Mutation-induced LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome.

Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. In this study, we investigate the mechanistic consequences of a homozygous variant LZTR1L580P by using patient-specific and CRISPR-Cas9-corrected induced pluripotent stem cell (iPSC) cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction identify an LZTR1L580P-specific disease mechanism provoking cardiac hypertrophy. The variant is predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymers. LZTR1 complex dysfunction results in the accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Furthermore, our data show that cardiomyocyte-specific MRAS degradation is mediated by LZTR1 via non-proteasomal pathways, whereas RIT1 degradation is mediated by both LZTR1-dependent and LZTR1-independent pathways. Uni- or biallelic genetic correction of the LZTR1L580P missense variant rescues the molecular and cellular disease phenotype, providing proof of concept for CRISPR-based therapies.

Generation of a genetically-modified induced pluripotent stem cell line harboring an oncogenic gene variant KRAS p.G12V.

Activating KRAS codon 12 gene variants are known to cause severe RAS-MAPK and PI3K-AKT signaling pathway hyperactivity and are frequently involved in the development of various carcinomas. Here, we describe the generation of a human iPSC line harboring the common oncogenic KRAS p.G12V variant by using CRISPR/Cas9 technology. The established KRASG12V iPSC line allows the study of oncogenic KRAS-induced signaling dysregulation and its impact on cell physiology in various iPSC-derived cell types and tissues. Furthermore, it might serve as a powerful platform for drug and toxicity screenings to identify new chemotherapeutic drugs.

In vitro models of human development and their potential application in developmental toxicity testing.

Recent publications describe the development of in vitro models of human development, for which applications in developmental toxicity testing can be envisaged. To date, these regulatory assessments have exclusively been performed in animal studies, the relevance of which to adverse reactions in humans may be questioned. Recently developed cell culture-based models of embryo-fetal development, however, do not yet exhibit sufficient levels of standardisation and reproducibility. Here, the advantages and shortcomings of both in vivo and in vitro developmental toxicity testing are addressed, as well as the possibility of integrated testing strategies as a viable option in the near future.

A Preclinical Study on Brugada Syndrome with a CACNB2 Variant Using Human Cardiomyocytes from Induced Pluripotent Stem Cells.

Aims: Some gene variants in the sodium channels, as well as calcium channels, have been associated with Brugada syndrome (BrS). However, the investigation of the human cellular phenotype and the use of drugs for BrS in presence of variant in the calcium channel subunit is still lacking. Objectives: The objective of this study was to establish a cellular model of BrS in the presence of a CACNB2 variant of uncertain significance (c.425C > T/p.S142F) using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and test drug effects using this model. Methods and results: This study recruited cells from a patient with Brugada syndrome (BrS) and recurrent ventricular fibrillation carrying a missense variant in CACNB2 as well as from three healthy independent persons. These cells (hiPSC-CMs) generated from skin biopsies of healthy persons and the BrS patient (BrS-hiPSC-CMs) as well as CRISPR/Cas9 corrected cells (isogenic control, site-variant corrected) were used for this study. The hiPSC-CMs from the BrS patient showed a significantly reduced L-type calcium channel current (ICa-L) compared with the healthy control hiPSC-CMs. The inactivation curve was shifted to a more positive potential and the recovery from inactivation was accelerated. The protein expression of CACNB2 of the hiPSC-CMs from the BrS-patient was significantly decreased compared with healthy hiPSC-CMs. Moreover, the correction of the CACNB2 site-variant rescued the changes seen in the hiPSC-CMs of the BrS patient to the normal state. These data indicate that the CACNB2 gene variant led to loss-of-function of L-type calcium channels in hiPSC-CMs from the BrS patient. Strikingly, arrhythmia events were more frequently detected in BrS-hiPSC-CMs. Bisoprolol (beta-blockers) at low concentration and quinidine decreased arrhythmic events. Conclusions: The CACNB2 variant (c.425C > T/p.S142F) causes a loss-of-function of L-type calcium channels and is pathogenic for this type of BrS. Bisoprolol and quinidine may be effective for treating BrS with this variant.

Subtype-Directed Differentiation of Human iPSCs into Atrial and Ventricular Cardiomyocytes.

The generation of homogeneous populations of subtype-specific cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial in cardiovascular disease modeling as well as in drug discovery and cardiotoxicity screenings. This protocol describes a simple, robust, and efficient monolayer-based differentiation of hiPSCs into defined atrial and ventricular cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Cyganek et al., 2018.

Intronic CRISPR Repair in a Preclinical Model of Noonan Syndrome-Associated Cardiomyopathy.

BACKGROUND: Noonan syndrome (NS) is a multisystemic developmental disorder characterized by common, clinically variable symptoms, such as typical facial dysmorphisms, short stature, developmental delay, intellectual disability as well as cardiac hypertrophy. The underlying mechanism is a gain-of-function of the RAS-mitogen-activated protein kinase signaling pathway. However, our understanding of the pathophysiological alterations and mechanisms, especially of the associated cardiomyopathy, remains limited and effective therapeutic options are lacking. METHODS: Here, we present a family with two siblings displaying an autosomal recessive form of NS with massive hypertrophic cardiomyopathy as clinically the most prevalent symptom caused by biallelic mutations within the leucine zipper-like transcription regulator 1 (LZTR1). We generated induced pluripotent stem cell-derived cardiomyocytes of the affected siblings and investigated the patient-specific cardiomyocytes on the molecular and functional level. RESULTS: Patients' induced pluripotent stem cell-derived cardiomyocytes recapitulated the hypertrophic phenotype and uncovered a so-far-not-described causal link between LZTR1 dysfunction, RAS-mitogen-activated protein kinase signaling hyperactivity, hypertrophic gene response and cellular hypertrophy. Calcium channel blockade and MEK inhibition could prevent some of the disease characteristics, providing a molecular underpinning for the clinical use of these drugs in patients with NS, but might not be a sustainable therapeutic option. In a proof-of-concept approach, we explored a clinically translatable intronic CRISPR (clustered regularly interspaced short palindromic repeats) repair and demonstrated a rescue of the hypertrophic phenotype. CONCLUSIONS: Our study revealed the human cardiac pathogenesis in patient-specific induced pluripotent stem cell-derived cardiomyocytes from NS patients carrying biallelic variants in LZTR1 and identified a unique disease-specific proteome signature. In addition, we identified the intronic CRISPR repair as a personalized and in our view clinically translatable therapeutic strategy to treat NS-associated hypertrophic cardiomyopathy.

Studying Brugada Syndrome With an SCN1B Variants in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: Among rare channelopathies BrS patients are at high risk of sudden cardiac death (SCD). SCN5A mutations are found in a quarter of patients. Other rare gene mutations including SCN1B have been implicated to BrS. Studying the human cellular phenotype of BrS associated with rare gene mutation remains lacking. OBJECTIVES: We sought to study the cellular phenotype of BrS with the SCN1B gene variants using human-induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: A BrS patient suffering from recurrent syncope harboring a two variants (c.629T > C and c.637C > A) in SCN1B, which encodes the function-modifying sodium channel beta1 subunit, and three independent healthy subjects were recruited and their skin biopsies were used to generate hiPSCs, which were differentiated into cardiomyocytes (hiPSC-CMs) for studying the cellular electrophysiology. A significantly reduced peak and late sodium channel current (INa) and a shift of activation curve to more positive potential as well as a shift of inactivation curve to more negative potential were detected in hiPSC-CMs of the BrS patient, indicating that the SCN1B variants impact the function of sodium channels in cardiomyocytes. The reduced INa led to a reduction of amplitude (APA) and upstroke velocity (V max ) of action potentials. Ajmaline, a sodium channel blocker, showed a stronger effect on APA and Vmax in BrS cells as compared to cells from healthy donors. Furthermore, carbachol was able to increase arrhythmia events and the beating frequency in BrS. CONCLUSION: Our hiPSC-CMs from a BrS-patient with two variants in SCN1B recapitulated some key phenotypic features of BrS and can provide a platform for studies on BrS with SCN1B variants.

A cellular model of Brugada syndrome with SCN10A variants using human-induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Brugada syndrome (BrS) is associated with a pronounced risk to develop sudden cardiac death (SCD). Up to 21% of patients are related to mutations in SCN5A. Studies identified SCN10A as a contributor of BrS. However, the investigation of the human cellular phenotype of BrS in the presence of SCN10A mutations remains lacking. The objective of this study was to establish a cellular model of BrS in presence of SCN10A mutations using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: Dermal fibroblasts obtained from a BrS patient suffering from SCD harbouring the SCN10A double variants (c.3803G>A and c.3749G>A) and three independent healthy control subjects were reprogrammed to hiPSCs. Human-induced pluripotent stem cells were differentiated into cardiomyocytes (hiPSC-CMs).The hiPSC-CMs from the BrS patient showed a significantly reduced peak sodium channel current (INa) and a significantly reduced ATX II (sea anemone toxin, an enhancer of late INa) sensitive as well as A-887826 (a blocker of SCN10A channel) sensitive late sodium channel current (INa) when compared with the healthy control hiPSC-CMs, indicating loss-of-function of sodium channels. Consistent with reduced INa the action potential amplitude and upstroke velocity (Vmax) were significantly reduced, which may contribute to arrhythmogenesis of BrS. Moreover, Ajmaline effects on action potentials were stronger in BrS-hiPSC-CMs than in healthy control cells. This is in agreement with the higher susceptibility of patients to sodium channel blocking drugs in unmasking BrS. CONCLUSION: Patient-specific hiPSC-CMs are able to recapitulate single-cell phenotype features of BrS with SCN10A mutations and may provide novel opportunities to further elucidate the cellular disease mechanism.

In vitro modelling of familial amyloidotic polyneuropathy allows quantitative detection of transthyretin amyloid fibril-like structures in hepatic derivatives of patient-specific induced pluripotent stem cells.

The transthyretin protein is thermodynamically destabilised by mutations in the transthyretin gene, promoting the formation of amyloid fibrils in various tissues. Consequently, impaired autonomic organ function is observed in patients suffering from transthyretin-related familial amyloidotic polyneuropathy (FAP). The influence of individual genetic backgrounds on fibril formation as a potential cause of genotype-phenotype variations needs to be investigated in order to ensure efficient patient-specific therapies. We reprogrammed FAP patient fibroblasts to induced pluripotent stem (iPS) cells and differentiated these cells into transthyretin-expressing hepatocyte-like cells (HLCs). HLCs differentiated from FAP iPS cells and healthy control iPS cells secreted the transthyretin protein in similar concentrations. Mass spectrometry revealed the presence of mutant transthyretin protein in FAP HLC supernatants. In comparison to healthy control iPS cells, we demonstrated the formation of transthyretin amyloid fibril-like structures in FAP HLC supernatants using the amyloid-specific dyes Congo red and thioflavin T. These dyes were also applicable for the quantitative determination of in vitro formed transthyretin fibril-like structures. Moreover, we confirmed the inhibition of fibril formation by the TTR kinetic stabiliser diclofenac. Thioflavin T fluorescence intensity measurements even allowed the quantification of amyloid fibril-like structures in 96-well plate formats as a prerequisite for patient-specific drug screening approaches.

Biphasic modulation of Wnt signaling supports efficient foregut endoderm formation from human pluripotent stem cells.

Pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are of great promise in regenerative medicine, including molecular studies of disease mechanisms, if the affected cell type can be authentically generated during in vitro differentiation. Most existing protocols aim to mimic embryonic development steps by the supplementation of specific cytokines and small molecules, but the involved signaling pathways need further exploration. In this study, we investigated enhanced initial activation of Wnt signaling for definitive endoderm formation and subsequent rapid shutdown of Wnt signaling for proper foregut endoderm specification using 3 µM CHIR99021 and 0.5 µg/mL of secreted frizzled-related protein 5 (sFRP-5) for biphasic modulation of the Wnt pathway. The definitive endoderm and foregut endoderm differentiation capabilities of Wnt pathway-modulated cells were determined based on the expression levels of the endodermal transcription factors SOX17 and FOXA2 and those of the transcription activator GATA4 and the α-fetoprotein (AFP) gene, respectively. Furthermore, the resulting biphasic Wnt pathway modulation was investigated at the protein level by analyzing phosphorylation of glycogen synthase kinase 3 beta (GSK3ß) and ß-catenin. Finally, Wnt target gene expression was determined using an improved lentiviral reporter construct that enabled robust T-cell transcription factor 4 (TCF4)/lymphoid enhancer-binding factor 1 (LEF1)-mediated luciferase expression in differentiating pluripotent stem cells. In conclusion, we demonstrated robust, homogeneous, and efficient derivation of foregut endodermal cells by inducing a biphasic modulation of the Wnt signaling pathway.

Human Mesenchymal Stem Cells Display Reduced Expression of CD105 after Culture in Serum-Free Medium.

Human Mesenchymal Stem Cells (hMSCs) present a promising tool for regenerative medicine. However, ex vivo expansion is necessary to obtain sufficient cells for clinical therapy. Conventional growth media usually contain the critical component fetal bovine serum. For clinical use, chemically defined media will be required. In this study, the capability of two commercial, chemically defined, serum-free hMSC growth media (MSCGM-CD and PowerStem) for hMSC proliferation was examined and compared to serum-containing medium (MSCGM). Immunophenotyping of hMSCs was performed using flow cytometry, and they were tested for their ability to differentiate into a variety of cell types. Although the morphology of hMSCs cultured in the different media differed, immunophenotyping displayed similar marker patterns (high expression of CD29, CD44, CD73, and CD90 cell surface markers and absence of CD45). Interestingly, the expression of CD105 was significantly lower for hMSCs cultured in MSCGM-CD compared to MSCGM. Both groups maintained mesenchymal multilineage differentiation potential. In conclusion, the serum-free growth medium is suitable for hMSC culture and comparable to its serum-containing counterpart. As the expression of CD105 has been shown to positively influence hMSC cardiac regenerative potential, the impact of CD105 expression onto clinical use after expansion in MSCGM-CD will have to be tested.