and hand function were further evaluated based on 8-m walk and 9-hole peg test performance times, respectively. To determine the safety of tDCS, the incidences of adverse effects were analyzed using risk differences. Out of 293 citations, 5 randomized controlled trials involving a total of 72 participants with cerebellar ataxia were included. Meta-analysis indicated a 26.1% (p = 0.003) improvement in ataxia immediately after tDCS with sustained efficacy over months (28.2% improvement after 3 months, p = 0.04) when compared with sham stimulation. tDCS seems to be domain-specific as the current analysis suggested a positive effect on gait (16.3% improvement, p = 0.04) and failed to reveal differences for hand function (p = 0.10) with respect to sham. The incidence of adverse events in tDCS and sham groups was similar. tDCS is an effective intervention for mitigating ataxia symptoms with lasting results that can be sustained for months. This treatment shows preferential effects on gait ataxia and is relatively safe.

The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64-69% overlap, depletion of proteins >70 kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies. SIGNIFICANCE: Here we analyzed the performance of two sample preparation methods for shotgun proteomic analysis of FFPE tissues to give a comprehensive overview of the obtained proteomes and the resemblance to its matching fresh frozen counterparts. These findings give us better understanding towards competent proteomics analysis of FFPE tissues. It is hoped that it will encourage further assessments of available protocols before establishing the most effective protocol for shotgun proteomic FFPE tissue analysis.

Skeletal muscle is a heterogeneous tissue comprised of muscle fiber and mononuclear cell types that, in addition to movement, influences immunity, metabolism and cognition. We investigated the gene expression patterns of skeletal muscle cells using RNA-seq of subtype-pooled single human muscle fibers and single cell RNA-seq of mononuclear cells from human vastus lateralis, mouse quadriceps, and mouse diaphragm. We identified 11 human skeletal muscle mononuclear cell types, including two fibro-adipogenic progenitor (FAP) cell subtypes. The human FBN1+ FAP cell subtype is novel and a corresponding FBN1+ FAP cell type was also found in single cell RNA-seq analysis in mouse. Transcriptome exercise studies using bulk tissue analysis do not resolve changes in individual cell-type proportion or gene expression. The cell-type gene signatures provide the means to use computational methods to identify cell-type level changes in bulk studies. As an example, we analyzed public transcriptome data from an exercise training study and revealed significant changes in specific mononuclear cell-type proportions related to age, sex, acute exercise and training. Our single-cell expression map of skeletal muscle cell types will further the understanding of the diverse effects of exercise and the pathophysiology of muscle disease.

LβT2 and αT3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. LβT2 cells are more mature gonadotrope precursors than αT3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat (STR) profiling was consistent with a mixed C57BL/6J × BALB/cJ genetic background, with distinct patterns for each cell type. Spectral karyotyping in αT3-1 cells revealed cell-to-cell variation in chromosome composition and pseudodiploidy. In LβT2 cells, chromosome counting and karyotyping demonstrated pseudotriploidy and high chromosomal variation among cells. Chromosome copy number variation was confirmed by single-cell DNA sequencing. Chromosomal compositions were consistent with a male sex for αT3-1 and a female sex for LβT2 cells. Among LβT2 stocks used in multiple laboratories, we detected two genetically similar but distinguishable lines via STR authentication, LβT2a and LβT2b. The two lines differed in morphological appearance, with LβT2a having significantly smaller cell and nucleus areas. Analysis of immediate early gene and gonadotropin subunit gene expression revealed variations in basal expression and responses to continuous and pulsatile GnRH stimulation. LβT2a showed higher basal levels of Egr1, Fos, and Lhb but lower Fos induction. Fshb induction reached significance only in LβT2b cells. Our study highlights the heterogeneity in gonadotrope cell line genomes and provides reference STR authentication patterns that can be monitored to improve experimental reproducibility and facilitate comparisons of results within and across laboratories.

We investigated serological responses following a SARS-CoV-2 outbreak in spring 2020 on a US Marine recruit training base. 147 participants that were isolated during an outbreak of respiratory illness were enrolled in this study, with visits approximately 6 and 10 weeks post-outbreak (PO). This cohort is comprised of young healthy adults, ages 18-26, with a high rate of asymptomatic infection or mild symptoms, and therefore differs from previously reported longitudinal studies on humoral responses to SARS-CoV-2, which often focus on more diverse age populations and worse clinical presentation. 80.9% (119/147) of the participants presented with circulating IgG antibodies against SARS-CoV-2 spike (S) receptor-binding domain (RBD) at 6 weeks PO, of whom 97.3% (111/114) remained positive, with significantly decreased levels, at 10 weeks PO. Neutralizing activity was detected in all sera from SARS-CoV-2 IgG positive participants tested (n=38) at 6 and 10 weeks PO, without significant loss between time points. IgG and IgA antibodies against SARS-CoV-2 RBD, S1, S2, and the nucleocapsid (N) protein, as well neutralization activity, were generally comparable between those participants that had asymptomatic infection or mild disease. A multiplex assay including S proteins from SARS-CoV-2 and related zoonotic and human endemic betacoronaviruses revealed a positive correlation for polyclonal cross-reactivity to S after SARS-CoV-2 infection. Overall, young adults that experienced asymptomatic or mild SARS-CoV-2 infection developed comparable humoral responses, with no decrease in neutralizing activity at least up to 10 weeks after infection.

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus RNA-seq and ATAC-seq resource from pediatric, adult, and aged postmortem pituitaries (snpituitaryatlas.princeton.edu) and characterize cell-type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early-life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors that are significantly associated with gene expression in PSCs compared with other cell types and within PSCs. We identify distinct deterministic mechanisms that contribute to heterogeneous marker expression within PSCs. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes and cell type identity.

Transcriptome studies have revealed age-, disease-, and region-associated microglial phenotypes reflecting changes in microglial function during development, aging, central nervous system homeostasis, and pathology. The molecular mechanisms that contribute to these transcriptomic changes are largely unknown. The aim of this study was to characterize the DNA methylation landscape of human microglia and the factors that contribute to variations in the microglia methylome. We hypothesized that both age and brain region would have a large impact on DNA methylation in microglia.

The most common genetic risk factors for Parkinson's disease (PD) are a set of heterozygous mutant (MT) alleles of the GBA1 gene that encodes β-glucocerebrosidase (GCase), an enzyme normally trafficked through the ER/Golgi apparatus to the lysosomal lumen. We found that half of the GCase in lysosomes from postmortem human GBA-PD brains was present on the lysosomal surface and that this mislocalization depends on a pentapeptide motif in GCase used to target cytosolic protein for degradation by chaperone-mediated autophagy (CMA). MT GCase at the lysosomal surface inhibits CMA, causing accumulation of CMA substrates including α-synuclein. Single-cell transcriptional analysis and proteomics of brains from GBA-PD patients confirmed reduced CMA activity and proteome changes comparable to those in CMA-deficient mouse brain. Loss of the MT GCase CMA motif rescued primary substantia nigra dopaminergic neurons from MT GCase-induced neuronal death. We conclude that MT GBA1 alleles block CMA function and produce α-synuclein accumulation.

Alcohol increases inhibitory neurotransmission, an effect mediated through GABA receptors. With chronic alcohol exposure, the inhibitory effects diminish. Glutamic acid decarboxylase (GAD) catalyzes glutamate in the synthesis of GABA. We sought to determine the amount of GAD65/67 mRNA in anterior cingulate cortex (BA24) and orbital prefrontal cortex (BA45) of medication-free alcoholics and nonpsychiatric controls postmortem. Studies were performed in 16 pairs of nonpsychiatric controls and alcoholics, matched for age, sex and PMI. DSM-IV diagnosis of alcohol use disorder (AUD) was made by the SCID I in a psychological autopsy. Frozen blocks of BA24 or BA45 were sectioned (10 µm) for in situ hybridization of 35S-labelled riboprobe for GAD65/67 mRNA and autoradiograms were analyzed by quantitative densitometry. Three isodensity bands of labeling were evident, with different relative amounts of GAD65 and GAD67 (outer and inner, predominantly GAD65, intermediate predominantly GAD67), and the isodensity bands were analyzed separately. GAD65/67 mRNA levels were not different between alcoholics and controls in the gray matter of BA24 (p = 0.53) or BA45 (p = 0.84) or in any of the three isodensity bands in which the GAD65/67 mRNA was distributed. GAD65/67 mRNA in white matter underlying either region was also not different in alcoholics (p > 0.05). GAD65/67 mRNA levels did not correlate with age, sex or duration of alcoholism in either BA24 or BA45. Effects on inhibitory neurotransmission in alcoholics do not appear to be associated with change in the levels of GAD65 or GAD67 mRNA.

MicroRNA-137 (miR-137) has been shown to play an important role in the differentiation of neural stem cells. Embryonic stem (ES) cells have the potential to differentiate into different cell types including neurons; however, the contribution of miR-137 in the maintenance and differentiation of ES cells remains unknown. Here, we show that miR-137 is mainly expressed in ES cells at the mitotic phase of the cell cycle and highly upregulated during differentiation. We identify that ES cell transcription factors, Klf4 and Tbx3, are downstream targets of miR-137, and we show that endogenous miR-137 represses the 3' untranslated regions of Klf4 and Tbx3. Transfection of ES cells with mature miR-137 RNA duplexes led to a significant reduction in cell proliferation and the expression of Klf4, Tbx3, and other self-renewal genes. Furthermore, we demonstrate that increased miR-137 expression accelerates differentiation of ES cells in vitro. Loss of miR-137 during ES cell differentiation significantly impeded neuronal gene expression and morphogenesis. Taken together, our results suggest that miR-137 regulates ES cell proliferation and differentiation by repressing the expression of downstream targets, including Klf4 and Tbx3.