Jennifer C. Moore

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc-Brn1b and linc-Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr(-/-) neonates, whereas linc-Brn1b(-/-) mutants displayed distinct abnormalities in the generation of upper layer II-IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001.

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.

Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget’s disease, or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP.

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6–10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.

Spinobulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by expansion of a polyglutamine tract in the androgen receptor (AR). This mutation confers toxic function to AR through unknown mechanisms. Mutant AR toxicity requires binding of its hormone ligand, suggesting that pathogenesis involves ligand-induced changes in AR. However, whether toxicity is mediated by native AR function or a novel AR function is unknown. We systematically investigated events downstream of ligand-dependent AR activation in a Drosophila model of SBMA. We show that nuclear translocation of AR is necessary, but not sufficient, for toxicity and that DNA binding by AR is necessary for toxicity. Mutagenesis studies demonstrated that a functional AF-2 domain is essential for toxicity, a finding corroborated by a genetic screen that identified AF-2 interactors as dominant modifiers of degeneration. These findings indicate that SBMA pathogenesis is mediated by misappropriation of native protein function, a mechanism that may apply broadly to polyglutamine diseases.

DDX3X is a DEAD-box RNA helicase that has been implicated in multiple aspects of RNA metabolism including translation initiation and the assembly of stress granules (SGs). Recent genomic studies have reported recurrent DDX3X mutations in numerous tumors including medulloblastoma (MB), but the physiological impact of these mutations is poorly understood. Here we show that a consistent feature of MB-associated mutations is SG hyper-assembly and concomitant translation impairment. We used CLIP-seq to obtain a comprehensive assessment of DDX3X binding targets and ribosome profiling for high-resolution assessment of global translation. Surprisingly, mutant DDX3X expression caused broad inhibition of translation that impacted DDX3X targeted and non-targeted mRNAs alike. Assessment of translation efficiency with single-cell resolution revealed that SG hyper-assembly correlated precisely with impaired global translation. SG hyper-assembly and translation impairment driven by mutant DDX3X were rescued by a genetic approach that limited SG assembly and by deletion of the N-terminal low complexity domain within DDX3X. Thus, in addition to a primary defect at the level of translation initiation caused by DDX3X mutation, SG assembly itself contributes to global translation inhibition. This work provides mechanistic insights into the consequences of cancer-related DDX3X mutations, suggesting that globally reduced translation may provide a context-dependent survival advantage that must be considered as a possible contributor to tumorigenesis.

Abstract Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo . Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance.

Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option. Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Abstract Follicular lymphoma is a rare lymphoid malignancy in pediatric patients and consequently remains poorly characterized, particularly with respect to its immunophenotype and molecular pathogenesis. A total of 23 pediatric patients with follicular lymphoma were identified, with a median age of 11 years and a male-to-female ratio of 2.3:1. Of the 19 patients for whom presenting clinical features were available, 15 patients had stage I, 1 had stage II, and 3 had stage III or IV disease. All tumors had a follicular architecture, and 74% of cases had grade 2 or 3 histologic features. All patients expressed CD20 and bcl-6, and 80% were positive for CD10. Bcl-2 expression was detected in only 5 of 16 cases. Consistent with this finding, bcl-2 gene rearrangements were detected in only 2 of 16 cases by polymerase chain reaction. These patients were treated primarily with cyclophosphamide, doxorubicin, vincristine, and prednisone–based chemotherapy; 4 patients also received involved-field irradiation. Of the 13 patients with available clinical follow-up, all but 2 achieved durable clinical remission. Importantly, all 4 patients with tumors diffusely positive for bcl-2 either presented with stage III/IV disease or had disease refractory to therapy, whereas patients with bcl-2–negative tumors uniformly had stage I disease, achieved complete remission, and experienced no relapses. These findings indicate that, in contrast to adult follicular lymphomas, dysregulated bcl-2 expression does not play a significant pathogenetic role in most pediatric follicular lymphomas. However, bcl-2 expression in pediatric follicular lymphoma identifies a subset of patients in whom disease is often disseminated at clinical presentation and is more refractory to combination chemotherapy.

Abstract The Runx1/core binding factor-β (CBFβ) transcriptional complex is required for the establishment of hematopoiesis during development. Despite its critical role during development, a detailed analysis of Runx1 expression within specific lineages and developmental stages of the adult hematopoietic system is lacking. To address this, we have developed a Runx1—green fluorescent protein (GFP) knock-in mouse. We show that Runx1 is expressed in several hematopoietic lineages, including myeloid, B-lymphoid, and T-lymphoid cells. By contrast, Runx1 is weakly expressed in early erythroid cells, and its expression is rapidly extinguished during later stages of erythropoiesis. Runx1 expression is induced during early B-cell development and is expressed at a uniform level during all subsequent stages of B-cell development. Within the thymus, Runx1 is expressed at the highest level in CD4-CD8- double-negative thymocytes. In peripheral T cells, Runx1 is differentially expressed, with CD4+ T cells expressing 2- to 3-fold higher levels of Runx1 than CD8+ cells. Taken together, these findings indicate that although widely expressed in the hematopoietic system, the expression of Runx1 is regulated in a cell type— and maturation stage—specific manner. In addition, the Runx1-IRES-GFP knock-in mouse strain should prove valuable for investigation of Runx1 function in adult hematopoiesis.

MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation.SOLiD ultra-deep sequencing identified >10(7) unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs.Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.

B7-H3 is actively being explored as an immunotherapy target for pediatric patients with solid tumors using monoclonal antibodies or T cells expressing chimeric antigen receptors (CARs). B7-H3-CARs containing a 41BB costimulatory domain are currently favored by several groups based on preclinical studies. In this study, we initially performed a detailed analysis of T cells expressing B7-H3-CARs with different hinge/transmembrane (CD8α versus CD28) and CD28 or 41BB costimulatory domains (CD8α/CD28, CD8α/41BB, CD28/CD28, CD28/41BB). Only subtle differences in effector function were observed between CAR T cell populations in vitro. However, CD8α/CD28-CAR T cells consistently outperformed other CAR T cell populations in three animal models, resulting in a significant survival advantage. We next explored whether adding 41BB signaling to CD8α/CD28-CAR T cells would further enhance effector function. Surprisingly, incorporating 41BB signaling into the CAR endodomain had detrimental effects, while expressing 41BBL on the surface of CD8α/CD28-CAR T cells enhanced their ability to kill tumor cells in repeat stimulation assays. Furthermore, 41BBL expression enhanced CD8α/CD28-CAR T cell expansion in vivo and improved antitumor activity in one of four evaluated models. Thus, our study highlights the intricate interplay between CAR hinge/transmembrane and costimulatory domains. Based on our study, we selected CD8α/CD28-CAR T cells expressing 41BBL for early phase clinical testing.

Although both the H1 and HES2 human embryonic stem cell lines (NIH codes: WA01 and ES02, respectively) are capable of forming all three germ layers and their derivatives, various lines of evidence including the need to use different protocols to induce cardiac differentiation hint that they have distinct preferences to become chamber-specific heart cells. However, a direct systematic comparison has not been reported. Here we electrophysiologically demonstrated that the distributions of ventricular-, atrial- and pacemaker-like derivatives were indeed different (ratios = 39:61:0 and 64:33:3 for H1 and HES2, respectively). Based on these results, we hypothesized the differences in their cardiogenic potentials are imprinted in the proteomes of undifferentiated H1 and HES2. Using multiplexing, high-resolution 2-D Differential In Gel Electrophoresis (DIGE) to minimize gel-to-gel variations that are common in conventional 2-D gels, a total of 2000 individual protein spots were separated. Of which, 55 were >2-fold differentially expressed in H1 and HES2 (p < 0.05) and identified by mass spectrometery. Bioinformatic analysis of these protein differences further revealed candidate pathways that contribute to the H1 and HES2 phenotypes. We conclude that H1 and HES2 have predetermined preferences to become ventricular, atrial, and pacemaker cells due to discrete differences in their proteomes. These results improve our basic understanding of hESCs and may lead to mechanism-based methods for their directed cardiac differentiation into chamber-specific cardiomyocytes.

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self-renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3-D microgeometries can support hESC colony formation, self-renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions.

Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs), but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived VCMs, human fetal and human adult ventricular and atrial CMs.Using two-dimensional-differential-in-gel electrophoresis, 121 differentially expressed (>1.5-fold; P<0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator-activated receptor α signaling in cardiac maturation. Consistently, WY-14643, a peroxisome proliferator-activated receptor α agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold, signifying their maturation.We conclude that the peroxisome proliferator-activated receptor α and thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.

Background:Herpes simplex virus, type 1 (HSV-1) commonly produces lytic mucosal lesions. It invariably initiates latent infection in sensory ganglia enabling persistent, lifelong infection. Acute HSV-1 encephalitis is rare and definitive evidence of latent infection in the brain is lacking. However, exposure untraceable to encephalitis has been repeatedly associated with impaired working memory and executive functions, particularly among schizophrenia patients.

Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC-Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC-Heps generated using a chemically defined four-step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D poly(ethylene glycol)-diacrylate fabricated inverted colloid crystal scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune-privileged material alginate and remained viable as well as functional upon transplantation into immune competent mice. This is the first report we are aware of demonstrating cGMP-compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications. Stem Cells Translational Medicine 2019;8:124&14.

Importance Current Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) ( DSM-5 ) diagnoses of substance use disorders rely on criterion count–based approaches, disregarding severity grading indexed by individual criteria. Objective To examine correlates of alcohol use disorder (AUD) across count-based severity groups (ie, mild, moderate, mild-to-moderate, severe), identify specific diagnostic criteria indicative of greater severity, and evaluate whether specific criteria within mild-to-moderate AUD differentiate across relevant correlates and manifest in greater hazards of severe AUD development. Design, Setting, and Participants This cohort study involved 2 cohorts from the family-based Collaborative Study on the Genetics of Alcoholism (COGA) with 7 sites across the United States: cross-sectional (assessed 1991-2005) and longitudinal (assessed 2004-2019). Statistical analyses were conducted from December 2022 to June 2023. Main Outcomes and Measures Sociodemographic, alcohol-related, psychiatric comorbidity, brain electroencephalography (EEG), and AUD polygenic score measures as correlates of DSM-5 AUD levels (ie, mild, moderate, severe) and criterion severity–defined mild-to-moderate AUD diagnostic groups (ie, low-risk vs high-risk mild-to-moderate). Results A total of 13 110 individuals from the cross-sectional COGA cohort (mean [SD] age, 37.8 [14.2] years) and 2818 individuals from the longitudinal COGA cohort (mean baseline [SD] age, 16.1 [3.2] years) were included. Associations with alcohol-related, psychiatric, EEG, and AUD polygenic score measures reinforced the role of increasing criterion counts as indexing severity. Yet within mild-to-moderate AUD (2-5 criteria), the presence of specific high-risk criteria (eg, withdrawal) identified a group reporting heavier drinking and greater psychiatric comorbidity even after accounting for criterion count differences. In longitudinal analyses, prior mild-to-moderate AUD characterized by endorsement of at least 1 high-risk criterion was associated with more accelerated progression to severe AUD (adjusted hazard ratio [aHR], 11.62; 95% CI, 7.54-17.92) compared with prior mild-to-moderate AUD without endorsement of high-risk criteria (aHR, 5.64; 95% CI, 3.28-9.70), independent of criterion count. Conclusions and Relevance In this cohort study of a combined 15 928 individuals, findings suggested that simple count-based AUD diagnostic approaches to estimating severe AUD vulnerability, which ignore heterogeneity among criteria, may be improved by emphasizing specific high-risk criteria. Such emphasis may allow better focus on individuals at the greatest risk and improve understanding of the development of AUD.

Oxytocin (OT), a hormone recently identified in the heart, induces embryonic and cardiac somatic stem cells to differentiate into cardiomyocytes (CM), possibly through nitric oxide (NO). We verified this hypothesis using P19 cells and P19 Clone 6 derivatives expressing a green fluorescent protein (GFP) reporter linked to cardiac myosin light chain-2v promoter. OT treatment of these cells induced beating cell colonies that were fully inhibited by N,G-nitro-L-arginine-methyl-ester (L-NAME), an inhibitor of NO synthases (NOS), partially reduced by 1400W, an inhibitor of inducible NOS, and ODQ, an inhibitor of NO-sensitive guanylyl cyclases. The NO generator S-nitroso-N-acetylpenicillamine (SNAP) reversed the L-NAME inhibition of cell beating and GFP expression. In OT-induced cells, L-NAME significantly decreased transcripts of the cardiac markers Nkx2.5, MEF2c, alpha-myosin heavy chain, and less, GATA4, endothelial NOS, and atrial natriuretic peptide, as well as the skeletal myocyte (SM) marker myogenin. Image analysis of OT-induced P19Cl6-GFP cells revealed ventricular CM coexpressing sarcomeric alpha-actinin and GFP, with some cells exclusively expressing alpha-actinin, most likely of the SM phenotype. The OT-mediated production of CM, but not SM, was diminished by L-NAME. In P19 cells, exogenously added OT stimulated the expression of its own transcript, which was reduced in the presence of L-NAME. Surprisingly, L-NAME alone decreased the expression of anti-stage specific embryonic antigen-1 marker of the undifferentiated state and induced some beating colonies as well as GFP in P19Cl6-GFP cells. Collectively, our data suggest that the pleiotropic action of NO is involved in the initiation of CM differentiation of P19 cells and maintenance of their undifferentiated state.

Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. We investigated the ability of two commercially available electroporation systems, the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However, the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency, producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally, we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. Our results indicate that these electroporation methods provide a reliable, efficient, and high-throughput approach to the genetic manipulation of hESC.

A preclinical therapy to treat neurodegeneration is developed that selectively targets the AF-2 domain of the androgen receptor while sparing other functions of this receptor. Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by toxic gain of function of the androgen receptor (AR). Previously, we found that co-regulator binding through the activation function-2 (AF2) domain of AR is essential for pathogenesis, suggesting that AF2 may be a potential drug target for selective modulation of toxic AR activity. We screened previously identified AF2 modulators for their ability to rescue toxicity in a Drosophila model of SBMA. We identified two compounds, tolfenamic acid (TA) and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole (MEPB), as top candidates for rescuing lethality, locomotor function and neuromuscular junction defects in SBMA flies. Pharmacokinetic analyses in mice revealed a more favorable bioavailability and tissue retention of MEPB compared with TA in muscle, brain and spinal cord. In a preclinical trial in a new mouse model of SBMA, MEPB treatment yielded a dose-dependent rescue from loss of body weight, rotarod activity and grip strength. In addition, MEPB ameliorated neuronal loss, neurogenic atrophy and testicular atrophy, validating AF2 modulation as a potent androgen-sparing strategy for SBMA therapy.

Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk of addiction-associated phenotypes in humans yet little is known the underlying neural basis. Induced pluripotent stem cells (iPSCs) were derived from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in CHRNA5. To understand the impact of these nicotinic receptor variants in humans, we differentiated these iPSCs to dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Results show that N398 variant human DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. While both variants exhibited physiological properties consistent with mature neuronal function, the N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of nicotine in both DA and glutamatergic neurons. Glutamatergic N398 neurons responded to lower nicotine doses (0.1 μM) with greater frequency and amplitude but they also exhibited rapid desensitization, consistent with previous analyses of N398-associated nicotinic receptor function. This study offers a proof-of-principle for utilizing human neurons to study gene variants contribution to addiction.

The OPRM1 A118G single nucleotide polymorphism (SNP rs1799971) gene variant encoding the N40D µ-opioid receptor (MOR) has been associated with dependence on opiates and other drugs of abuse but its mechanism is unknown. The frequency of G-allele carriers is ~40% in Asians, ~16% in Europeans, and ~3% in African-Americans. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy. Here we generated homozygous N40D subject-specific induced inhibitory neuronal cells (iNs) from seven human-induced pluripotent stem (iPS) cell lines from subjects of European descent (both male and female) and probed the impact of N40D MOR regulation on synaptic transmission. We found that D40 iNs exhibit consistently stronger suppression (versus N40) of spontaneous inhibitory postsynaptic currents (sIPSCs) across multiple subjects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were recapitulated in two sets of independently engineered isogenic N40D iNs. In addition, we employed biochemical analysis and observed differential N-linked glycosylation of human MOR N40D. This study identifies neurophysiological and molecular differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.

<h3>Objective</h3> Dystonia is a complex movement disorder. Research progress has been difficult, particularly in developing widely effective therapies. This is a review of the current state of knowledge, research gaps, and proposed research priorities. <h3>Methods</h3> The NIH convened leaders in the field for a 2-day workshop. The participants addressed the natural history of the disease, the underlying etiology, the pathophysiology, relevant research technologies, research resources, and therapeutic approaches and attempted to prioritize dystonia research recommendations. <h3>Results</h3> The heterogeneity of dystonia poses challenges to research and therapy development. Much can be learned from specific genetic subtypes, and the disorder can be conceptualized along clinical, etiology, and pathophysiology axes. Advances in research technology and pooled resources can accelerate progress. Although etiologically based therapies would be optimal, a focus on circuit abnormalities can provide a convergent common target for symptomatic therapies across dystonia subtypes. The discussions have been integrated into a comprehensive review of all aspects of dystonia. <h3>Conclusion</h3> Overall research priorities include the generation and integration of high-quality phenotypic and genotypic data, reproducing key features in cellular and animal models, both of basic cellular mechanisms and phenotypes, leveraging new research technologies, and targeting circuit-level dysfunction with therapeutic interventions. Collaboration is necessary both for collection of large data sets and integration of different research methods.

Almost 7 years after their first derivation from human embryos, a pressing urgency to deliver the promises of therapies based on human embryonic stem cells (hESC) has arisen. Protocols have been developed to support long-term growth of undifferentiated cells and partially direct differentiation to specific cell lineages. The stage has almost been set for the next step: transplantation in animal models of human disease. Here, we review the state-of-the-art with respect to the transplantation of embryonic stem cell-derived heart cells in animals. One problem affecting progress in this area and functional analysis in vivo in general, is the availability of genetically marked hESC. There are only a few cell lines that express reporter genes ubiquitously, and none is associated with particular lineages; a major hurdle has been the resistance of hESC to established infection and chemical transfection methodologies to introduce ectopic genes. The methods that have been successful are reviewed. We also describe the processes for generating a new, genetically-modified hESC line that constitutively expresses GFP as well as some of its characteristics, including its ability to form cardiomyocytes with electrophysiological properties of ventricular-like cells.

&lt;b&gt;&lt;i&gt;Background:&lt;/i&gt;&lt;/b&gt; Copy number variation on chromosome 15q11.2 (BP1-BP2) causes a deletion of &lt;i&gt;CYFIP1&lt;/i&gt;, &lt;i&gt;NIPA1&lt;/i&gt;, &lt;i&gt;NIPA2&lt;/i&gt; and &lt;i&gt;TUBGCP5&lt;/i&gt;. Furthermore, it also affects brain structure and elevates the risk for several neurodevelopmental disorders that are associated with dendritic spine abnormalities. In rodents, altered &lt;i&gt;cyfip1 &lt;/i&gt;expression changes dendritic spine morphology, motivating analyses of human neuronal cells derived from induced pluripotent stem cells (iPSCs; iPSC-neurons). &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; iPSCs were generated from a mother and her offspring, both carrying the 15q11.2 (BP1-BP2) deletion, and a non-deletion control. Gene expression in the deletion region was estimated using quantitative real-time PCR assays. Neural progenitor cells (NPCs) and iPSC-neurons were characterized using immunocytochemistry. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt;&lt;i&gt;CYFIP1&lt;/i&gt;, &lt;i&gt;NIPA1&lt;/i&gt;, &lt;i&gt;NIPA2&lt;/i&gt; and &lt;i&gt;TUBGCP5&lt;/i&gt; gene expression was lower in iPSCs, NPCs and iPSC-neurons from the mother and her offspring in relation to control cells. CYFIP1 and PSD-95 protein levels were lower in iPSC-neurons derived from the copy number variant-bearing individuals using Western blot analysis. Ten weeks after differentiation, iPSC-neurons appeared to show dendritic spines, and qualitative analysis suggested that dendritic morphology was altered in 15q11.2-deletion subjects compared with control cells. &lt;b&gt;&lt;i&gt;Conclusions:&lt;/i&gt;&lt;/b&gt; The 15q11.2 (BP1-BP2) deletion is associated with a reduced expression of four genes in iPSC-derived neuronal cells; it may also be associated with altered iPSC-neuron dendritic morphology.

Alcohol abuse produces an enormous impact on health, society, and the economy. Currently, there are very limited therapies available, largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs, but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3+ (LC3B+) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition, a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover, a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells. This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models.

Non-coding RNA, including microRNA (miRNA) serves critical regulatory functions in the developing brain. The let-7 family of miRNAs has been shown to regulate neuronal differentiation, neural subtype specification, and synapse formation in animal models. However, the regulatory role of human let-7c (hsa-let-7c) in human neuronal development has yet to be examined. Let-7c is encoded on chromosome 21 in humans and therefore may be overexpressed in human brains in Trisomy 21 (T21), a complex neurodevelopmental disorder. Here, we employ recent developments in stem cell biology to show that hsa-let-7c mediates important regulatory epigenetic functions that control the development and functional activity of human induced neuronal cells (iNs). We show that overexpression of hsa-let-7c in human iNs derived from induced pluripotent stem (iPS), as well as embryonic stem (ES), cells leads to morphological as well as functional deficits including impaired neuronal morphologic development, synapse formation and synaptic strength, as well as a marked reduction of neuronal excitability. Importantly, we have assessed these findings over three independent genetic backgrounds, showing that some of these effects are subject to influence by background genetic variability with the most robust and reproducible effect being a striking reduction in spontaneous neural firing. Collectively, these results suggest an important function for let-7 family miRNAs in regulation of human neuronal development and raise implications for understanding the complex molecular etiology of neurodevelopmental disorders, such as T21, where let-7c gene dosage is increased.

The clinical application of stem cell therapies is still limited by the ability to produce defined, differentiated cell populations in large numbers in culture. High throughput screens to identify factors which enhance differentiation to particular lineages and promote expansion of precursors in culture are dependent on the development of sensitive and reproducible assays for screening. Here we describe a bioassay to identify factors with cardiomyogenic activity which enhance the yield of cardiomyocytes from undifferentiated stem cells. The assay is based on a Green Fluorescent Protein (GFP) reporter under the transcriptional control of the 250 bp MLC-2v promoter expressed in pluripotent P19 embryonal carcinoma cells. We show that reporter expression is limited to developing cardiomyocytes and can be used to determine quantitatively the number of ventricular cardiomyocytes formed in cultures under inducing or non-inducing conditions. This assay differs from all others described previously in that it has an easily quantifiable readout, there is negligible background differentiation in the absence of exogenous cardiogenic factors and it is carried out feeder cell-free. Thus, it is entirely independent of competing differentiation inhibitory factors, such as leukemia inhibitory factor. Patch clamp electrophysiology of the GFP-positive cells confirmed their functional ventricular phenotype and indicated that selection on the basis of GFP would provide cells suitable for transplantation.

RNA interference (RNAi), a post-transcriptional gene silencing mechanism originally described in Caenorhabditis elegans, involves sequence-specific mRNA degradation mediated by double-stranded RNAs (dsRNAs). Passive dsRNA uptake has been uniquely observed in C. elegans due to the expression of systemic RNA interference defective-1 (SID-1). Here we investigated the ability of ectopic SID-1 expression to enable passive cellular uptake of short interfering RNA (siRNA) or double stranded RNA (dsRNA) in pluripotent mouse embryonic stem cells (mESCs). When SID-1-GFP and the Firefly luciferase reporter gene (luc(Fir)) were co-expressed in mESCs, luc(Fir) activity could be suppressed by simple incubation with dsRNAs/siRNAs that were designed to specifically target luc(Fir). By contrast, suppression was not observed in mESCs expressing luc(Fir) and GFP alone or when either GFP- or SID-1-GFP-expressing cells were incubated with control dsRNAs/siRNAs (non-silencing or Renilla luciferase-specific). These results may lead to high-throughput experimental strategies for studying ESC differentiation and novel approaches to genetically inhibit or eliminate the tumorigenicity of ESCs.

Cardiovascular diseases remain the leading cause of mortality and morbidity worldwide. Despite substantial improvements in acute management, survivors of myocardial infarction often progress to heart failure. Since adult cardiomyocytes (CMs) do not regenerate, their loss permanently compromises myocardial contractile function. Heart transplantation is currently the last resort for end-stage heart failure, but is hampered by a severe shortage of donor organs and rejection. Cell-based therapies are a promising alternative: Various cell types such as human fetal CMs, skeletal muscle myoblasts and smooth muscle cells have been tested but these approaches are also limited by cell availability or side effects (e.g. due to their non-cardiac identity). In recent years, clinical studies exploiting adult bone marrow mesenchymal stem cells for transplantation in patients with coronary artery disease have reported favorable outcomes but their cardiomyogenic ability is limited. By contrast, human embryonic stem cells (hESCs), derived from the inner cell mass of blastocyst-stage embryos, are pluripotent and can self-renew and differentiate into all cell types including CMs. Furthermore, hESC-derived CMs (hESC-CMs) are viable human heart cells that can functionally integrate with the recipient organ after transplantation. This article reviews the current state and hurdles of hESC-CM research, as well as their therapeutic potentials and limitations.

Chimeric antigen receptor (CAR) T cells targeting CD123, an acute myeloid leukemia (AML) antigen, hold the promise of improving outcomes for patients with refractory/recurrent disease. We generated five lentiviral vectors encoding CD20, which may serve as a target for CAR T cell depletion, and 2^nd or 3^rd generation CD123-CARs since the benefit of two costimulatory domains is model dependent. Four CARs were based on the CD123-specific single-chain variable fragment (scFv) 26292 (292) and one CAR on the CD123-specific scFv 26716 (716), respectively. We designed CARs with different hinge/transmembrane (H/TM) domains and costimulatory domains, in combination with the zeta (z) signaling domain: 292.CD8aH/TM.41BBz (8.41BBz), 292.CD8aH/TM.CD28z (8.28z), 716.CD8aH/TM.CD28z (716.8.28z), 292.CD28H/TM. CD28z (28.28z), and 292.CD28H/TM.CD28.41BBz (28.28.41BBz). Transduction efficiency, expansion, phenotype, and target cell recognition of the generated CD123-CAR T cells did not significantly differ. CAR constructs were eliminated for the following reasons: (1) 8.41BBz CARs induced significant baseline signaling, (2) 716.8.28z CAR T cells had decreased anti-AML activity, and (3) CD28.41BBz CAR T cells had no improved effector function in comparison to CD28z CAR T cells. We selected the 28.28z CAR since CAR expression on the cell surface of transduced T cells was higher in comparison to 8.28z CARs. The clinical study (NCT04318678) evaluating 28.28z CAR T cells is now open for patient accrual.

Two whales were observed closely for an hour off Sarasota, Florida, by residents who provided observations of structural details which identify only the right whale, Eubalaena glacialis, a temperate and subpolar species previously known to range to the Florida east coast, but not to enter the Gulf of Mexico.

A spontaneously reverted iPSC line was identified from an A-T subject with heterozygous ATM truncation mutations. The reverted iPSC line expressed ATM protein and was capable of radiation-induced phosphorylation of CHK2 and H2A.X. Genome-wide SNP analysis confirmed a match to source T cells and also to a distinct, non-reverted iPSC line from the same subject. Rearranged T cell receptor sequences predict that the iPSC culture originated as several independently reprogrammed cells that resolved into a single major clone, suggesting that gene correction likely occurred early in the reprogramming process. Gene expression analysis comparing ATM−/− iPSC lines to unrelated ATM+/− cells identifies a large number of differences, but comparing only the isogenic pair of A-T iPSC lines reveals that the primary pathway affected by loss of ATM is a diminished expression of p53-related mRNAs. Gene reversion in culture, although likely a rare event, provided a novel, reverted cell line for studying ATM function.

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Strongylocentrotus purpuratus. Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity.

Gap junctions, encoded by the connexin (Cx) multi-gene family, couple adjacent cells and underlie cell–cell communications. Previous mouse studies suggest that Cxs play an important role in development but their role in human cardiogenesis is undefined. Human embryonic stem cells (hESC) provide a unique model for studying human differentiation. Lentivirus-mediated stable overexpression of Cx43 in hESC (Cx43-hESC) did not affect colony morphology, karyotype and expression of pluripotency genes such as Oct4 but completely suppressed the formation of spontaneously beating, cardiomyocyte-containing clusters in embryoid bodies (EBs). Unlike control hEBs, the transcripts of several mesodermal markers (kallikrein, δ-globin, and CMP), ventricular myosin light chain and cardiac troponin I were absent or delayed. Transcriptomic and pathway analyses showed that 194 genes crucial for movement, growth, differentiation and maintenance were differentially expressed in Cx43-hESC. We conclude that Cx43 mediates the expression of an array of genes involved in human cardiogenesis, in addition to intercellular communication.

We have generated two iPSC lines from skin biopsies of two healthy individuals. Skin fibroblasts were derived and reprogrammed using a Sendai virus-based approach. The resulting iPSC lines have normal karyotype, express stemness markers and can generate endoderm, mesoderm and ectoderm in vitro. These iPSC lines can be used as healthy controls in differentiation paradigms as well as backbone for gene editing experiments.

cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development.In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases.Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis.An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA.Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo.In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae.These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.

SUMMARY A major impediment to the actualization of the induced pluripotent stem cell (iPSC)-based personalized medicine revolution is the lack of widely accepted standard operating procedures (SOPs) across different groups and institutions. The various methods employed can include choice of starting materials, reprogramming agents, and culture conditions, with each of these factors hypothesized to influence the reprogramming efficiency and transcriptional identity of iPSCs. As such, we systematically compared iPSC reprogramming procedures using cells derived from the somatic cells of three patients with 16p11.2 deletion syndrome (16p11.2del) and found remarkable similarity among the different methods. FACS analysis revealed that regardless of somatic cell type (fibroblast, lymphocyte, erythroblast), route of reprogramming factor introduction (mRNA, Sendai virus, episome), donor sex, or facility (Rutgers, NYSCF), 16p11.2del patient iPSCs were viable as high purity cultures expressing pluripotency marker proteins. This observation was supported at the transcript level by qPCR analysis, which demonstrated the ability for the iPSCs to differentiate into all three embryonic germ cell lineages after 12 days in culture as embryoid bodies. NGN2-mediated differentiation of these iPSCs produced functional neurons that formed active synaptic networks as revealed by multi-electrode array (MEA) recordings. Importantly, no group-wise comparisons among the reprogramming methods yielded consistent statistically significant differences, indicating that these procedures are equally capable of producing pluripotent stem cells that can efficiently differentiate into mature, functional neurons. This work highlights the utility of these reprogramming methods and supports the use of differentially reprogrammed iPSCs for direct comparative studies of human neurodevelopment.

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies, particularly in view of the diverse methods for derivation and maintenance of these cell lines. However, characterization methods are generally not standardized and many currently used assays are subjective, making dependable and direct comparison of cell lines difficult. In order to address this problem, we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines, derived in two different laboratories using different derivation techniques, as pathogen free, genetically stable, and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control, a crucial element of successful hESC research and clinical translation.

ABSTRACT Download Free Sample The study of mental health disorders and the genetics behind these disorders can be greatly enhanced by the use of induced pluripotent stem cells (iPSC). Since many mental health disorders develop after puberty, the only way in which to study the genetic mechanism of these diseases previously was through cellular surrogates, such as blood or cultured fibroblasts. Having the ability to reprogram adult cells to the pluripotent stage provides the capacity to study the onset of these disorders during a culture model of neural development and to include the impact of genetic risk factors and potential environmental triggers. Working with the National Institute of Mental Health (NIMH), the Rutgers Cell and DNA Repository (RUCDR) has begun banking iPSC source cells and converting those source cells into iPSC for distribution to the scientific community. Although initial protocols were developed to reprogram fibroblasts, the ability to reprogram blood cells has several advantages inc...

Multipotent P19CL6 cells differentiate into cardiac myocytes or neural lineages when stimulated with dimethyl sulfoxide (DMSO) or retinoic acid (RA), respectively. Expression of the transcription factor Tbx6 was found to increase during cardiac myocyte differentiation and to decrease during neural differentiation. Overexpression of Tbx6 was not sufficient to drive P19CL6 cells to a cardiac myocyte fate or to accelerate DMSO-induced differentiation. In contrast, knockdown of Tbx6 dramatically inhibited DMSO-induced differentiation of P19CL6 cells to cardiac myocytes, as evidenced by the loss of striated muscle-specific markers and spontaneous beating. Tbx6 knockdown was also accompanied by almost complete loss of Nkx2.5, a transcription factor involved in the specification of the cardiac myocyte lineage, indicating that Nkx2.5 is downstream of Tbx6. In distinction to its positive role in cardiac myocyte differentiation, Tbx6 knockdown augmented RA-induced differentiation of P19CL6 cells to both neurons and glia, and accelerated the rate of neurite formation. Conversely, Tbx6 overexpression attenuated differentiation to neural lineages. Thus, in the P19CL6 model, Tbx6 is required for cardiac myocyte differentiation and represses neural differentiation. We propose a model in which Tbx6 is a part of a molecular switch that modulates divergent differentiation programs within a single progenitor cell.

Human somatic cells can be reprogrammed to the pluripotent state to become human-induced pluripotent stem cells (hiPSC). This reprogramming is achieved by activating signaling pathways that are expressed during early development. These pathways can be induced by ectopic expression of four transcription factors—Oct4, Sox2, Klf4, and c-Myc. Although there are many ways to deliver these transcription factors into the somatic cells, this chapter will provide protocols that can be used to generate hiPSC from lentiviruses.

Abstract Background The OPRM1 A118G gene variant (N40D) encoding the µ-opioid receptor (MOR) has been associated with dependence on opiates and other abused drugs but its mechanism is unknown. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy. Methods Seven human induced pluripotent stem (iPS) cell lines from homozygous N40D subjects (4 with N40 and 3 with D40 variants) were generated and human induced neuronal cells (iNs) were derived from these iPS cell lines. Morphological, gene expression as well as synaptic physiology analyses were conducted in human iN cells carrying N40D MOR variants; Two pairs of isogenic pluripotent stem cells carrying N40D were generated using CRISPR/Cas9 genome-editing and iN cells derived from them were analyzed. Results Inhibitory human neurons generated from subjects carrying N40D MOR gene variants show mature properties in morphological and functional analyses. Gene expression revealed that they express mature neuronal marker and MORs. Activation of MORs suppressed inhibitory synaptic transmission in human neurons carrying both N40 or D40 MOR variants but D40 show stronger effects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were validated in two sets of independently generated isogenic N40D iNs. Conclusions Activations of N40D MOR variants show different regulatory effects on synaptic transmission in inhibitory human neurons. This study identifies neurophysiological differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.

Abnormal dendritic arbor development has been implicated in a number of neurodevelopmental disorders, such as autism and Rett syndrome, and the neuropsychiatric disorder schizophrenia. Postmortem brain samples from subjects with schizophrenia show elevated levels of NOS1AP in the dorsolateral prefrontal cortex, a region of the brain associated with cognitive function. We previously reported that the long isoform of NOS1AP (NOS1AP-L), but not the short isoform (NOS1AP-S), negatively regulates dendrite branching in rat hippocampal neurons. To investigate the role that NOS1AP isoforms play in human dendritic arbor development, we adapted methods to generate human neural progenitor cells and neurons using induced pluripotent stem cell (iPSC) technology. We found that increased protein levels of either NOS1AP-L or NOS1AP-S decrease dendrite branching in human neurons at the developmental time point when primary and secondary branching actively occurs. Next, we tested whether pharmacological agents can decrease the expression of NOS1AP isoforms. Treatment of human iPSC-derived neurons with d-serine, but not clozapine, haloperidol, fluphenazine, or GLYX-13, results in a reduction in endogenous NOS1AP-L, but not NOS1AP-S, protein expression; however, d-serine treatment does not reverse decreases in dendrite number mediated by overexpression of NOS1AP isoforms. In summary, we demonstrate how an in vitro model of human neuronal development can help in understanding the etiology of schizophrenia and can also be used as a platform to screen drugs for patients.

Driven maturation of embryonic stem cell-derived cardiomyocytes confers post-transplantation safety

Drug use disorders (DUDs) related to prescription opioids (narcotic pain medications) have a huge socioeconomical impact; however, the molecular and cellular mechanism of how opioids function in human is largely unknown. The Single Nucleotide Polymorphism (SNP) rs1799971 (OPRM1 A118G), produces a non-synonymous amino acid substitution, replacing Asparagine (N40) with Aspartate (D40), in the μ-opioid receptor (MOR), and is strongly associated with Drug use disorders (DUDs) and alcoholism. The impact of these genetic variants on neural function, including synaptic transmission and its molecular/cellular underpinnings, has not been established in humans. To lay the foundation for addressing these important and outstanding questions, we are using the induced neuronal (iN) cell technology that we have pioneered to generate human neurons carrying homozygous N40 or D40 MORs. Specifically, we have generated multiple induced pluripotent stem (iPS) cell lines carrying either homozygous MOR N40 or MOR D40 alleles from human subjects. Additionally, in order to mitigate the possible confounding issues related to the genetic background variations among these subjects, we also used CRISPR/Cas9 mediated gene targeting to create two isogenic pairs of human pluripotent stem cell lines: one introducing homozygous D40 alleles into a human embryonic stem (ES) cell line and the other converting one of the D40 homozygous iPS cell lines to homozygous N40. Using human neurons as a model system, our compelling preliminary data suggest that: 1) activation of both N40 and D40 MORs by agonist DAMGO in human neurons suppresses inhibitory synaptic release and the D40 MORs show stronger inhibitory effects; 2) exposure of human neurons to DAMGO for 1 day leads to loss of the DAMGO-induced suppression of synaptic release, possibly due to internalization of MORs or altered intracellular signaling; and 3) D40 human neurons show defective recovery from the loss of MOR regulation on synaptic transmission induced by prolonged agonist exposure. The objective of this proposal is to determine the functional impact and the underlying cellular/molecular mechanisms of OPRM1 allelic variants. The central hypothesis is that the N40 to D40 substitution leads to aberrant MOR expression and/or downstream signaling, resulting in altered cellular responses. The proposed research is innovative, because we will combine recent developments in stem cell biology with synaptic physiology to directly probe the impact of OPRM1 gene variants on synaptic function in human neurons. Our expectations are that cellular and synaptic function is altered in neurons carrying the MOR D40 variant. This study will provide fundamental biological insight into the regulatory functions of the MOR in human neurons.

Several years ago the stranding of a rare species of whale on the Florida Keys stimulated one of us to attempt to discover just what ranges of marine mammals could be shown to extend into Florida waters. When the results of this investigation were recorded (Moore, 1953), one of the interesting items revealed was the occurrence of the little piked whale, Balaenoptera acutorostrata, in the Gulf of Mexico. This midget member of the finback family of the whalebone whales was known before this to occur only as far south as Long Beach (lat. 39°30′ N.), New Jersey, where one stranded in the fall of 1866 (Rhoads, 1903). Its previously known range on the eastern continental shelf of North America, Anderson (1946) has succinctly described as, “… rare in Baffin Bay, common on South Greenland coast and in Davis Strait, and on Labrador and Newfoundland coasts; [occurs in] Gulf of St. Lawrence, and south to New York and New Jersey.” Jonsgaard's (1951) reference to a record from Cape Hatteras is a misinterpretation. This indicates that off the eastern North American coast it was known to be common between 70° and 50° north latitude and to occur south to about 40° until our above-mentioned report extended this south to just below 25°. This southernmost record of the species for the North Atlantic Ocean was based upon the February, 1949, stranding of an adult piked whale in the Florida Keys. A further extension of the animal's known range up into the Gulf of Mexico at 30° latitude was reported on the basis of an adult's skeleton from near Spring Creek, Florida.

Abstract Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC-Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC-Heps generated using a chemically defined four-step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D PEG-DA fabricated inverted colloid crystal (ICC) scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune-privileged material alginate. This is the first report we are aware of demonstrating cGMP-compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications.

April 28, 2014April 8, 2014Free AccessAndrogen-Mediated Effects On Ictal Behavior Among Male Rats Is Mediated Through Pregnane Xenobiotic Receptors, Which Mediated Neurosteroidognesis (P1.265)Carolyn Koonce, Jennifer Moore, Swarup Mitra, and Cheryl FryeAuthors Info & AffiliationsApril 8, 2014 issue82 (10_supplement)https://doi.org/10.1212/WNL.82.10_supplement.P1.265 Letters to the Editor

This chapter contains sections titled: Introduction Background Electroporation Methods for Stem Cell Transfection Perspectives References

OBJECTIVE: Standard slow-cool/rapid-thaw methods of cryopreservation have produced poor results when applied to human embryonic stem (hES) cells. Vitrification results have been encouraging but a survey of the literature as well as the standard operating practices of one large stem cell bank show that vitrification has not been widely adopted as the method of choice for cryopreservation of hES cells. Here, a commercially available hES cell line was used to evaluate the efficiency of a new vitrification method and compare it to results with slow-cooling. DESIGN: At the time of passage, single colonies were dissected into pieces and either vitrified or slow-cooled. They were thawed after 1-2 weeks. MATERIALS AND METHODS: To vitrify, colony pieces were exposed to glycerol and ethylene glycol-based vitrification solutions and loaded into 0.25cc straws which were then heat sealed at both ends, cooled in liquid nitrogen (LN2) vapor and stored in LN2. Straws were thawed in air for 3sec and submerged in a 20°C water bath for 7sec. The cryoprotectant was removed through serial dilutions. Slow-cooling entailed exposure of colonies to 10% DMSO, loading into 2.0ml cryo-vials, and overnight storage at -70°C before LN2 storage. Vials were thawed in air for 30sec and submerged in a 37°C water bath until no ice was visible. Cells were removed, washed, and placed in culture. Post-thaw survival, expansion, and differentiation were evaluated. RESULTS: In four thaw experiments, 27/30 (90%) of the vitrified colony pieces had attached and re-established colonies in mTeSR1 medium/matrigel within 24hrs. In the same period, none of the slow-cooled colony pieces (0/23) had visibly attached. After culture and daily medium exchange for 12 days, one colony (1/23 or 4%) had emerged. Vitrified colonies were ready for passage by 7 days post thaw, while the slow-cooled colony required a total of 21 days of culture before passage. No appreciable differentiation was seen in any of the colonies in either treatment immediately after thaw or at first post-thaw passage. CONCLUSIONS: Compared to a standard slow-cooling method, the vitrification method examined here showed significantly higher post-thaw cell survival; the vitrified colonies also proliferated faster and required a considerably shorter time before they could be passaged. This new method provides other advantages by avoiding the use of DMSO and using a sealed container which avoids contamination issues.

Ectopic expression of systemic RNA interference defective protein (SID-1) in embryonic stem cells: Implications for high-throughput gene screening

DNA transfer into human embryonic stem cells (hESC), derived from the inner cell mass of a four day old embryo and able to differenti - ate into derivatives of all 3 germ layers, is limited by their general resistance to common methods used to introduce exogenous DNA. Here we describe a transfection method using the Nucleofector® 96-well Shuttle® System that can be used with hESC to express plasmid-encoded protein or to inhibit protein production with siRNAs in a reliable, efficient, and high-throughput manner. Nucleofection® was found to yield a large fraction of transfected cells with minimal effect on cell viability or loss of pluripotency marker.

The discovery that human primary cells such as nucleated blood cells or cultured skin fibroblasts can be reprogrammed into induced pluripotent stem cells (hiPSC) has ushered in a new era for research on the genetic etiology of neuropsychiatric disorders. Such hiPSC can be differentiated into several types of neurons, which may provide a primitive model for studying cellular variation in neuronal function due to underlying genetic variants causing the disorder. It is critical that source cells for possible reprogramming and their derived hiPSC be banked in an accredited facility capable of proper quality assurance that includes a genetic profile for future authentication of secondary biomaterials (e.g., differentiated cellular derivatives). Nucleated blood cells are more easily obtained compared to skin fibroblasts and can be cryopreserved for many years before they are reprogrammed to hiPSC. However, to enable all possible future uses of biosamples, some of which may not yet even be contemplated, researchers and biobanks must obtain clear informed consent from subjects for broad use of their biosamples in research.

ABSTRACT Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation predisposing to schizophrenia. Previous studies showed that engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multi-center effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. We show that in neurons that were trans-differentiated from induced pluripotent stem cells derived from three NRXN1 -deletion patients, the same impairment in neurotransmitter release was observed as in engineered NRXN1 -deficient neurons. This impairment manifested as a decrease in spontaneous synaptic events and in evoked synaptic responses, and an alteration in synaptic paired-pulse depression. Nrxn1 -deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1 -binding protein, and were associated with characteristic gene expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Abstract Hem1 is a hematopoietic specific member of the HEM family proteins, which have been identified as components of the WAVE regulatory complex. To further characterize the role of Hem1 in hematopoietic cell function, we have generated mice deficient in Hem1 using gene targeting methodology. Hem1-deficient mice manifest several phenotypic abnormalities, including peripheral blood lymphopenia and neutrophilia, splenomegaly, premature mortality, and with variable penetrance thymic hypoplasia and alopecia. Flow cytometric analysis of peripheral blood and spleen demonstrated that Hem1−/ − mice have a marked reduction in peripheral B cells, changes that were evident in both adult and neonatal mice; the splenomegaly in Hem1−/ − mice was attributable to increased extramedullary hematopoiesis. The frequency of bone marrow B cell progenitors was also markedly reduced in Hem1−/ − mice. To assess the role of Hem1 in hematopoietic stem cell (HSC) function, competitive bone marrow transplantation assays were performed. In contrast to wild-type HSCs, Hem1-deficient HSCs had poor competitive repopulating activity in irradiated recipient mice. KSL cell analysis demonstrated no significant difference in the frequency of lin-c-kit+Sca1+ HSCs between wild-type and Hem1−/ − bone marrow, suggesting that the defective competitive repopulating activity of Hem1−/ − HSCs is attributable to defective bone marrow homing or stem cell niche interaction. Given the biochemical evidence implicating the HEM proteins as a component of the WAVE regulatory complex, we also assessed the ability of Hem1-deficient leukocytes to undergo cytoskeletal remodeling in vitro. Using a transwell assay, Hem1−/ − bone marrow storage pool neutrophils demonstrated markedly blunted chemotactic responses to formylated peptide which was attributable to defective f-actin formation. Hem1−/ − peripheral CD4+ T cells similarly manifested chemotactic defects in response to SDF-1, and showed blunted proliferation when stimulated with antibodies against CD3 and CD28. Finally, a model of Streptococcus pneumoniae infection was employed to test the role of Hem1 in the in vivo function of neutrophils. Hem1−/ − mice were dramatically more sensitive to S. pneumoniae than wild-type littermates, as manifested by the inability to eliminate S. pneumoniae organisms in vivo and higher mortality. In summary, Hem1 deficiency results in deficiencies and functional defects in multiple hematopoietic lineages due to defective signaling to the actin cytoskeleton, and importantly, Hem1 plays a critical role in innate immunity to S. pneumoniae in vivo.

Abstract The Runx1/Core Binding Factor- β (CBF β) transcriptional complex is required for the establishment of hematopoiesis during development. To permit the analysis of Runx1 expression in hematopoietic cell subsets, we have recently developed a novel murine line in which the expression of both full-length Runx1 and GFP is driven by the Runx1 promoter [Runx1-internal ribosomal entry site-green fluorescent protein (Runx1-GFP) knock-in mouse; Blood 103:2522]. Analysis of these mice has revealed that Runx1 is expressed in all hematopoietic lineages with the exception of erythroid cells. During our analysis, we identified in the bone marrow of these mice a cell population that expresses GFP at levels 2–5 fold higher than any other cell type (GFPhi). These cells have low forward and side scatter properties and do not express c-kit or several lineage-associated cell surface markers (lin−). In comparison to c-kit+lin− cells, these GFPhic-kit−lin− cells possess little colony forming activity in vitro. While they lack primary CFU-S activity in contrast to c-kit+lin− cells, GFPhic-kit−lin− cells possess secondary CFU-S activity. A c-kit−lin− hematopoietic stem cell (HSC) has been described by others that contributes to long-term, but not short-term, hematopoietic reconstitution in lethally irradiated recipients and may represent progenitors of c-kit+lin− HSCs in vivo (Ortiz et al. Immunity 10:173). The GFPhi bone marrow cells that we have identified share many of the properties of those c-kit−lin− cells identified by others; consequently, they likely represent the same cell population. Our ability to isolate these cells based on differential GFP expression should enable us to highly purify these GFPhic-kit−lin− cells and further characterize their immunophenotypic and biologic properties.

A specialty conference (Coastal Zone 78) will be held March 14, 15 and 16, 1978 at the Jack Tar Hotel in San Francisco, California.This will be a symposium on technical, environmental socioeconomic and regulatory aspects of coastal zone planning and management.COASTAL ZONE 78 will be a multidisciplinary specialty conference to provide an opportunity for those scientists, engineers, planners, and other involved professionals to convene and exchange information and views.The purpose of the conference is to provide a forum for discussion of coastal zone management, beneficial use, protection and development leading hopefully to a better understanding of the interrelationships between the environmental, socio-economic, engineering, and regulatory decisions involved.The conference will foster more effective and meaningful jurisdictional arrangements, conservation considerations, regulations, enforcement policies, planning activities, and design parameters in the development and implementation of coastal plans.This conference will include, in addition to the standard presentation of papers and social functions, (1) a short course on the history and implementation of Coastal Zone Management preceding the conference, (2) a bus tour to several nearby coastal areas following the conference, and (3) a one-day Gordon Conference Type workshop (predicted on availability of a sponsor).Conference proceedings will be published and made available to participants at the conference.Subsequent to the conference, they will be for sale on a permanent basis.

PGM1 subtypes and ESD phenotypes of 600 mothers and their respective newborn infants residing in the northern part of the island of Tasmania were examined. The allele frequencies of PGM1 in the mothers were 1+ = 0.6525, 1- = 0.1250, 2+ = 0.1758 and 2- = 0.0467, and in the newborns were 1+ = 0.6675, 1- = 0.1275, 2+ = 0.01600 and 2- = 0.0450. Both samples were found to exhibit Hardy-Weinberg equilibrium conditions and there was no significant difference between them. Also, the frequencies of PGM1 alleles were overall similar to frequencies in neighbouring populations on the Australian mainland. The frequency of ESD*2 in both samples was similar (0.105) but the mothers' genotypes were not in Hardy-Weinberg equilibrium (due to an excess of type 2). The ESD allele frequencies in Tasmania are similar to those reported in other white Australian populations.