Electrophysiological Properties of Tetraploid Cardiomyocytes Derived from Murine Pluripotent Stem Cells Generated by Fusion of Adult Somatic Cells with Embryonic Stem Cells.

Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.

G3BP1-dependent mechanism suppressing protein aggregation in Huntington's models and its demise upon stress granule assembly.

Stress granules are membrane-less ribonucleoprotein organelles that assemble upon exposure to stress conditions, but rapidly disassemble upon removal of stress. However, chronic stress can lead to persistent stress granules, a feature of distinct age-related neurodegenerative disorders. Among them, Huntington's disease (HD), which is caused by mutant expansion of the polyglutamine (polyQ) repeats of huntingtin protein (HTT), leading to its aggregation. To identify modulators of mutant HTT aggregation, we define its interactome in striatal neurons differentiated from patient-derived induced pluripotent stem cells (HD-iPSCs). We find that HTT interacts with G3BP1, a characteristic component of stress granules. Knockdown of G3BP1 increases mutant HTT protein levels and abolishes the ability of iPSCs as well as their differentiated neural counterparts to suppress mutant HTT aggregation. Moreover, loss of G3BP1 hastens polyQ-expanded aggregation and toxicity in the neurons of HD C. elegans models. Likewise, the assembly of G3BP1 into stress granules upon distinct stress conditions also reduces its interaction with HTT in human cells, promoting mutant HTT aggregation. Notably, enhancing the levels of G3BP1 is sufficient to induce proteasomal degradation of mutant HTT and prevent its aggregation, whereas the formation of stress granules blocks these ameliorative effects. In contrast, a mutant G3BP1 variant that cannot accumulate into granules retains its capacity to prevent mutant HTT aggregation even when the cells assemble stress granules. Thus, our findings indicate a direct role of G3BP1 and stress granule assembly in mutant HTT aggregation that may have implications for HD.

The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells.

Histones modulate gene expression by chromatin compaction, regulating numerous processes such as differentiation. However, the mechanisms underlying histone degradation remain elusive. Human embryonic stem cells (hESCs) have a unique chromatin architecture characterized by low levels of trimethylated histone H3 at lysine 9 (H3K9me3), a heterochromatin-associated modification. Here we assess the link between the intrinsic epigenetic landscape and ubiquitin-proteasome system of hESCs. We find that hESCs exhibit high expression of the ubiquitin-conjugating enzyme UBE2K. Loss of UBE2K upregulates the trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation. Besides H3K9 trimethylation, UBE2K binds histone H3 to induce its polyubiquitination and degradation by the proteasome. Notably, ubc-20, the worm orthologue of UBE2K, also regulates histone H3 levels and H3K9 trimethylation in Caenorhabditis elegans germ cells. Thus, our results indicate that UBE2K crosses evolutionary boundaries to promote histone H3 degradation and reduce H3K9me3 repressive marks in immortal cells.

DNAJB6, a Key Factor in Neuronal Sensitivity to Amyloidogenesis.

CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington's disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed in vivo, explaining why stem cells are intrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.

Author Correction: The ubiquitin ligase UBR5 suppresses proteostasis collapse in pluripotent stem cells from Huntington's disease patients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanism suppressing H3K9 trimethylation in pluripotent stem cells and its demise by polyQ-expanded huntingtin mutations.

Pluripotent stem cells are invaluable resources to study development and disease, holding a great promise for regenerative medicine. Here we use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) from patients with Huntington's disease (HD-iPSCs) to shed light into the normal function of huntingtin (HTT) and its demise in disease. We find that HTT binds ATF7IP, a regulator of the histone H3 methyltransferase SETDB1. HTT inhibits the interaction of the ATF7IP-SETDB1 complex with other heterochromatin regulators and transcriptional repressors, maintaining low levels of H3K9 trimethylation (H3K9me3) in hESCs. Loss of HTT promotes global increased H3K9me3 levels and enrichment of H3K9me3 marks at distinct genes, including transcriptional regulators of neuronal differentiation. Although these genes are normally expressed at low amounts in hESCs, HTT knockdown (KD) reduces their induction during neural differentiation. Notably, mutant expanded polyglutamine repeats in HTT diminish its interaction with ATF7IP-SETDB1 complex and trigger H3K9me3 in HD-iPSCs. Conversely, KD of ATF7IP in HD-iPSCs reduces H3K9me3 alterations and ameliorates gene expression changes in their neural counterparts. Taken together, our results indicate ATF7IP as a potential target to correct aberrant H3K9me3 levels induced by mutant HTT.

The ubiquitin ligase UBR5 suppresses proteostasis collapse in pluripotent stem cells from Huntington's disease patients.

Induced pluripotent stem cells (iPSCs) undergo unlimited self-renewal while maintaining their potential to differentiate into post-mitotic cells with an intact proteome. As such, iPSCs suppress the aggregation of polyQ-expanded huntingtin (HTT), the mutant protein underlying Huntington's disease (HD). Here we show that proteasome activity determines HTT levels, preventing polyQ-expanded aggregation in iPSCs from HD patients (HD-iPSCs). iPSCs exhibit high levels of UBR5, a ubiquitin ligase required for proteasomal degradation of both normal and mutant HTT. Conversely, loss of UBR5 increases HTT levels and triggers polyQ-expanded aggregation in HD-iPSCs. Moreover, UBR5 knockdown hastens polyQ-expanded aggregation and neurotoxicity in invertebrate models. Notably, UBR5 overexpression induces polyubiquitination and degradation of mutant HTT, reducing polyQ-expanded aggregates in HD-cell models. Besides HTT levels, intrinsic enhanced UBR5 expression determines global proteostasis of iPSCs preventing the aggregation of misfolded proteins ensued from normal metabolism. Thus, our findings indicate UBR5 as a modulator of super-vigilant proteostasis of iPSCs.

Proteostasis of Huntingtin in Health and Disease.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by motor dysfunction, cognitive deficits and psychosis. HD is caused by mutations in the Huntingtin (HTT) gene, resulting in the expansion of polyglutamine (polyQ) repeats in the HTT protein. Mutant HTT is prone to aggregation, and the accumulation of polyQ-expanded fibrils as well as intermediate oligomers formed during the aggregation process contribute to neurodegeneration. Distinct protein homeostasis (proteostasis) nodes such as chaperone-mediated folding and proteolytic systems regulate the aggregation and degradation of HTT. Moreover, polyQ-expanded HTT fibrils and oligomers can lead to a global collapse in neuronal proteostasis, a process that contributes to neurodegeneration. The ability to maintain proteostasis of HTT declines during the aging process. Conversely, mechanisms that preserve proteostasis delay the onset of HD. Here we will review the link between proteostasis, aging and HD-related changes.

Somatic increase of CCT8 mimics proteostasis of human pluripotent stem cells and extends C. elegans lifespan.

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and, therefore, are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex, a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover, increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality.

Murine transgenic iPS cell line for monitoring and selection of cardiomyocytes.

We report here a transgenic murine induced pluripotent stem cell (iPSC) line expressing puromycin N-acetyltransferase (PAC) and enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain promoter. This transgenic cell line reproducibly differentiates into EGFP-expressing cardiomyocytes (CMs) which can be generated at high purity with puromycin treatment and exhibit molecular and functional properties of immature heart muscle cells. This genetically modified iPSC line can be used for assessment of the utility of CMs for myocardial repair, pharmacological and toxicological applications and development of improved cardiac differentiation protocols.

Generation of human induced pluripotent stem cell line from a patient with a long QT syndrome type 2.

We report here the generation of human iPS cell line UKKi009-A from dermal fibroblasts of a patient carrying heterozygous mutation c.3035-3045delTCCCTCGATGC, p.Leu1012Pro (fs*55) in KCNH2 gene leading to long QT syndrome type 2 (LQT2). We used the Sleeping Beauty transposon-based plasmids expressing OSKM along with microRNAs 307/367 to reprogram the fibroblasts. The iPS cells possess pluripotent stem cell characteristics and differentiate to cell lineages of all three germ layers. This cell line can serve as a source for in vitro modeling of LQT2. This cell line is distributed by the European Collection of Authenticated Cell Cultures (ECACC).

Chromosome tracking in fused cells by single nucleotide polymorphisms.

Single nucleotide polymorphisms (SNP) refer to single-base differences in DNA sequence between individuals of the same species. In experimental setting, inbred mouse strains can easily be distinguished by their typical SNPs. Therefore, if cell fusion partners are selected to originate from two different genotypes the detection of strain specific SNPs in the genome of fused cells can be utilized as a complimentary method to traditional karyotyping and cell ploidy analyses to monitor the success of the cell fusion procedure and identification of chromosomes from both genotypes in established fusion cell lines. In this chapter, we describe the method for selection and detection of SNPs on each of the 23 pairs of murine chromosome in cell hybrids generated by fusion of murine somatic cells originating from DBA/2J female mice and murine embryonic stem (ES) cells originating from 129/Ola male mice. While parental fusing partners show the presence of only a single strain specific allele the tetraploid fusion hybrid cells harbor alleles originating from both fusing partners indicating that the fusion clones retained both parental nuclei and at least one of each pair of parental autosomes, which were not lost in the course of cell expansion.

Ascorbic Acid-Induced Cardiac Differentiation of Murine Pluripotent Stem Cells: Transcriptional Profiling and Effect of a Small Molecule Synergist of Wnt/ß-Catenin Signaling Pathway.

BACKGROUND: Reproducible and efficient differentiation of pluripotent stem cells (PSCs) to cardiomyocytes (CMs) is essential for their use in regenerative medicine, drug testing and disease modeling. The aim of this study was to evaluate the effect of some previously reported cardiogenic substances on cardiac differentiation of mouse PSCs. METHODS: Differentiation was performed by embryoid body (EB)-based method using three different murine PSC lines. The differentiation efficiency was monitored by RT-qPCR, immunocytochemistry and flow cytometry, and the effect mechanistically evaluated by transcriptome analysis of treated EBs. RESULTS: Among the five tested compounds (ascorbic acid, dorsomorphin, cyclic adenosine 3',5'-monophosphate, cardiogenol C, cyclosporin A) only ascorbic acid (AA) exerted a strong and reproducible cardiogenic effect in CGR8 cells which was less consistent in other two PSC lines. AA induced only minor changes in transcriptome of CGR8 cells after administration during the initial two days of differentiation. Cardiospecific genes and transcripts involved in angiogenesis, erythropoiesis and hematopoiesis were up-regulated on day 5 but not on days 2 or 3 of differentiation. The cardiac differentiation efficiency was improved when QS11, a small-molecule synergist of Wnt/ß-catenin signaling pathway, was added to cultures after AA-treatment. CONCLUSION: This study demonstrates that only minor transcriptional changes are sufficient for enhancement of cardiogenesis of murine PSCs by AA and that AA and QS11 exhibit synergistic effects and enhance the efficiency of CM differentiation of murine PSCs.

Monitoring and reporting transfusion reactions as a quality indicator - a clinical audit.

BACKGROUND AND OBJECTIVE: This audit was conducted as a part of a quality assurance activity to assess the frequency of receiving completely filled out blood transfusion reaction forms which were accompanied by the required samples. Once this information is known, we will elevate the bar each year to achieve 100% compliance. The sub-aim was to evaluate the frequency of the reported transfusion reactions. MATERIALS AND METHODS: The study was conducted from 1st April 2010 to 30th April 2011. The information was evaluated and the frequency of receiving completely filled blood transfusion reaction forms was assessed. The variables identified were the type of transfusion reaction, the blood component transfused, the health care personnel filling the form, and whether there was legible handwriting and a completely filled form. Transfusion reactions were reported as a percentage of the total number of units transfused. RESULTS: During the study period, 17,880 packed red cells, 13,200 platelets, 13,620 fresh frozen plasma and 2256 cryoprecipitate were transfused and 106 transfusion reactions (0.23%) were reported. Of these, febrile non hemolytic transfusion reaction was the most common (47%), the majority caused by packed red cells. CONCLUSION: Eighty-four percent of the transfusion reaction forms were completely filled as per our criteria. Febrile non hemolytic transfusion reactions were the most common reactions reported.

Mesenchymal stem cells and their conditioned medium improve integration of purified induced pluripotent stem cell-derived cardiomyocyte clusters into myocardial tissue.

Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) might become therapeutically relevant to regenerate myocardial damage. Purified iPS-CMs exhibit poor functional integration into myocardial tissue. The aim of this study was to investigate whether murine mesenchymal stem cells (MSCs) or their conditioned medium (MScond) improves the integration of murine iPS-CMs into myocardial tissue. Vital or nonvital embryonic murine ventricular tissue slices were cocultured with purified clusters of iPS-CMs in combination with murine embryonic fibroblasts (MEFs), MSCs, or MScond. Morphological integration was assessed by visual scoring and functional integration by isometric force and field potential measurements. We observed a moderate morphological integration of iPS-CM clusters into vital, but a poor integration into nonvital, slices. MEFs and MSCs but not MScond improved morphological integration of CMs into nonvital slices and enabled purified iPS-CMs to confer force. Coculture of vital slices with iPS-CMs and MEFs or MSCs resulted in an improved electrical integration. A comparable improvement of electrical coupling was achieved with the cell-free MScond, indicating that soluble factors secreted by MSCs were involved in electrical coupling. We conclude that cells such as MSCs support the engraftment and adhesion of CMs, and confer force to noncontractile tissue. Furthermore, soluble factors secreted by MSCs mediate electrical coupling of purified iPS-CM clusters to myocardial tissue. These data suggest that MSCs may increase the functional engraftment and therapeutic efficacy of transplanted iPS-CMs into infarcted myocardium.

The disease-specific phenotype in cardiomyocytes derived from induced pluripotent stem cells of two long QT syndrome type 3 patients.

Long QT syndromes (LQTS) are heritable diseases characterized by prolongation of the QT interval on an electrocardiogram, which often leads to syncope and sudden cardiac death. Here we report the generation of induced pluripotent stems (iPS) cells from two patients with LQTS type 3 carrying a different point mutation in a sodium channel Nav1.5 (p.V240M and p.R535Q) and functional characterization of cardiomyocytes (CM) derived from them. The iPS cells exhibited all characteristic properties of pluripotent stem cells, maintained the disease-specific mutation and readily differentiated to CM. The duration of action potentials at 50% and 90% repolarization was longer in LQTS-3 CM as compared to control CM but this difference did not reach statistical significance due to high variations among cells. Sodium current recordings demonstrated longer time to peak and longer time to 90% of inactivation of the Na(+) channel in the LQTS-3 CM. This hints at a defective Na(+) channel caused by deficiency in open-state inactivation of the Na(+) channel that is characteristic of LQTS-3. These analyses suggest that the effect of channel mutation in the diseased CM is demonstrated in vitro and that the iPS cell-derived CM can serve as a model system for studying the pathophysiology of LQTS-3, toxicity testing and design of novel therapeutics. However, further improvements in the model are still required to reduce cell-to-cell and cell line-to-cell line variability.

The novel desmin mutant p.A120D impairs filament formation, prevents intercalated disk localization, and causes sudden cardiac death.

BACKGROUND: The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. METHODS AND RESULTS: We identified the 2 novel variants DES-p.A120D (c.359C>A) and DES-p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in cell lines and of the isolated recombinant protein. Model variants of codon 120 indicated that ionic interactions contribute to this filament formation defect. Ex vivo analysis of ventricular tissue slices revealed a loss of desmin staining within the intercalated disk and severe cytoplasmic aggregate formation, whereas z-band localization was not affected. The functional experiments of desmin-p.H326R did not demonstrate any differences from wild type. CONCLUSIONS: Because of the functional in vivo and in vitro characterization, DES-p.A120D has to be regarded as a pathogenic mutation and DES-p.H326R as a rare variant with unknown significance. Presumably, the loss of the desmin-p. A120D filament localization at the intercalated disk explains its clinical arrhythmogenic potential.

Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells.

AIMS: Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as promising cell type for cardiac cell replacement therapy. We investigated long-term electrophysiological integration and maturation of transplanted iPSCM, which are essential for therapeutic benefit. METHODS AND RESULTS: Murine iPSCM expressing enhanced green fluorescent protein and a puromycin resistance under control of the α-myosin heavy chain promoter were purified by antibiotic selection and injected into adult mouse hearts. After 6-12 days, 3-6 weeks, or 6-8 months, viable slices of recipient hearts were prepared. Slices were focally stimulated by a unipolar electrode placed in host tissue, and intracellular action potentials (APs) were recorded with glass microelectrodes in transplanted cells and neighbouring host tissue within the slices. Persistence and electrical integration of transplanted iPSCM into recipient hearts could be demonstrated at all time points. Quality of coupling improved, as indicated by a maximal stimulation frequency without conduction blocks of 5.77 ± 0.54 Hz at 6-12 days, 8.98 ± 0.38 Hz at 3-6 weeks and 10.82 ± 1.07 Hz at 6-8 months after transplantation. AP properties of iPSCM became more mature from 6-12 days to 6-8 months after transplantation, but still differed significantly from those of host APs. CONCLUSION: Transplanted iPSCM can persist in the long term and integrate electrically into host tissue, supporting their potential for cell replacement therapy. Quality of electrical integration improves between 6-12 days and 6-8 months after transplantation, and there are signs of an electrophysiological maturation. However, even after 6-8 months, AP properties of transplanted iPSCM differ from those of recipient cardiomyocytes.

Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants.

Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.