Preclinical evaluation of CRISPR-based therapies for Noonan syndrome caused by deep-intronic LZTR1 variants.

Gene variants in LZTR1 are implicated to cause Noonan syndrome associated with a severe and early-onset hypertrophic cardiomyopathy. Mechanistically, LZTR1 deficiency results in accumulation of RAS GTPases and, as a consequence, in RAS-MAPK signaling hyperactivity, thereby causing the Noonan syndrome-associated phenotype. Despite its epidemiological relevance, pharmacological as well as invasive therapies remain limited. Here, personalized CRISPR-Cas9 gene therapies might offer a novel alternative for a curative treatment in this patient cohort. In this study, by utilizing a patient-specific screening platform based on iPSC-derived cardiomyocytes from two Noonan syndrome patients, we evaluated different clinically translatable therapeutic approaches using small Cas9 orthologs targeting a deep-intronic LZTR1 variant to cure the disease-associated molecular pathology. Despite high editing efficiencies in cardiomyocyte cultures transduced with lentivirus or all-in-one adeno-associated viruses, we observed crucial differences in editing outcomes in proliferative iPSCs vs. non-proliferative cardiomyocytes. While editing in iPSCs rescued the phenotype, the same editing approaches did not robustly restore LZTR1 function in cardiomyocytes, indicating critical differences in the activity of DNA double-strand break repair mechanisms between proliferative and non-proliferative cell types and highlighting the importance of cell type-specific screens for testing CRISPR-Cas9 gene therapies.

Genome engineering of a neuronal specific, optogenetic, induced pluripotent stem cell line.

Control of neuronal activity by optogenetic tools is increasingly explored in disease modelling and optogenetics and holds great promise for regenerative therapy. To investigate neuronal connectivity with other excitable cells we established an optogenetic induced pluripotent stem cell line. The SynfChrimson line harbors a stably integrated, fast, red light-activatable channel (f-Chrimson), under the control of synapsin promotor in the AAVS1 locus. Multielectrode array analysis showed that SynfChrimson derived neurons are light-activatable. The specificity of the SynfChrimson function in neurons was validated by cardiomyocyte differentiations which do not respond to light stimulations.

4D-Printable Photocrosslinkable Polyurethane-Based Inks for Tissue Scaffold and Actuator Applications.

4D printing recently emerges as an exciting evolution of conventional 3D printing, where a printed construct can quickly transform in response to a specific stimulus to switch between a temporary variable state and an original state. In this work, a photocrosslinkable polyethylene-glycol polyurethane ink is synthesized for light-assisted 4D printing of smart materials. The molecular weight distribution of the ink monomers is tunable by adjusting the copolymerization reaction time. Digital light processing (DLP) technique is used to program a differential swelling response in the printed constructs after humidity variation. Bioactive microparticles are embedded into the ink and the improvement of biocompatibility of the printed constructs is demonstrated for tissue engineering applications. Cell studies reveal above 90% viability in 1 week and ≈50% biodegradability after 4 weeks. Self-folding capillary scaffolds, dynamic grippers, and film actuators are made and activated in a humid environment. The approach offers a versatile platform for the fabrication of complex constructs. The ink can be used in tissue engineering and actuator applications, making the ink a promising avenue for future research.

Protocol to develop force-generating human skeletal muscle organoids.

Force generation is an essential property of skeletal muscle models in vitro. We describe a versatile 1-step procedure to direct undifferentiated human pluripotent stem cells (PSCs) into contractile skeletal muscle organoids (SMOs). Our protocol provides detailed steps for 3D casting of PSCs using either collagen-I/Matrigel- or fibrin/Geltrex-based hydrogels, SMO differentiation, and application of different culture platforms for mechanical loading and contractility analysis. The SMO model may be particularly useful to study human muscle development and developmental skeletal muscle disorders in vitro. For complete details on the use and execution of this protocol, please refer to Shahriyari et al.1.

Atrial fibrillation-associated electrical remodelling in human induced pluripotent stem cell-derived atrial cardiomyocytes: a novel pathway for antiarrhythmic therapy development.

AIMS: Atrial fibrillation (AF) is associated with tachycardia-induced cellular electrophysiology alterations which promote AF chronification and treatment resistance. Development of novel antiarrhythmic therapies is hampered by the absence of scalable experimental human models that reflect AF-associated electrical remodelling. Therefore, we aimed to assess if AF-associated remodelling of cellular electrophysiology can be simulated in human atrial-like cardiomyocytes derived from induced pluripotent stem cells in the presence of retinoic acid (iPSC-aCM), and atrial-engineered human myocardium (aEHM) under short term (24 h) and chronic (7 days) tachypacing (TP). METHODS AND RESULTS: First, 24-h electrical pacing at 3 Hz was used to investigate whether AF-associated remodelling in iPSC-aCM and aEHM would ensue. Compared to controls (24 h, 1 Hz pacing) TP-stimulated iPSC-aCM presented classical hallmarks of AF-associated remodelling: (i) decreased L-type Ca2+ current (ICa,L) and (ii) impaired activation of acetylcholine-activated inward-rectifier K+ current (IK,ACh). This resulted in action potential shortening and an absent response to the M-receptor agonist carbachol in both iPSC-aCM and aEHM subjected to TP. Accordingly, mRNA expression of the channel-subunit Kir3.4 was reduced. Selective IK,ACh blockade with tertiapin reduced basal inward-rectifier K+ current only in iPSC-aCM subjected to TP, thereby unmasking an agonist-independent constitutively active IK,ACh. To allow for long-term TP, we developed iPSC-aCM and aEHM expressing the light-gated ion-channel f-Chrimson. The same hallmarks of AF-associated remodelling were observed after optical-TP. In addition, continuous TP (7 days) led to (i) increased amplitude of inward-rectifier K+ current (IK1), (ii) hyperpolarization of the resting membrane potential, (iii) increased action potential-amplitude and upstroke velocity as well as (iv) reversibly impaired contractile function in aEHM. CONCLUSIONS: Classical hallmarks of AF-associated remodelling were mimicked through TP of iPSC-aCM and aEHM. The use of the ultrafast f-Chrimson depolarizing ion channel allowed us to model the time-dependence of AF-associated remodelling in vitro for the first time. The observation of electrical remodelling with associated reversible contractile dysfunction offers a novel platform for human-centric discovery of antiarrhythmic therapies.

Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy is a severe cardiac disorder characterized by lethal arrhythmias and sudden cardiac death, with currently no effective treatment. Plakophilin 2 (PKP2) is the most frequently affected gene. Here we show that adeno-associated virus (AAV)-mediated delivery of PKP2 in PKP2c.2013delC/WT induced pluripotent stem cell-derived cardiomyocytes restored not only cardiac PKP2 levels but also the levels of other junctional proteins, found to be decreased in response to the mutation. PKP2 restoration improved sodium conduction, indicating rescue of the arrhythmic substrate in PKP2 mutant induced pluripotent stem cell-derived cardiomyocytes. Additionally, it enhanced contractile function and normalized contraction kinetics in PKP2 mutant engineered human myocardium. Recovery of desmosomal integrity and cardiac function was corroborated in vivo, by treating heterozygous Pkp2c.1755delA knock-in mice. Long-term treatment with AAV9-PKP2 prevented cardiac dysfunction in 12-month-old Pkp2c.1755delA/WT mice, without affecting wild-type mice. These findings encourage clinical exploration of PKP2 gene therapy for patients with PKP2 haploinsufficiency.

Somatic mosaicism in STAG2-associated cohesinopathies: Expansion of the genotypic and phenotypic spectrum.

STAG2 is a component of the large, evolutionarily highly conserved cohesin complex, which has been linked to various cellular processes like genome organization, DNA replication, gene expression, heterochromatin formation, sister chromatid cohesion, and DNA repair. A wide spectrum of germline variants in genes encoding subunits or regulators of the cohesin complex have previously been identified to cause distinct but phenotypically overlapping multisystem developmental disorders belonging to the group of cohesinopathies. Pathogenic variants in STAG2 have rarely been implicated in an X-linked cohesinopathy associated with undergrowth, developmental delay, and dysmorphic features. Here, we describe for the first time a mosaic STAG2 variant in an individual with developmental delay, microcephaly, and hemihypotrophy of the right side. We characterized the grade of mosaicism by deep sequencing analysis on DNA extracted from EDTA blood, urine and buccal swabs. Furthermore, we report an additional female with a novel de novo splice variant in STAG2. Interestingly, both individuals show supernumerary nipples, a feature that has not been reported associated to STAG2 before. Remarkably, additional analysis of STAG2 transcripts in both individuals showed only wildtype transcripts, even after blockage of nonsense-mediated decay using puromycin in blood lymphocytes. As the phenotype of STAG2-associated cohesinopathies is dominated by global developmental delay, severe microcephaly, and brain abnormalities, we investigated the expression of STAG2 and other related components of the cohesin complex during Bioengineered Neuronal Organoids (BENOs) generation by RNA sequencing. Interestingly, we observed a prominent expression of STAG2, especially between culture days 0 and 15, indicating an essential function of STAG2 in early brain development. In summary, we expand the genotypic and phenotypic spectrum of STAG2-associated cohesinopathies and show that BENOs represent a promising model to gain further insights into the critical role of STAG2 in the complex process of nervous system development.

Engineered skeletal muscle recapitulates human muscle development, regeneration and dystrophy.

BACKGROUND: Human pluripotent stem cell-derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for the derivation of skeletal muscle cells and their utility in skeletal muscle tissue engineering with the aim to model skeletal muscle regeneration and dystrophy in vitro. METHODS: Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors followed by primary and secondary foetal myogenesis into three-dimensional muscle. To simulate Duchenne muscular dystrophy (DMD), a patient-specific induced pluripotent stem cell line was compared to a CRISPR/Cas9-edited isogenic control line. RESULTS: The established skeletal muscle differentiation protocol robustly and faithfully recapitulates critical steps of embryonic myogenesis in two-dimensional and three-dimensional cultures, resulting in functional human skeletal muscle organoids (SMOs) and engineered skeletal muscles (ESMs) with a regeneration-competent satellite-like cell pool. Tissue-engineered muscle exhibits organotypic maturation and function (up to 5.7 ± 0.5 mN tetanic twitch tension at 100 Hz in ESM). Contractile performance could be further enhanced by timed thyroid hormone treatment, increasing the speed of contraction (time to peak contraction) as well as relaxation (time to 50% relaxation) of single twitches from 107 ± 2 to 75 ± 4 ms (P < 0.05) and from 146 ± 6 to 100 ± 6 ms (P < 0.05), respectively. Satellite-like cells could be documented as largely quiescent PAX7+ cells (75 ± 6% Ki67- ) located adjacent to muscle fibres confined under a laminin-containing basal membrane. Activation of the engineered satellite-like cell niche was documented in a cardiotoxin injury model with marked recovery of contractility to 57 ± 8% of the pre-injury force 21 days post-injury (P < 0.05 compared to Day 2 post-injury), which was completely blocked by preceding irradiation. Absence of dystrophin in DMD ESM caused a marked reduction of contractile force (-35 ± 7%, P < 0.05) and impaired expression of fast myosin isoforms resulting in prolonged contraction (175 ± 14 ms, P < 0.05 vs. gene-edited control) and relaxation (238 ± 22 ms, P < 0.05 vs. gene-edited control) times. Restoration of dystrophin levels by gene editing rescued the DMD phenotype in ESM. CONCLUSIONS: We introduce human muscle models with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease and repair.

Using different geometries to modulate the cardiac fibroblast phenotype and the biomechanical properties of engineered connective tissues.

Tissue engineering with human cardiac fibroblasts (CF) allows identifying novel mechanisms and anti-fibrotic drugs in the context of cardiac fibrosis. However, substantial knowledge on the influences of the used materials and tissue geometries on tissue properties and cell phenotypes is necessary to be able to choose an appropriate model for a specific research question. As there is a clear lack of information on how CF react to the mold architecture in engineered connective tissues (ECT), we first compared the effect of two mold geometries and materials with different hardnesses on the biomechanical properties of ECT. We could show that ECT, which formed around two distant poles (non-uniform model) were less stiff and more strain-resistant than ECT, which formed around a central rod (uniform model), independent of the materials used for poles and rods. Next, we investigated the cell state and could demonstrate that in the uniform versus non-uniform model, the embedded cells have a higher cell cycle activity and display a more pronounced myofibroblast phenotype. Differential gene expression analysis revealed that uniform ECT displayed a fibrosis-associated gene signature similar to the diseased heart. Furthermore, we were able to identify important relationships between cell and tissue characteristics, as well as between biomechanical tissue parameters by implementing cells from normal heart and end-stage heart failure explants from patients with ischemic or dilated cardiomyopathy. Finally, we show that the application of pro- and anti-fibrotic factors in the non-uniform and uniform model, respectively, is not sufficient to mimic the effect of the other geometry. Taken together, we demonstrate that modifying the mold geometry in tissue engineering with CF offers the possibility to compare different cellular phenotypes and biomechanical tissue properties.

Impact of Immunosuppressive Drugs on Fibroblasts: An In Vitro Study.

Background: The aim of this study was to compare the direct impact of different agents for immunosuppressive therapy on mouse fibroblasts as a possible cause of drug-induced gingival overgrowth (DIGO). Methods: 3T3 mouse fibroblasts were cultivated in cell-specific media (2 × 104 cells/mL) and treated for 6, 24, 48 and 72 h with one of three immunosuppressive drugs (IsDs): cyclosporin a (CsA), tacrolimus (TaC) and sirolimus (SiR). Different concentrations (10−750 ng/mL) were used to mimic serum levels under active immunosuppressive therapy conditions. Cell population characteristics (cell number, viability and morphology) were assessed using computer-assisted cell analysis. Expression of pro-collagen type I carboxy-terminal propeptide (PICP) was identified using an ELISA assay. Results: The influence of IsDs on the biological status of 3T3 fibroblasts was time- and dose-dependent. Comparing CsA and TaC, the total cell amount was enhanced using concentrations in the range of 10−150 ng/mL (p > 0.05). In contrast, treatment with SiR resulted in a decrease in the average cell number (p < 0.01). PICP and cell diameter of fibroblasts were not susceptible to IsD treatment (p > 0.05). Conclusions: Our results revealed time-dependent effects of IsDs, with distinct influences on cell number. The cell morphology and the PICP balance of the investigated fibroblast cell line remained unaffected. Hence, the potential role of IsDs is not a unilateral mechanism of action but rather a multifactorial process.

Defined Engineered Human Myocardium for Disease Modeling, Drug Screening, and Heart Repair.

Different engineered heart muscle formats have been developed for applications in disease modeling, drug screening, and heart repair. The advantage of 3D engineered versus 2D monolayer and 3D aggregate cardiomyocyte cultures is a clearly advanced degree of maturation, which in many aspects resembles the postnatal rather than the embryonic or fetal heart, in the most advanced 3D culture formats. According to the desired in vitro (disease modeling or drug screening) and in vivo (heart repair) application, scale and geometry of tissue engineered heart muscle must be adapted. In this updated methods paper, we report a simple and scalable (up and down) collagen-based protocol for the construction of Engineered Human Myocardium (EHM) under defined, serum-free conditions.

Remote control of the heart and beyond.

A resorbable closed-loop sensor-actuator implant can temporarily control heart rate.

Transmural myocardial repair with engineered heart muscle in a rat model of heterotopic heart transplantation - A proof-of-concept study.

Engineered heart muscle (EHM) can be implanted epicardially to remuscularize the failing heart. In case of a severely scarred ventricle, excision of scar followed by transmural heart wall replacement may be a more desirable application. Accordingly, we tested the hypothesis that allograft (rat) and xenograft (human) EHM can also be administered as transmural heart wall replacement in a heterotopic, volume-loaded heart transplantation model. We first established a novel rat model model to test surgical transmural left heart wall repair. Subsequently and in continuation of our previous allograft studies, we tested outcome after implantation of contractile engineered heart muscle (EHM) and non-contractile engineered connective tissue (ECT) as well as engineered mesenchymal tissue (EMT) allografts as transmural heart wall replacement. Finally, proof-of-concept for the application of human EHM was obtained in an athymic nude rat model. Only in case of EHM implantation, remuscularization of the surgically created transmural defect was observed with palpable graft vascularization. Taken together, feasibility of transmural heart repair using bioengineered myocardial grafts could be demonstrated in a novel rat model of heterotopic heart transplantation.

Regenerative potential of epicardium-derived extracellular vesicles mediated by conserved miRNA transfer.

AIMS: After a myocardial infarction, the adult human heart lacks sufficient regenerative capacity to restore lost tissue, leading to heart failure progression. Finding novel ways to reprogram adult cardiomyocytes into a regenerative state is a major therapeutic goal. The epicardium, the outermost layer of the heart, contributes cardiovascular cell types to the forming heart and is a source of trophic signals to promote heart muscle growth during embryonic development. The epicardium is also essential for heart regeneration in zebrafish and neonatal mice and can be reactivated after injury in adult hearts to improve outcome. A recently identified mechanism of cell-cell communication and signalling is that mediated by extracellular vesicles (EVs). Here, we aimed to investigate epicardial signalling via EV release in response to cardiac injury and as a means to optimize cardiac repair and regeneration. METHODS AND RESULTS: We isolated epicardial EVs from mouse and human sources and targeted the cardiomyocyte population. Epicardial EVs enhanced proliferation in H9C2 cells and in primary neonatal murine cardiomyocytes in vitro and promoted cell cycle re-entry when injected into the injured area of infarcted neonatal hearts. These EVs also enhanced regeneration in cryoinjured engineered human myocardium (EHM) as a novel model of human myocardial injury. Deep RNA-sequencing of epicardial EV cargo revealed conserved microRNAs (miRs) between human and mouse epicardial-derived exosomes, and the effects on cell cycle re-entry were recapitulated by administration of cargo miR-30a, miR-100, miR-27a, and miR-30e to human stem cell-derived cardiomyocytes and cryoinjured EHM constructs. CONCLUSION: Here, we describe the first characterization of epicardial EV secretion, which can signal to promote proliferation of cardiomyocytes in infarcted mouse hearts and in a human model of myocardial injury, resulting in enhanced contractile function. Analysis of exosome cargo in mouse and human identified conserved pro-regenerative miRs, which in combination recapitulated the therapeutic effects of promoting cardiomyocyte proliferation.

Truncated titin proteins and titin haploinsufficiency are targets for functional recovery in human cardiomyopathy due to TTN mutations.

Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cell­derived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9­generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.

Basic and Translational Research in Cardiac Repair and Regeneration: JACC State-of-the-Art Review.

This paper aims to provide an important update on the recent preclinical and clinical trials using cell therapy strategies and engineered heart tissues for the treatment of postinfarction left ventricular remodeling and heart failure. In addition to the authors' own works and opinions on the roadblocks of the field, they discuss novel approaches for cardiac remuscularization via the activation of proliferative mechanisms in resident cardiomyocytes or direct reprogramming of somatic cells into cardiomyocytes. This paper's main mindset is to present current and future strategies in light of their implications for the design of future patient trials with the ultimate objective of facilitating the translation of discoveries in regenerative myocardial therapies to the clinic.

A microRNA signature that correlates with cognition and is a target against cognitive decline.

While some individuals age without pathological memory impairments, others develop age-associated cognitive diseases. Since changes in cognitive function develop slowly over time in these patients, they are often diagnosed at an advanced stage of molecular pathology, a time point when causative treatments fail. Thus, there is great need for the identification of inexpensive and minimal invasive approaches that could be used for screening with the aim to identify individuals at risk for cognitive decline that can then undergo further diagnostics and eventually stratified therapies. In this study, we use an integrative approach combining the analysis of human data and mechanistic studies in model systems to identify a circulating 3-microRNA signature that reflects key processes linked to neural homeostasis and inform about cognitive status. We furthermore provide evidence that expression changes in this signature represent multiple mechanisms deregulated in the aging and diseased brain and are a suitable target for RNA therapeutics.

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Establishment of a second generation homozygous CRISPRa human induced pluripotent stem cell (hiPSC) line for enhanced levels of endogenous gene activation.

CRISPR/Cas9 technology based on nuclease inactive dCas9 and fused to the heterotrimeric VPR transcriptional activator is a powerful tool to enhance endogenous transcription by targeting defined genomic loci. We generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying dCas9 fused to VPR along with a WPRE element at the AAVS1 locus (CRISPRa2). We demonstrated pluripotency, genomic integrity and differentiation potential into all three germ layers. CRISPRa2 cells showed increased transgene expression and higher transcriptional induction in hiPSC-derived cardiomyocytes compared to a previously described CRISPRa line. Both lines allow studying endogenous transcriptional modulation with lower and higher transcript abundance.