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The dry mass and the orientation of biomolecules can be imaged without a label by measuring their permittivity tensor (PT), which describes how biomolecules affect the phase and polarization of light. Three-dimensional (3D) imaging of PT has been challenging. We present a label-free computational microscopy technique, PT imaging (PTI), for the 3D measurement of PT. PTI encodes the invisible PT into images using oblique illumination, polarization-sensitive detection and volumetric sampling. PT is decoded from the data with a vectorial imaging model and a multi-channel inverse algorithm, assuming uniaxial symmetry in each voxel. We demonstrate high-resolution imaging of PT of isotropic beads, anisotropic glass targets, mouse brain tissue, infected cells and histology slides. PTI outperforms previous label-free imaging techniques such as vector tomography, ptychography and light-field imaging in resolving the 3D orientation and symmetry of organelles, cells and tissue. We provide open-source software and modular hardware to enable the adoption of the method., (© 2024. The Author(s).)

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing., Competing Interests: DECLARATION OF INTERESTS MK is a co-scientific founder of Montara Therapeutics and serves on the Scientific Advisory Boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, Alector, and Montara Therapeutics, and is an advisor to Modulo Bio and Recursion Therapeutics. MK is an inventor on US Patent 11,254,933 related to CRISPRi and CRISPRa screening, and on a US Patent application on in vivo screening methods. JRH is a co-founder of Azalea Therapeutics. CF is a co-founder of Mirimus, Inc. B.A. is an advisory board member with options for Arbor Biotechnologies and Tessera Therapeutics. BA holds equity in Celsius Therapeutics. The Regents of the University of California have patents issued and pending for CRISPR technologies (on which JAD is an inventor) and delivery technologies (on which JAD and JRH are co-inventors). JAD is a cofounder of Azalea Therapeutics, Caribou Biosciences, Editas Medicine, Evercrisp, Scribe Therapeutics, Intellia Therapeutics, and Mammoth Biosciences. JAD is a scientific advisory board member at Evercrisp, Caribou Biosciences, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, The Column Group and Inari. JAD is Chief Science Advisor to Sixth Street, a Director at Johnson & Johnson, Altos and Tempus, and has a research project sponsored by Apple Tree Partners.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing., Competing Interests: Competing interests statement:B.R.C. is a founder of Tenaya Therapeutics, a company focused on finding treatments for heart failure, including genetic cardiomyopathies. B.R.C. holds equity in Tenaya. C.D.C. is a founder, with equity, in Ciznor Co., a CNS therapeutics company. B.R.C. and C.D.C. are inventors on a patent application for this work. Y.-C.T. is a full-time employee at PacBio, a company developing single-molecule sequencing technologies. The other authors declare no competing interests.

Science museums play an important role in science education, engaging the public with science concepts and building support for scientific research. Here, we describe Give Heart Cells a Beat, an interactive exhibit that lets museum visitors synchronize the beating of live stem cell-derived cardiomyocytes to their own heart rate in real time. The beat rate of cells accurately matched the beat rate of visitors and responded dynamically to changes such as exercise. Visitor evaluation revealed that engagement with the specimen prompted curiosity in heart biology and stem cells. Give Heart Cells a Beat is the product of a close collaboration between a museum and an academic research laboratory, and to our knowledge, it is the first interactive exhibit to use live human heart cells. We hope this exhibit serves as an example for the implementation of stem cell technology in informal science education and inspires future relationships between academia and public science venues., Competing Interests: Declaration of interests B.R.C. is a founder of and holds equity in Tenaya Therapeutics (tenayatherapeutics.com), a company focused on finding treatments for heart failure, including genetic cardiomyopathies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism remains elusive. In this study, we used induced pluripotent stem cells to test if the only coding variant in this allele block, BAG3 C151R , alters protein and cellular function in human cardiomyocytes. Quantitative protein interaction analysis identified changes in BAG3 C151R protein partners specific to cardiomyocytes. Knockdown of genes encoding for BAG3-interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3 C151R associates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3 C151R variant have improved response to proteotoxic stress in a dose-dependent manner. This study suggests that BAG3 C151R could be responsible for the cardioprotective effect of the haplotype block, by increasing cardiomyocyte protection from stress. Preferential binding partners of BAG3 C151R may reveal potential targets for cardioprotective therapies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Exon-skipping is a powerful genetic tool, especially when delivering genes using an AAV-mediated full-length gene supplementation strategy is difficult owing to large length of genes. Here, we used engineered human induced pluripotent stem cells and artificial intelligence to evaluate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9-based exon-skipping vectors targeting genes of the retinal pigment epithelium (RPE). The model system was choroideremia; this is an X-linked inherited retinal disease caused by mutation of the CHM gene., Methods: We explored whether artificial intelligence detected differentiation of human OTX2, PAX6 and MITF (hOPM) cells, in which OTX2, PAX6 and MITF expression was induced by doxycycline treatment, into RPE. Plasmid encoding CHM exon-skipping modules targeting the splice donor sites of exons 6 were constructed. A clonal hOPM cell line with a frameshift mutation in exon 6 was generated and differentiated into RPE. CHM exon 6-skipping was induced, and the effects of skipping on phagocytic activity, cell death and prenylation of Rab small GTPase (RAB) were evaluated using flow cytometry, an in vitro prenylation assay and western blotting., Results: Artificial intelligence-based evaluation of RPE differentiation was successful. Retinal pigment epithelium cells with a frameshift mutation in exon 6 showed increased cell death, reduced phagocytic activity and increased cytosolic unprenylated RABs only when oxidative stress was in play. The latter two phenotypes were partially rescued by exon 6-skipping of CHM., Conclusions: CHM exon 6-skipping contributed to RPE phagocytosis probably by increasing RAB38 prenylation under oxidative stress., (© 2022 John Wiley & Sons Ltd.)

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field., Competing Interests: Declaration of interests S.W.S. is on the scientific advisory council of the Lewy Body Dementia Association and the MSA Coalition. S.W.S. is an editorial board member for the Journal of Parkinson Disease and JAMA Neurology. S.W.S. received research support from Cerevel Therapeutics. M.K. serves on the scientific advisory boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, and Alector and is a consultant to Modulo Bio and Recursion Therapeutics. Participation by researchers from Data Tecnica International, LLC in this project was part of a competitive contract awarded to Data Tecnica International, LLC by the National Institutes of Health to support open science research. M.A.N. also currently serves on the scientific advisory board for Clover Therapeutics and is an advisor to Neuron23 Inc. E.A. is founder, shareholder, and scientific advisor of Cholestenix, Ltd., (Published by Elsevier Inc.)

Background: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors., Methods: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors., Results: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms., Conclusions: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.

Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease., Competing Interests: Declaration of interests D.S. is scientific co-founder, shareholder, and director of Tenaya Therapeutics. B.G.B. and B.R.C. are scientific co-founders and shareholders of Tenaya Therapeutics. K.S.P. is a shareholder of Tenaya Therapeutics. N.J.K. has received research support from Vir Biotechnology and F. Hoffmann-La Roche. N.J.K. has consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics, and Interline Therapeutics; is a shareholder of Tenaya Therapeutics, Maze Therapeutics, and Interline Therapeutics; and is financially compensated by GEn1E Lifesciences, Inc. and Twist Bioscience Corp., (Copyright © 2022 Elsevier Inc. All rights reserved.)

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways., (© 2021. The Author(s).)

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene ( NEFL , CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases., Competing Interests: BC is a founder of Tenaya Therapeutics (https://www.tenayatherapeutics.com/), a company focused on finding treatments for heart failure, including genetic cardiomyopathies. BC holds equity in Tenaya, and Tenaya provides research support for heart failure related research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Feliciano, Wu, Watry, Marley, Ramadoss, Ghanim, Liu, Zholudeva, McDevitt, Saporta, Conklin and Judge.)

Age-related macular degeneration and genetic forms of blindness such as Best Disease and Retinitis Pigmentosa can be caused by degeneration of the Retinal Pigment Epithelium (RPE). RPE generated from patient-derived induced pluripotent stem cells (iPSCs) is valuable for both the study of disease mechanisms and development of therapeutic strategies. However, protocols to produce iPSC-derived RPE in vitro are often inefficient, labor-intensive, low-throughput, and highly variable between cell lines and within batches. Here, we report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in Best Disease patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Although coronavirus disease 2019 (COVID-19) causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human induced pluripotent stem cell (iPSC)-derived heart cells to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural genes corroborates adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and nuclear disruption. Human autopsy specimens from patients with COVID-19 reflected similar alterations, particularly sarcomeric fragmentation. These notable cytopathic features in cardiomyocytes provide insights into SARS-CoV-2-induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise concerns about the long-term consequences of COVID-19 in asymptomatic and severe cases., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (> 20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

Previous studies have suggested that the loss of the translation initiation factor eIF4G1 homolog NAT1 induces excessive self-renewability of naive pluripotent stem cells (PSCs); yet the role of NAT1 in the self-renewal and differentiation of primed PSCs is still unclear. Here, we generate a conditional knockout of NAT1 in primed PSCs and use the cells for the functional analyses of NAT1. Our results show that NAT1 is required for the self-renewal and neural differentiation of primed PSCs. In contrast, NAT1 deficiency in naive pluripotency attenuates the differentiation to all cell types. We also find that NAT1 is involved in efficient protein expression of an RNA uridyltransferase, TUT7. TUT7 is involved in the neural differentiation of primed PSCs via the regulation of human endogenous retrovirus accumulation. These data demonstrate the essential roles of NAT1 and TUT7 in the precise transition of stem cell fate., Competing Interests: Declaration of Interests K.T. is on the scientific advisory board of I Peace, without salary. B.R.C. is a scientific advisor to Tenaya Therapeutics. S.Y. is a scientific advisor (without salary) to iPS Academia Japan., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

DISCOVER-seq (discovery of in situ Cas off-targets and verification by sequencing) is a broadly applicable approach for unbiased CRISPR-Cas off-target identification in cells and tissues. It leverages the recruitment of DNA repair factors to double-strand breaks (DSBs) after genome editing with CRISPR nucleases. Here, we describe a detailed experimental protocol and analysis pipeline with which to perform DISCOVER-seq. The principle of this method is to track the precise recruitment of MRE11 to DSBs by chromatin immunoprecipitation followed by next-generation sequencing. A customized open-source bioinformatics pipeline, BLENDER (blunt end finder), then identifies off-target sequences genome wide. DISCOVER-seq is capable of finding and measuring off-targets in primary cells and in situ. The two main advantages of DISCOVER-seq are (i) low false-positive rates because DNA repair enzyme binding is required for genome edits to occur and (ii) its applicability to a wide variety of systems, including patient-derived cells and animal models. The whole protocol, including the analysis, can be completed within 2 weeks.

Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.

Cardiac tissues are able to adjust their contractile behavior to adapt to the local mechanical environment. Nonuniformity of the native tissue mechanical properties contributes to the development of heart dysfunctions, yet the current in vitro cardiac tissue models often fail to recapitulate the mechanical nonuniformity. To address this issue, a 3D cardiac microtissue model is developed with engineered mechanical nonuniformity, enabled by 3D-printed hybrid matrices composed of fibers with different diameters. When escalating the complexity of tissue mechanical environments, cardiac microtissues start to develop maladaptive hypercontractile phenotypes, demonstrated in both contractile motion analysis and force-power analysis. This novel hybrid system could potentially facilitate the establishment of "pathologically-inspired" cardiac microtissue models for deeper understanding of heart pathology due to nonuniformity of the tissue mechanical environment., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human pluripotent stem cells (hPSCs) have the intrinsic ability to self-organize into complex multicellular organoids that recapitulate many aspects of tissue development. However, robustly directing morphogenesis of hPSC-derived organoids requires novel approaches to accurately control self-directed pattern formation. Here, we combined genetic engineering with computational modeling, machine learning, and mathematical pattern optimization to create a data-driven approach to control hPSC self-organization by knock down of genes previously shown to affect stem cell colony organization, CDH1 and ROCK1. Computational replication of the in vitro system in silico using an extended cellular Potts model enabled machine learning-driven optimization of parameters that yielded emergence of desired patterns. Furthermore, in vitro the predicted experimental parameters quantitatively recapitulated the in silico patterns. These results demonstrate that morphogenic dynamics can be accurately predicted through model-driven exploration of hPSC behaviors via machine learning, thereby enabling spatial control of multicellular patterning to engineer human organoids and tissues. A record of this paper's Transparent Peer Review process is included in the Supplemental Information., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. Target sites might carry genetic variations that are not distinguishable by sgRNA designing tools based on one reference genome. AlleleAnalyzer is an open-source software that incorporates single-nucleotide variants and short insertions and deletions to design sgRNAs for precisely editing 1 or multiple haplotypes of a sequenced genome, currently supporting 11 Cas proteins. It also leverages patterns of shared genetic variation to optimize sgRNA design for different human populations. AlleleAnalyzer is available at https://github.com/keoughkath/AlleleAnalyzer .

For long QT syndrome (LQTS), recent progress in genome-sequencing technologies enabled the identification of rare genomic variants with diagnostic, prognostic, and therapeutic implications. However, pathogenic stratification of the identified variants remains challenging, especially in variants of uncertain significance. This study aimed to propose a phenotypic cell-based diagnostic assay for identifying LQTS to recognize pathogenic variants in a high-throughput manner suitable for screening. We investigated the response of LQT2-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) following I Kr blockade using a multi-electrode array, finding that the response to I Kr blockade was significantly smaller than in Control-iPSC-CMs. Furthermore, we found that LQT1-iPSC-CMs and LQT3-iPSC-CMs could be distinguished from Control-iPSC-CMs by I Ks blockade and I Na blockade, respectively. This strategy might be helpful in compensating for the shortcomings of genetic testing of LQTS patients., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in gene regulatory elements have been associated with a wide range of complex neuropsychiatric disorders. However, due to their cell-type specificity and difficulties in characterizing their regulatory targets, the ability to identify causal genetic variants has remained limited. To address these constraints, we perform an integrative analysis of chromatin interactions, open chromatin regions and transcriptomes using promoter capture Hi-C, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing, respectively, in four functionally distinct neural cell types: induced pluripotent stem cell (iPSC)-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus-like neurons and primary astrocytes. We identify hundreds of thousands of long-range cis-interactions between promoters and distal promoter-interacting regions, enabling us to link regulatory elements to their target genes and reveal putative processes that are dysregulated in disease. Finally, we validate several promoter-interacting regions by using clustered regularly interspaced short palindromic repeats (CRISPR) techniques in human excitatory neurons, demonstrating that CDK5RAP3, STRAP and DRD2 are transcriptionally regulated by physically linked enhancers.

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

MESP1 is a key transcription factor in development of early cardiovascular tissue and it is required for induction of the cardiomyocyte (CM) gene expression program, but its role in vascular development is unclear. Here, we used inducible CRISPRi knock-down of MESP1 to analyze the molecular processes of the early differentiation stages of human induced pluripotent stem cells into mesoderm and subsequently vascular progenitor cells. We found that expression of the mesodermal marker, BRACHYURY (encoded by T) was unaffected in MESP1 knock-down cells as compared to wild type cells suggesting timely movement through the primitive streak whereas another mesodermal marker MIXL1 was slightly, but significantly decreased. In contrast, the expression of the vascular cell surface marker KDR was decreased and CD31 and CD34 expression were substantially reduced in MESP1 knock-down cells supporting inhibition or delay of vascular specification. In addition, mRNA microarray data revealed several other altered gene expressions including the EMT regulating transcription factors SNAI1 and TWIST1, which were both significantly decreased indicating that MESP1 knock-down cells are less likely to undergo EMT during vascular progenitor differentiation. Our study demonstrates that while leaving primitive streak markers unaffected, MESP1 expression is required for timely vascular progenitor specification. Thus, MESP1 expression is essential for the molecular features of early CM, EC and VSMC lineage specification., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The integration of in vitro cardiac tissue models, human induced pluripotent stem cells (hiPSCs) and genome-editing tools allows for the enhanced interrogation of physiological phenotypes and recapitulation of disease pathologies. Here, using a cardiac tissue model consisting of filamentous three-dimensional matrices populated with cardiomyocytes derived from healthy wild-type (WT) hiPSCs (WT hiPSC-CMs) or isogenic hiPSCs deficient in the sarcomere protein cardiac myosin-binding protein C (MYBPC3 -/- hiPSC-CMs), we show that the WT microtissues adapted to the mechanical environment with increased contraction force commensurate to matrix stiffness, whereas the MYBPC3 -/- microtissues exhibited impaired force development kinetics regardless of matrix stiffness and deficient contraction force only when grown on matrices with high fibre stiffness. Under mechanical overload, the MYBPC3 -/- microtissues had a higher degree of calcium transient abnormalities, and exhibited an accelerated decay of calcium dynamics as well as calcium desensitization, which accelerated when contracting against stiffer fibres. Our findings suggest that MYBPC3 deficiency and the presence of environmental stresses synergistically lead to contractile deficits in cardiac tissues.

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis., Competing Interests: AL, DJ, PS, JM, FM, VW No competing interests declared, MM Currently an employee of Tenaya Therapeutics. BC On the Scientific Advisory Boards of: CODA Therapeutics, making therapies related to pain relief; Cellogy Inc., a cell dynamics measurement company; and Scientist.com, a scientific services company. Also a founder of Tenaya Therapeutics (https://www.tenayatherapeutics.com/), a company focused on finding treatments for heart failure, including the use of CRISPR interference to interrogate genetic cardiomyopathies. Holds equity in Tenaya, and Tenaya provides research support for heart failure-related research. TM A consultant for Tenaya Therapeutics., (© 2018, Libby et al.)

Quantification of abnormal contractile motions of cardiac tissue has been a noteworthy challenge and significant limitation in assessing and classifying the drug-induced arrhythmias (i.e., Torsades de pointes). To overcome these challenges, researchers have taken advantage of computational image processing tools to measure contractile motion from cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). However, the amplitude and frequency analysis of contractile motion waveforms does not produce sufficient information to objectively classify the degree of variations between two or more sets of cardiac contractile motions. In this paper, we generated contractile motion data from beating hiPSC-CMs using motion tracking software based on optical flow analysis, and then implemented a computational algorithm, phase space reconstruction (PSR), to derive parameters (embedding, regularity, and fractal dimensions) to further characterize the dynamic nature of the cardiac contractile motions. Application of drugs known to cause cardiac arrhythmia induced significant changes to these resultant dimensional parameters calculated from PSR analysis. Integrating this new computational algorithm with the existing analytical toolbox of cardiac contractile motions will allow us to expand current assessments of cardiac tissue physiology into an automated, high-throughput, and quantifiable manner which will allow more objective assessments of drug-induced proarrhythmias., (© 2018 Wiley Periodicals, Inc.)

The creation of human induced pluripotent stem cells (hiPSCs) has provided an unprecedented opportunity to study tissue morphogenesis and organ development through 'organogenesis-in-a-dish'. Current approaches to cardiac organoid engineering rely on either direct cardiac differentiation from embryoid bodies (EBs) or generation of aligned cardiac tissues from predifferentiated cardiomyocytes from monolayer hiPSCs. To experimentally model early cardiac organogenesis in vitro, our protocol combines biomaterials-based cell patterning with stem cell organoid engineering. 3D cardiac microchambers are created from 2D hiPSC colonies; these microchambers approximate an early-development heart with distinct spatial organization and self-assembly. With proper training in photolithography microfabrication, maintenance of human pluripotent stem cells, and cardiac differentiation, a graduate student with guidance will likely be able to carry out this experimental protocol, which requires ∼3 weeks. We envisage that this in vitro model of human early heart development could serve as an embryotoxicity screening assay in drug discovery, regulation, and prescription for healthy fetal development. We anticipate that, when applied to hiPSC lines derived from patients with inherited diseases, this protocol can be used to study the disease mechanisms of cardiac malformations at an early stage of embryogenesis.

Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel.

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

Rationale: During each beat, cardiac myocytes (CMs) generate the mechanical output necessary for heart function through contractile mechanisms that involve shortening of sarcomeres along myofibrils. Human-induced pluripotent stem cells (hiPSCs) can be differentiated into CMs (hiPSC-CMs) that model cardiac contractile mechanical output more robustly when micropatterned into physiological shapes. Quantifying the mechanical output of these cells enables us to assay cardiac activity in a dish., Objective: We sought to develop a computational platform that integrates analytic approaches to quantify the mechanical output of single micropatterned hiPSC-CMs from microscopy videos., Methods and Results: We micropatterned single hiPSC-CMs on deformable polyacrylamide substrates containing fluorescent microbeads. We acquired videos of single beating cells, of microbead displacement during contractions, and of fluorescently labeled myofibrils. These videos were independently analyzed to obtain parameters that capture the mechanical output of the imaged single cells. We also developed novel methods to quantify sarcomere length from videos of moving myofibrils and to analyze loss of synchronicity of beating in cells with contractile defects. We tested this computational platform by detecting variations in mechanical output induced by drugs and in cells expressing low levels of myosin-binding protein C., Conclusions: Our method can measure the cardiac function of single micropatterned hiPSC-CMs and determine contractile parameters that can be used to elucidate mechanisms that underlie variations in CM function. This platform will be amenable to future studies of the effects of mutations and drugs on cardiac function., (© 2017 American Heart Association, Inc.)

The human genome produces thousands of long noncoding RNAs (lncRNAs)-transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs., (Copyright © 2017, American Association for the Advancement of Science.)

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes., Competing Interests: S.Y. is a scientific advisor of iPS Academia Japan without salary. E.C.H. receives research funding from Clementia Pharmaceuticals to support clinical trials in FOP that are unrelated to this work.

This protocol is designed to detect single-nucleotide substitutions generated by genome editing in a highly sensitive and quantitative manner. It uses a combination of allele-specific hydrolysis probes and a new digital polymerase chain reaction (dPCR) technology called droplet digital PCR (ddPCR). ddPCR partitions a reaction into more than 10,000 nanoliter-scale water-in-oil droplets. As a result, each droplet contains only a few copies of the genome so that ddPCR is able to detect rare genome-editing events without missing them., (© 2016 Cold Spring Harbor Laboratory Press.)

The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells., (© 2016 Cold Spring Harbor Laboratory Press.)

Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.

Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements. Here we describe an approach that combines features of EHM and cardiospheres: Micro-Heart Muscle (μHM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form μHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior "cranial" NCC form craniofacial bone, whereas solely posterior "trunk" NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages.

Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site-specific double-strand break (DSB)-mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA., (Copyright © 2016 John Wiley & Sons, Inc.)

G-protein-coupled receptors (GPCRs) are key regulators of skeletal homeostasis and are likely important in fracture healing. Because GPCRs can activate multiple signaling pathways simultaneously, we used targeted disruption of G(i) -GPCR or activation of G(s) -GPCR pathways to test how each pathway functions in the skeleton. We previously demonstrated that blockade of G(i) signaling by pertussis toxin (PTX) transgene expression in maturing osteoblastic cells enhanced cortical and trabecular bone formation and prevented age-related bone loss in female mice. In addition, activation of G(s) signaling by expressing the G(s) -coupled engineered receptor Rs1 in maturing osteoblastic cells induced massive trabecular bone formation but cortical bone loss. Here, we test our hypothesis that the G(i) and G(s) pathways also have distinct functions in fracture repair. We applied closed, nonstabilized tibial fractures to mice in which endogenous G(i) signaling was inhibited by PTX, or to mice with activated G(s) signaling mediated by Rs1. Blockade of endogenous G(i) resulted in a smaller callus but increased bone formation in both young and old mice. PTX treatment decreased expression of Dkk1 and increased Lef1 mRNAs during fracture healing, suggesting a role for endogenous G(i) signaling in maintaining Dkk1 expression and suppressing Wnt signaling. In contrast, adult mice with activated Gs signaling showed a slight increase in the initial callus size with increased callus bone formation. These results show that G(i) blockade and G(s) activation of the same osteoblastic lineage cell can induce different biological responses during fracture healing. Our findings also show that manipulating the GPCR/cAMP signaling pathway by selective timing of G(s) and G(i) -GPCR activation may be important for optimizing fracture repair., (© 2015 American Society for Bone and Mineral Research.)

Tissue morphogenesis and organ formation are the consequences of biochemical and biophysical cues that lead to cellular spatial patterning in development. To model such events in vitro, we use PEG-patterned substrates to geometrically confine human pluripotent stem cell colonies and spatially present mechanical stress. Modulation of the WNT/β-catenin pathway promotes spatial patterning via geometric confinement of the cell condensation process during epithelial-mesenchymal transition, forcing cells at the perimeter to express an OCT4+ annulus, which is coincident with a region of higher cell density and E-cadherin expression. The biochemical and biophysical cues synergistically induce self-organizing lineage specification and creation of a beating human cardiac microchamber confined by the pattern geometry. These highly defined human cardiac microchambers can be used to study aspects of embryonic spatial patterning, early cardiac development and drug-induced developmental toxicity.