Derivation of Naïve Human Embryonic Stem Cells Using a CHK1 Inhibitor.

Embryonic development is a continuum in vivo. Transcriptional analysis can separate established human embryonic stem cells (hESC) into at least four distinct developmental pluripotent stages, two naïve and two primed, early and late relative to the intact epiblast. In this study we primarily show that exposure of frozen human blastocysts to an inhibitor of checkpoint kinase 1 (CHK1) upon thaw greatly enhances establishment of karyotypically normal late naïve hESC cultures. These late naïve cells are plastic and can be toggled back to early naïve and forward to early primed pluripotent stages. The early primed cells are transcriptionally equivalent to the post inner cell mass intermediate (PICMI) stage seen one day following transfer of human blastocysts into in vitro culture and are stable at an earlier stage than conventional primed hESC.

General and Specific Aversive Modulation of Active Avoidance Require Central Amygdala.

Three studies provide evidence that the central nucleus of the amygdala, a structure with a well-established role in conditioned freezing, is also required for conditioned facilitation of instrumental avoidance in rats. First, the immediate early gene c-Fos was measured following the presentation of a previously shock-paired tone in subjects trained either on an unsignaled avoidance task or not (in addition to tone only presentations in naïve controls). Significantly elevated expression of c-Fos was found in both the avoidance trained and Pavlovian trained conditions relative to naïve controls (but with no difference between the two trained conditions). In a subsequent study, intracranial infusions of muscimol into the central amygdala significantly attenuated the facilitation of shock-avoidance by a shock-paired Pavlovian cue relative to pre-operative responding. The final study used a virogenetic approach to inhibit the central amygdala prior to testing. This treatment eliminated the transfer of motivational control over shock-avoidance by both a shock-paired Pavlovian stimulus, as well as a cue paired with a perceptually distinct aversive event (i.e., klaxon). These findings provide compelling support for a role of central amygdala in producing aversive Pavlovian-instrumental transfer.

Multiplex CRISPR/Cas9 genome editing in hematopoietic stem cells for fetal hemoglobin reinduction generates chromosomal translocations.

Sickle cell disease and ß-thalassemia are common monogenic disorders that cause significant morbidity and mortality globally. The only curative treatment currently is allogeneic hematopoietic stem cell transplantation, which is unavailable to many patients due to a lack of matched donors and carries risks including graft-versus-host disease. Genome editing therapies targeting either the BCL11A erythroid enhancer or the HBG promoter are already demonstrating success in reinducing fetal hemoglobin. However, where a single locus is targeted, reliably achieving levels high enough to deliver an effective cure remains a challenge. We investigated the application of a CRISPR/Cas9 multiplex genome editing approach, in which both the BCL11A erythroid enhancer and HBG promoter are disrupted within human hematopoietic stem cells. We demonstrate superior fetal hemoglobin reinduction with this dual-editing approach without compromising engraftment or lineage differentiation potential of edited cells post-xenotransplantation. However, multiplex editing consistently resulted in the generation of chromosomal rearrangement events that persisted in vivo following transplantation into immunodeficient mice. The risk of oncogenic events resulting from such translocations therefore currently prohibits its clinical translation, but it is anticipated that, in the future, alternative editing platforms will help alleviate this risk.

Genetic mapping and phenotypic analysis of shotH.3.2 in Drosophila melanogaster.

Genetic screens are used to identify genes involved in specific biological processes. An EMS mutagenesis screen in Drosophila melanogaster identified growth control phenotypes in the developing eye. One mutant line from this screen, H.3.2, was phenotypically characterized using the FLP/FRT system and genetically mapped by complementation analysis and genomic sequencing by undergraduate students participating in the multi-institution Fly-CURE consortium. H.3.2 was found to have a nonsense mutation in short stop (shot), anortholog of the mammalian spectraplakin dystonin (DST). shot and DST are involved in cytoskeletal organization and play roles during cell growth and proliferation.

In Vivo Gene Therapy for Canine SCID-X1 Using Cocal-Pseudotyped Lentiviral Vector.

Hematopoietic stem and progenitor cell (HSPC)-based ex vivo gene therapy has demonstrated clinical success for X-linked severe combined immunodeficiency (SCID-X1) patients who lack a suitable donor for HSPC transplantation. Nevertheless, this form of treatment is associated with an increased risk of infectious disease complications and genotoxicity mainly due to the conditioning regimen. In addition, ex vivo gene therapy approaches require sophisticated facilities to manufacture gene-modified cells and to care for the patients after chemotherapy. Considering these impediments, we have developed an in vivo gene therapy approach to treat canine SCID-X1 after HSPC mobilization and systemic delivery of the therapeutic vector. Here, we investigated the use of the cocal envelope to pseudotype a lentiviral (LV) vector expressing a functional gammaC gene. The cocal envelope is resistant to serum inactivation compared with the commonly used vesicular stomatitis virus envelope glycoprotein (VSV-G) envelope and thus well suited for systemic delivery. Two SCID-X1 neonatal canines treated with this approach achieved long-term therapeutic immune reconstitution with no prior conditioning. Therapeutic levels of gene-corrected CD3+ T cells were demonstrated for at least 16 months, and all other correlates of T cell functionality were within normal range. Retroviral integration-site analysis demonstrated polyclonal T cell reconstitution. Comparative analysis of integration profiles of foamy viral (FV) vector and cocal LV vector after in vivo gene therapy found distinct integration-site patterns. These data demonstrate that clinically relevant and durable correction of canine SCID-X1 can be achieved with in vivo delivery of cocal LV. Since manufacturing of cocal LV is similar to VSV-G LV, this approach is easily translatable to a clinical setting, thus providing for a highly portable and accessible gene therapy platform for SCID-X1.

Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal.

To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.

Using DNase Hi-C techniques to map global and local three-dimensional genome architecture at high resolution.

The folding and three-dimensional (3D) organization of chromatin in the nucleus critically impacts genome function. The past decade has witnessed rapid advances in genomic tools for delineating 3D genome architecture. Among them, chromosome conformation capture (3C)-based methods such as Hi-C are the most widely used techniques for mapping chromatin interactions. However, traditional Hi-C protocols rely on restriction enzymes (REs) to fragment chromatin and are therefore limited in resolution. We recently developed DNase Hi-C for mapping 3D genome organization, which uses DNase I for chromatin fragmentation. DNase Hi-C overcomes RE-related limitations associated with traditional Hi-C methods, leading to improved methodological resolution. Furthermore, combining this method with DNA capture technology provides a high-throughput approach (targeted DNase Hi-C) that allows for mapping fine-scale chromatin architecture at exceptionally high resolution. Hence, targeted DNase Hi-C will be valuable for delineating the physical landscapes of cis-regulatory networks that control gene expression and for characterizing phenotype-associated chromatin 3D signatures. Here, we provide a detailed description of method design and step-by-step working protocols for these two methods.

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells.

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or "tipping point" at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations.

It's Her Fault: Student Acceptance of Rape Myths On Two College Campuses.

The present study examined factors that are associated with an individual's adherence to rape myths at two colleges located in the same town. Particularly, we examined sex, race, and participants' drinking behavior in relation to rape myth acceptance. We found that males and heavy drinkers are more likely than females and non/low drinkers to adhere to rape myths. An interaction between males and drinking was also found indicating a moderated effect of gender on rape myth acceptance. In addition, the college with sexual assault programming did not experience a lowered acceptance of rape myths compared with the college with no programming.

Fine-scale chromatin interaction maps reveal the cis-regulatory landscape of human lincRNA genes.

High-throughput methods based on chromosome conformation capture have greatly advanced our understanding of the three-dimensional (3D) organization of genomes but are limited in resolution by their reliance on restriction enzymes. Here we describe a method called DNase Hi-C for comprehensively mapping global chromatin contacts. DNase Hi-C uses DNase I for chromatin fragmentation, leading to greatly improved efficiency and resolution over that of Hi-C. Coupling this method with DNA-capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 998 large intergenic noncoding RNA (lincRNA) promoters in two human cell lines. Our results revealed that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the Polycomb repressive complex. Our results provide the first glimpse of the cell type-specific 3D organization of lincRNA genes.

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Derivation of naive human embryonic stem cells.

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.