Production of large, defined genome modifications in rats by targeting rat embryonic stem cells.

Rats were more frequently used than mice to model human disease before mouse embryonic stem cells (mESCs) revolutionized genetic engineering in mice. Rat ESCs (rESCs) were first reported over 10 years ago, yet they are not as frequently used as mESCs. CRISPR-based gene editing in zygotes is widely used in rats but is limited by the difficulty of inserting or replacing DNA sequences larger than about 10 kb.

Homozygous ALS-linked FUS P525L mutations cell- autonomously perturb transcriptome profile and chemoreceptor signaling in human iPSC microglia.

Amyotrophic lateral sclerosis is a fatal disease pathologically typified by motor and cortical neurodegeneration as well as microgliosis. The FUS P525L mutation is highly penetrant and causes ALS cases with earlier disease onset and more aggressive progression. To date, how P525L mutations may affect microglia during ALS pathogenesis had not been explored.

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo.

Assessment of the role of afucosylated glycoforms on the antibody-dependent phagocytosis activity of an antibody to Aβ aggregates.

The terminal sugars of Fc glycans can influence the Fc-dependent biological activities of monoclonal antibody therapeutics. Afucosylated N-glycans have been shown to significantly alter binding to FcγRIIIa and affect antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, in order to maintain and ensure safety and efficacy for antibodies whose predominant mechanism of action (MOA) is ADCC, afucosylation is routinely monitored and controlled within appropriate limits.

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage.

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 () causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery.

Using ovality to predict nonmutagenic, orally efficacious pyridazine amides as cell specific spleen tyrosine kinase inhibitors.

Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay.

A high-throughput screen for teratogens using human pluripotent stem cells.

There is need in the pharmaceutical and chemical industries for high-throughput human cell-based assays for identifying hazardous chemicals, thereby reducing the overall reliance on animal studies for predicting the risk of toxic responses in humans. Despite instances of human-specific teratogens such as thalidomide, the use of human cell-teratogenicity assays has just started to be explored. Herein, a human pluripotent stem cell test (hPST) for identifying teratogens is described, benchmarking the in vitro findings to traditional preclinical toxicology teratogenicity studies and when available to teratogenic outcomes in humans.

Refining the human iPSC-cardiomyocyte arrhythmic risk assessment model.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are capable of detecting drug-induced clinical arrhythmia, Torsade de Pointes (TdP), and QT prolongation. Efforts herein employ a broad set of structurally diverse drugs to optimize the predictive algorithm for applications in discovery toxicology and cardiac safety screening. The changes in the beat rhythm and rate of a confluent monolayer of hiPS-CMs by 88 marketed and 30 internal discovery compounds were detected with real-time cellular impedance measurement and quantified by measures of arrhythmic beating (IB20, lowest concentration inducing ≥ 20% arrhythmic [irregular, atypical] beats in 3 consecutive 20-s sweeps, and predicted proarrhythmic score [PPS]-IB20) or changes in beat rate (BR20, the lowest concentration inducing a reduction in beat rate of ≥ 20% at 3 consecutive sweeps compared with the time-matched vehicle control group, and PPS-BR20).

Pyrrolopyrazines as selective spleen tyrosine kinase inhibitors.

We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series.

The effects of jaspamide on human cardiomyocyte function and cardiac ion channel activity.

Jaspamide (jasplakinolide; NSC-613009) is a cyclodepsipeptide that has antitumor activity. A narrow margin of safety was observed between doses required for efficacy in mouse tumor models and doses that caused severe acute toxicity in rats and dogs. We explored the hypothesis that the observed toxicity was due to cardiotoxicity.

Skeletogenic phenotype of human Marfan embryonic stem cells faithfully phenocopied by patient-specific induced-pluripotent stem cells.

Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an extracellular matrix protein. MFS is inherited as an autosomal dominant trait and displays major manifestations in the ocular, skeletal, and cardiovascular systems. Here we report molecular and phenotypic profiles of skeletogenesis in tissues differentiated from human embryonic stem cells and induced pluripotent stem cells that carry a heritable mutation in FBN1.

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups.

Determination of the human cardiomyocyte mRNA and miRNA differentiation network by fine-scale profiling.

To gain insight into the molecular regulation of human heart development, a detailed comparison of the mRNA and miRNA transcriptomes across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and biopsies from fetal, adult, and hypertensive human hearts was performed. Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes revealed 3 distinct groups of genes: pluripotent specific, transitional cardiac specification, and mature cardiomyocyte specific. Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes largely stabilizes 20 days after initiation of differentiation.

Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes.

Improved in vitro systems for predicting drug-induced toxicity are needed in the pharmaceutical and biotechnology industries to decrease late-stage drug attrition. One unmet need is an early screen for cardiotoxicity, which accounts for about one third of safety-based withdrawn pharmaceuticals. Herein, the first published report of a high-throughput functional assay employing a monolayer of beating human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is described, detailing a model that accurately detects drug-induced cardiac abnormalities.

Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells.

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429, and miR-183-96-182 were downregulated in human BCSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells.

Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver.

To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome. Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives, and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation. First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2%-11%).

Viral infection of human progenitor and liver-derived cells encapsulated in three-dimensional PEG-based hydrogel.

We have studied the encapsulation of human progenitor cells into 3D PEG hydrogels. Replication-incompetent lentivirus promoter reporter vectors were found to efficiently detect the in vivo expression of human hepatic genes in hydrogel-encapsulated liver progenitor cells. Similarly, hydrogel-encapsulated cells could be efficiently infected with hepatitis C virus, and progeny infectious virus could be recovered from the media supernatants of the hydrogels.

Isolation and transcriptional profiling of purified hepatic cells derived from human embryonic stem cells.

The differentiation of human embryonic stem cells (hESCs) into functional hepatocytes provides a powerful in vitro model system for studying the molecular mechanisms governing liver development. Furthermore, a well-characterized renewable supply of hepatocytes differentiated from hESCs could be used for in vitro assays of drug metabolism and toxicology, screening of potential antiviral agents, and cell-based therapies to treat liver disease. In this study, we describe a protocol for the differentiation of hESCs toward hepatic cells with complex cellular morphologies.

High-throughput functional screen of mouse gastrula cDNA libraries reveals new components of endoderm and mesoderm specification.

This study describes a cross-species functional screen of mouse gastrula cDNA libraries for components of endoderm and mesoderm specification. Pools of 96 cDNAs from arrayed mouse gastrula cDNA libraries were transcribed into mRNA and injected into either the presumptive mesoderm or the ectoderm of one-cell Xenopus laevis embryos. Injected embryos were examined at gastrula stage by in situ hybridization with endoderm or mesoderm markers.

Overgrowth of a mouse model of the Simpson-Golabi-Behmel syndrome is independent of IGF signaling.

The type 1 Simpson-Golabi-Behmel overgrowth syndrome (SGBS1) is caused by loss-of-function mutations of the X-linked GPC3 gene encoding glypican-3, a cell-surface heparan sulfate proteoglycan that apparently plays a negative role in growth control by an unknown mechanism. Mice carrying a Gpc3 gene knockout exhibited several phenotypic features that resemble clinical hallmarks of SGBS1, including somatic overgrowth, renal dysplasia, accessory spleens, polydactyly, and placentomegaly. In Gpc3/DeltaH19 double mutants (lacking GPC3 and also carrying a deletion around the H19 gene region that causes bialellic expression of the closely linked Igf2 gene by imprint relaxation), the Gpc3-null phenotype was exacerbated, while additional SGBS1 features (omphalocele and skeletal defects) were manifested.