Inherited cardiac diseases, pluripotent stem cells, and genome editing combined-the past, present, and future.

Research on mechanisms underlying monogenic cardiac diseases such as primary arrhythmias and cardiomyopathies has until recently been hampered by inherent limitations of heterologous cell systems, where mutant genes are expressed in noncardiac cells, and physiological differences between humans and experimental animals. Human-induced pluripotent stem cells (hiPSCs) have proven to be a game changer by providing new opportunities for studying the disease in the specific cell type affected, namely the cardiomyocyte. hiPSCs are particularly valuable because not only can they be differentiated into unlimited numbers of these cells, but they also genetically match the individual from whom they were derived. The decade following their discovery showed the potential of hiPSCs for advancing our understanding of cardiovascular diseases, with key pathophysiological features of the patient being reflected in their corresponding hiPSC-derived cardiomyocytes (the past). Now, recent advances in genome editing for repairing or introducing genetic mutations efficiently have enabled the disease etiology and pathogenesis of a particular genotype to be investigated (the present). Finally, we are beginning to witness the promise of hiPSC in personalized therapies for individual patients, as well as their application in identifying genetic variants responsible for or modifying the disease phenotype (the future). In this review, we discuss how hiPSCs could contribute to improving the diagnosis, prognosis, and treatment of an individual with a suspected genetic cardiac disease, thereby developing better risk stratification and clinical management strategies for these potentially lethal but treatable disorders.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

RATIONALE: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. OBJECTIVE: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. METHODS AND RESULTS: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. CONCLUSIONS: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.

STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines.

Targeted integration of large DNA cargoes (>10 kb) or genomic replacements in mammalian cells, such as human pluripotent stem cells (hPS cells), remains challenging. Here we describe a platform termed serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN) to circumvent this. First, a landing pad cassette is precisely inserted or used to replace specific genomic regions. The site-specific integrase Bxb1 then enables DNA constructs, including those >50 kb, to be integrated into the genome, while Cre recombinase excises auxiliary DNA sequences to prevent postintegrative silencing. Using a strategy whereby the positive selection marker is only expressed if the donor plasmid carrying the payload is correctly targeted, we can obtain 100% enrichment for cells containing the DNA payload. Procedures for expressing Cre efficiently also mean that a clonal isolation step is no longer essential to derive the required genetically modified hPS cells containing the integrated DNA, potentially reducing clonal variability. Furthermore, STRAIGHT-IN facilitates rapid and multiplexed generation of genetically matched hPS cells when multiple donor plasmids are delivered simultaneously. STRAIGHT-IN has various applications, which include integrating complex genetic circuits for synthetic biology, as well as creating panels of hPS cells lines containing, as necessary, hundreds of disease-linked variants for disease modeling and drug discovery. After establishing the hPS cell line containing the landing pad, the entire procedure, including donor plasmid synthesis, takes 1.5-3 months, depending on whether single or multiple DNA payloads are integrated. This protocol only requires the researcher to be skilled in molecular biology and standard cell culture techniques.

Targeting colorectal cancer stem cells with inducible caspase-9.

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133(+) CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo.

CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells.

Advances in genome editing and our ability to derive and differentiate human induced pluripotent stem cells (hiPSCs) into a wide variety of cell types present in the body is revolutionizing how we model human diseases in vitro. Central to this has been the development of the CRISPR/Cas9 system as an inexpensive and highly efficient tool for introducing or correcting disease-associated mutations. However, the ease with which CRISPR/Cas9 enables genetic modification is a double-edged sword, with the challenge now being to introduce changes precisely to just one allele without disrupting the other.In this chapter, we describe strategies to introduce specific mutations into hiPSCs without enrichment steps. Monoallelic modification is contingent on the target activity of the guide RNA, delivery method of the CRISPR/Cas9 components and design of the oligonucleotide(s) transfected. As well as addressing these aspects, we detail high throughput culturing, freezing and screening methods to identify clonal hiPSCs with the desired nucleotide change. This set of protocols offers an efficient and ultimately time- and labor-saving approach for generating isogenic pairs of hiPSCs to detect subtle phenotypic differences caused by the disease variant.

STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells.

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively.

Cryopreservation of human pluripotent stem cell-derived cardiomyocytes is not detrimental to their molecular and functional properties.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a powerful platform for in vitro modelling of cardiac diseases, safety pharmacology and drug screening. All these applications require large quantities of well-characterised and standardised batches of hiPSC-CMs. Cryopreservation of hiPSC-CMs without affecting their biochemical or biophysical phenotype is essential for facilitating this, but ideally requires the cells being unchanged by the freeze-thaw procedure. We therefore compared the in vitro functional and molecular characteristics of fresh and cryopreserved hiPSC-CMs generated from multiple independent hiPSC lines. While the frozen hiPSC-CMs exhibited poorer replating than their freshly-derived counterparts, there was no difference in the proportion of cardiomyocytes retrieved from the mixed population when this was factored in, although for several lines a higher percentage of ventricular-like hiPSC-CMs were recovered following cryopreservation. Furthermore, cryopreserved hiPSC-CMs from one line exhibited longer action potential durations. These results provide evidence that cryopreservation does not compromise the in vitro molecular, physiological and mechanical properties of hiPSC-CMs, though can lead to an enrichment in ventricular myocytes. It also validates this procedure for storing hiPSC-CMs, thereby allowing the same batch of hiPSC-CMs to be used for multiple applications and evaluations.

Isogenic Sets of hiPSC-CMs Harboring Distinct KCNH2 Mutations Differ Functionally and in Susceptibility to Drug-Induced Arrhythmias.

Mutations in KCNH2 can lead to long QT syndrome type 2. Variable disease manifestation observed with this channelopathy is associated with the location and type of mutation within the protein, complicating efforts to predict patient risk. Here, we demonstrated phenotypic differences in cardiomyocytes derived from isogenic human induced pluripotent stem cells (hiPSC-CMs) genetically edited to harbor mutations either within the pore or tail region of the ion channel. Electrophysiological analysis confirmed that the mutations prolonged repolarization of the hiPSC-CMs, with differences between the mutations evident in monolayer cultures. Blocking the hERG channel revealed that the pore-loop mutation conferred greater susceptibility to arrhythmic events. These findings showed that subtle phenotypic differences related to KCNH2 mutations could be captured by hiPSC-CMs under genetically matched conditions. Moreover, the results support hiPSC-CMs as strong candidates for evaluating the underlying severity of individual KCNH2 mutations in humans, which could facilitate patient risk stratification.

Role of the human ST6GalNAc-I and ST6GalNAc-II in the synthesis of the cancer-associated sialyl-Tn antigen.

The Sialyl-Tn antigen (Neu5Acalpha2-6GalNAc-O-Ser/Thr) is highly expressed in several human carcinomas and is associated with carcinoma aggressiveness and poor prognosis. We characterized two human sialyltransferases, CMP-Neu5Ac:GalNAc-R alpha2,6-sialyltransferase (ST6GalNAc)-I and ST6GalNAc-II, that are candidate enzymes for Sialyl-Tn synthases. We expressed soluble recombinant hST6GalNAc-I and hST6GalNAc-II and characterized the substrate specificity of both enzymes toward a panel of glycopeptides, glycoproteins, and other synthetic glycoconjugates. The recombinant ST6GalNAc-I and ST6GalNAc-II showed similar substrate specificity toward glycoproteins and GalNAcalpha-O-Ser/Thr glycopeptides, such as glycopeptides derived from the MUC2 mucin and the HIVgp120. We also observed that the amino acid sequence of the acceptor glycopeptide contributes to the in vitro substrate specificity of both enzymes. We additionally established a gastric cell line, MKN45, stably transfected with the full length of either ST6GalNAc-I or ST6GalNAc-II and evaluated the carbohydrate antigens expression profile induced by each enzyme. MKN45 transfected with ST6GalNAc-I showed high expression of Sialyl-Tn, whereas MKN45 transfected with ST6GalNAc-II showed the biosynthesis of the Sialyl-6T structure [Galbeta1-3 (Neu5Acalpha2-6)GalNAc-O-Ser/Thr]. In conclusion, although both enzymes show similar in vitro activities when Tn antigen alone is available, whenever both Tn and T antigens are present, ST6GalNAc-I acts preferentially on Tn antigen, whereas the ST6GalNAc-II acts preferentially on T antigen. Our results show that ST6GalNAc-I is the major Sialyl-Tn synthase and strongly support the hypothesis that the expression of the Sialyl-Tn antigen in cancer cells is due to ST6GalNAc-I activity.

Autophagy in human embryonic stem cells.

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

Molecular identification and targeting of colorectal cancer stem cells.

Tumor initiating or cancer stem cells (CSCs) are suggested to be responsible for tumor initiation and growth. Moreover, therapy resistance and minimal residual disease are thought to result from selective resistance of CSCs. Isolation of CSCs from colon carcinomas can be accomplished by selection of a subpopulation of tumor cells based on expression of one or multiple cell surface markers associated with cancer stemness, like CD133, CD44, CD24, CD29, CD166 and Lgr5. Identification of colon CSCs will lead to a better rational for new therapies that aim to target this fraction specifically. In this review, we analyze known markers used for selection of colon CSCs and their potential function in CSC biology. Moreover, we discuss potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies as well as to address more fundamental questions like the actual role of CSCs in tumor growth.

Generation of human embryonic stem cell reporter knock-in lines by homologous recombination.

This unit describes a series of technical procedures to form clonal human embryonic stem cell (hESC) lines that are genetically modified by homologous recombination. To develop a reporter knock-in hESC line, a vector is configured to contain a reporter gene adjacent to a positive selection cassette. These core elements are flanked by homologous sequences that, following electroporation into hESCs, promote the integration of the vector into the appropriate genomic locus. The positive selection cassette facilitates the enrichment and isolation of genetically modified hESC colonies that are then screened by PCR to identify correctly targeted lines. The selection cassette, flanked by loxP sites, is subsequently excised from the positively targeted hESCs via the transient expression of Cre recombinase. This is necessary because the continued presence of the cassette may interfere with the regulation of the reporter or neighboring genes. Finally, these genetically modified hESCs are clonally isolated using single-cell deposition flow cytometry. Reporter knock-in hESC lines are valuable tools that allow easy and rapid identification and isolation of specific hESC derivatives.

A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis.

The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take approximately 1 month.

Transthyretin is involved in depression-like behaviour and exploratory activity.

Transthyretin (TTR), the major transporter of thyroid hormones and vitamin A in cerebrospinal fluid (CSF), binds the Alzheimer beta-peptide and thus might confer protection against neurodegeneration. In addition, altered TTR levels have been described in the CSF of patients with psychiatric disorders, yet its function in the CNS is far from understood. To determine the role of TTR in behaviour we evaluated the performance of TTR-null mice in standardized tasks described to assess depression, exploratory activity and anxiety. We show that the absence of TTR is associated with increased exploratory activity and reduced signs of depressive-like behaviour. In order to investigate the mechanism underlying these alterations, we measured the levels of catecholamines. We found that the levels of noradrenaline were significantly increased in the limbic forebrain of TTR-null mice. This report represents the first clear indication that TTR plays a role in behaviour, probably by modulation of the noradrenergic system.