Preliminary evidence that Merkel cells exert chemosensory functions in human epidermis.

The mechanotransduction of light-touch sensory stimuli is considered to be the main physiological function of epidermal Merkel cells (MCs). Recently, however, MCs have been demonstrated to be also thermo-sensitive, suggesting that their role in skin physiologically extends well beyond mechanosensation. Here, we demonstrate that in healthy human skin epidermal MCs express functional olfactory receptors, namely OR2AT4, just like neighbouring keratinocytes. Selective stimulation of OR2AT4 by topical application of the synthetic odorant, Sandalore®, significantly increased Piccolo protein expression in MCs, as assessed by quantitative immunohistomorphometry, indicating increased vesicle trafficking and recycling, and significantly reduced nerve growth factor (NGF) immunoreactivity within MCs, possibly indicating increased neurotrophin release upon OR2AT4 activation. Live-cell imaging showed that Sandalore® rapidly induces a loss of FFN206-dependent fluorescence in MCs, suggesting OR2AT4-dependent MC depolarization and subsequent vesicle secretion. Yet, in contrast to keratinocytes, OR2AT4 stimulation by Sandalore® altered neither the number nor the proliferation status of MCs. These preliminary ex vivo findings demonstrate that epidermal MCs also exert OR-dependent chemosensory functions in human skin, and invite one to explore whether these newly identified properties are dysregulated in selected skin disorders, for example, in pruritic dermatoses, and if these novel MC functions can be therapeutically targeted to maintain/promote skin health.

From iPSC towards cardiac tissue-a road under construction.

The possibility to generate induced pluripotent stem cells (iPSC) opens the way to generate virtually all cell types of our human body. In combination with modern gene editing techniques like CRISPR/CAS, a new set of powerful tools becomes available for life science. Scientific fields like genotype and cell type-specific pharmacology, disease modeling, stem cell biology, and developmental biology have been dramatically fostered and their faces have been changed. However, as golden as the age of iPSC-derived cells and their manipulation has started, the shine begins to tarnish. Researchers face more and more practical problems intrinsic to the system. These problems are related to the specific culturing conditions which are not yet sufficient to mimic the natural environment of native stem cells differentiating towards adult cells. However, researchers work hard to uncover these factors. Here, we review a common standard approach to generate iPSCs and transduce these to iPSC cardiomyocytes. Further, we review recent achievements and discuss their current limitations and future perspectives. We are on track, but the road is still under construction.

Human Lineage-Specific Transcriptional Regulation through GA-Binding Protein Transcription Factor Alpha (GABPa).

A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes.

Recessive cardiac phenotypes in induced pluripotent stem cell models of Jervell and Lange-Nielsen syndrome: disease mechanisms and pharmacological rescue.

Jervell and Lange-Nielsen syndrome (JLNS) is one of the most severe life-threatening cardiac arrhythmias. Patients display delayed cardiac repolarization, associated high risk of sudden death due to ventricular tachycardia, and congenital bilateral deafness. In contrast to the autosomal dominant forms of long QT syndrome, JLNS is a recessive trait, resulting from homozygous (or compound heterozygous) mutations in KCNQ1 or KCNE1. These genes encode the α and ß subunits, respectively, of the ion channel conducting the slow component of the delayed rectifier K(+) current, IKs. We used complementary approaches, reprogramming patient cells and genetic engineering, to generate human induced pluripotent stem cell (hiPSC) models of JLNS, covering splice site (c.478-2A>T) and missense (c.1781G>A) mutations, the two major classes of JLNS-causing defects in KCNQ1. Electrophysiological comparison of hiPSC-derived cardiomyocytes (CMs) from homozygous JLNS, heterozygous, and wild-type lines recapitulated the typical and severe features of JLNS, including pronounced action and field potential prolongation and severe reduction or absence of IKs. We show that this phenotype had distinct underlying molecular mechanisms in the two sets of cell lines: the previously unidentified c.478-2A>T mutation was amorphic and gave rise to a strictly recessive phenotype in JLNS-CMs, whereas the missense c.1781G>A lesion caused a gene dosage-dependent channel reduction at the cell membrane. Moreover, adrenergic stimulation caused action potential prolongation specifically in JLNS-CMs. Furthermore, sensitivity to proarrhythmic drugs was strongly enhanced in JLNS-CMs but could be pharmacologically corrected. Our data provide mechanistic insight into distinct classes of JLNS-causing mutations and demonstrate the potential of hiPSC-CMs in drug evaluation.

The Natural Plant Product Rottlerin Activates Kv7.1/KCNE1 Channels.

BACKGROUND/AIMS: Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. METHODS: Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. RESULTS: We show that currents carried by Kv7.1 (EC50 = 1.48 µM), Kv7.1/KCNE1 (EC50 = 4.9 µM), and Kv7.4 (EC50 = 0.148 µM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1. CONCLUSION: Rottlerin or an optimized derivative holds a potential as QT interval correcting drug.

Universal cardiac induction of human pluripotent stem cells in two and three-dimensional formats: implications for in vitro maturation.

Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large-scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high-efficiency procedure for generating CMs both in two-dimensional (2D) and three-dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost-efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin-free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time-course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture-induced maturation of the resulting CMs. This suggested that hPSC-CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC-CMs to functional readouts and thus define the cornerstones of culture-induced maturation.

Pathophysiological Mechanisms of Cardiac Dysfunction in Transgenic Mice with Viral Myocarditis.

Viral myocarditis is pathologically associated with RNA viruses such as coxsackievirus B3 (CVB3), or more recently, with SARS-CoV-2, but despite intensive research, clinically proven treatment is limited. Here, by use of a transgenic mouse strain (TG) containing a CVB3ΔVP0 genome we unravel virus-mediated cardiac pathophysiological processes in vivo and in vitro. Cardiac function, pathologic ECG alterations, calcium homeostasis, intracellular organization and gene expression were significantly altered in transgenic mice. A marked alteration of mitochondrial structure and gene expression indicates mitochondrial impairment potentially contributing to cardiac contractile dysfunction. An extended picture on viral myocarditis emerges that may help to develop new treatment strategies and to counter cardiac failure.

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

Intermediate Hair Follicles from Patients with Female Pattern Hair Loss Are Associated with Nutrient Insufficiency and a Quiescent Metabolic Phenotype.

Female pattern hair loss (FPHL) is a non-scarring alopecia resulting from the progressive conversion of the terminal (t) scalp hair follicles (HFs) into intermediate/miniaturized (i/m) HFs. Although data supporting nutrient deficiency in FPHL HFs are lacking, therapeutic strategies are often associated with nutritional supplementation. Here, we show by metabolic analysis that selected nutrients important for hair growth such as essential amino acids and vitamins are indeed decreased in affected iHFs compared to tHFs in FPHL scalp skin, confirming nutrient insufficiency. iHFs also displayed a more quiescent metabolic phenotype, as indicated by altered metabolite abundance in freshly collected HFs and release/consumption during organ culture of products/substrates of TCA cycle, aerobic glycolysis, and glutaminolysis. Yet, as assessed by exogenous nutrient supplementation ex vivo, nutrient uptake mechanisms are not impaired in affected FPHL iHFs. Moreover, blood vessel density is not diminished in iHFs versus tHFs, despite differences in tHFs from different FPHL scalp locations or versus healthy scalp or changes in the expression of angiogenesis-associated growth factors. Thus, our data reveal that affected iHFs in FPHL display a relative nutrient insufficiency and dormant metabolism, but are still capable of absorbing nutrients, supporting the potential of nutritional supplementation as an adjunct therapy for FPHL.

Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.

Experimentally Induced Epithelial-Mesenchymal Transition of Human Hair Follicle Stem Cells as a Model of Scarring Alopecia Ex Vivo.

Primary cicatricial alopecia is characterized by a permanent "scarring" alopecia. This condition is characterized by the irreversible loss of hair follicles (HF) as a result of apoptosis and epithelial-mesenchymal transition (EMT) of epithelial stem cells localized in the HF bulge.We here report the procedure for experimentally induced EMT in healthy human epidermal stem cells (eSCs) using full-length HF organ culture ex vivo. The present model can be used to recapitulate the complex processes observed in scarring alopecia patient tissues, to further investigate the mechanisms involved in EMT transformation of HFeSCs, and to test substances that could prevent and/or rescue HFeSCs from EMT for the management of scarring alopecias.

Human Wound Healing Ex Vivo Model with Focus on Molecular Markers.

Wound healing is a complex, multifactorial process that is divided in sequential and overlapping phases in order to restore the skin barrier. For the study of wound healing, different in vivo, in vitro, and ex vivo models have been used in the past. Here we describe in detail the methodology of the human skin punch-in-a-punch ex vivo wound healing model.

The first versatile human iPSC-based model of ectopic virus induction allows new insights in RNA-virus disease.

A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.

Cardiogenic programming of human pluripotent stem cells by dose-controlled activation of EOMES.

Master cell fate determinants are thought to induce specific cell lineages in gastrulation by orchestrating entire gene programs. The T-box transcription factor EOMES (eomesodermin) is crucially required for the development of the heart-yet it is equally important for endoderm specification suggesting that it may act in a context-dependent manner. Here, we define an unrecognized interplay between EOMES and the WNT signaling pathway in controlling cardiac induction by using loss and gain-of-function approaches in human embryonic stem cells. Dose-dependent EOMES induction alone can fully replace a cocktail of signaling molecules otherwise essential for the specification of cardiogenic mesoderm. Highly efficient cardiomyocyte programming by EOMES mechanistically involves autocrine activation of canonical WNT signaling via the WNT3 ligand, which necessitates a shutdown of this axis at a subsequent stage. Our findings provide insights into human germ layer induction and bear biotechnological potential for the robust production of cardiomyocytes from engineered stem cells.

Revised roles of ISL1 in a hES cell-based model of human heart chamber specification.

The transcription factor ISL1 is thought to be key for conveying the multipotent and proliferative properties of cardiac precursor cells. Here, we investigate its function upon cardiac induction of human embryonic stem cells. We find that ISL1 does not stabilize the transient cardiac precursor cell state but rather serves to accelerate cardiomyocyte differentiation. Conversely, ISL1 depletion delays cardiac differentiation and respecifies nascent cardiomyocytes from a ventricular to an atrial identity. Mechanistic analyses integrate this unrecognized anti-atrial function of ISL1 with known and newly identified atrial inducers. In this revised view, ISL1 is antagonized by retinoic acid signaling via a novel player, MEIS2. Conversely, ISL1 competes with the retinoic acid pathway for prospective cardiomyocyte fate, which converges on the atrial specifier NR2F1. This study reveals a core regulatory network putatively controlling human heart chamber formation and also bears implications for the subtype-specific production of human cardiomyocytes with enhanced functional properties.

Generation and cardiac subtype-specific differentiation of PITX2-deficient human iPS cell lines for exploring familial atrial fibrillation.

Loss-of-function mutations in the PITX2 transcription factor gene have been shown to cause familial atrial fibrillation (AF). To potentially model aspects of AF and unravel PITX2-regulated downstream genes for drug target discovery, we here report the generation of integration-free PITX2-deficient hiPS cell lines. We also show that both PITX2 knockout hiPS cells and isogenic wild-type controls can selectively be differentiated into human atrial cardiomyocytes, to potentially uncover differentially expressed gene sets between these groups.

Cardiac Subtype-Specific Modeling of Kv1.5 Ion Channel Deficiency Using Human Pluripotent Stem Cells.

The ultrarapid delayed rectifier K+ current (IKur), mediated by Kv1.5 channels, constitutes a key component of the atrial action potential. Functional mutations in the underlying KCNA5 gene have been shown to cause hereditary forms of atrial fibrillation (AF). Here, we combine targeted genetic engineering with cardiac subtype-specific differentiation of human induced pluripotent stem cells (hiPSCs) to explore the role of Kv1.5 in atrial hiPSC-cardiomyocytes. CRISPR/Cas9-mediated mutagenesis of integration-free hiPSCs was employed to generate a functional KCNA5 knockout. This model as well as isogenic wild-type control hiPSCs could selectively be differentiated into ventricular or atrial cardiomyocytes at high efficiency, based on the specific manipulation of retinoic acid signaling. Investigation of electrophysiological properties in Kv1.5-deficient cardiomyocytes compared to isogenic controls revealed a strictly atrial-specific disease phentoype, characterized by cardiac subtype-specific field and action potential prolongation and loss of 4-aminopyridine sensitivity. Atrial Kv1.5-deficient cardiomyocytes did not show signs of arrhythmia under adrenergic stress conditions or upon inhibiting additional types of K+ current. Exposure of bulk cultures to carbachol lowered beating frequencies and promoted chaotic spontaneous beating in a stochastic manner. Low-frequency, electrical stimulation in single cells caused atrial and mutant-specific early afterdepolarizations, linking the loss of KCNA5 function to a putative trigger mechanism in familial AF. These results clarify for the first time the role of Kv1.5 in atrial hiPSC-cardiomyocytes and demonstrate the feasibility of cardiac subtype-specific disease modeling using engineered hiPSCs.

Adrenergic Stress Protection of Human iPS Cell-Derived Cardiomyocytes by Fast Kv7.1 Recycling.

The fight-or-flight response (FFR), a physiological acute stress reaction, involves positive chronotropic and inotropic effects on heart muscle cells mediated through ß-adrenoceptor activation. Increased systolic calcium is required to enable stronger heart contractions whereas elevated potassium currents are to limit the duration of the action potentials and prevent arrhythmia. The latter effect is accomplished by an increased functional activity of the Kv7.1 channel encoded by KCNQ1. Current knowledge, however, does not sufficiently explain the full extent of rapid Kv7.1 activation and may hence be incomplete. Using inducible genetic KCNQ1 complementation in KCNQ1-deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we here reinvestigate the functional role of Kv7.1 in adapting human CMs to adrenergic stress. Under baseline conditions, Kv7.1 was barely detectable at the plasma membrane of hiPSC-CMs, yet it fully protected these from adrenergic stress-induced beat-to-beat variability of repolarization and torsade des pointes-like arrhythmia. Furthermore, isoprenaline treatment increased field potential durations specifically in KCNQ1-deficient CMs to cause these adverse macroscopic effects. Mechanistically, we find that the protective action by Kv7.1 resides in a rapid translocation of channel proteins from intracellular stores to the plasma membrane, induced by adrenergic signaling. Gene silencing experiments targeting RAB GTPases, mediators of intracellular vesicle trafficking, showed that fast Kv7.1 recycling under acute stress conditions is RAB4A-dependent.Our data reveal a key mechanism underlying the rapid adaptation of human cardiomyocytes to adrenergic stress. These findings moreover aid to the understanding of disease pathology in long QT syndrome and bear important implications for safety pharmacological screening.

Functional high-resolution time-course expression analysis of human embryonic stem cells undergoing cardiac induction.

Cardiac induction of human embryonic stem cells (hESCs) is a process bearing increasing medical relevance, yet it is poorly understood from a developmental biology perspective. Anticipated technological progress in deriving stably expandable cardiac precursor cells or in advancing cardiac subtype specification protocols will likely require deeper insights into this fascinating system. Recent improvements in controlling hESC differentiation now enable a near-homogeneous induction of the cardiac lineage. This is based on an optimized initial stimulation of mesoderm-inducing signaling pathways such as Activin and/or FGF, BMP, and WNT, followed by WNT inhibition as a secondary requirement. Here, we describe a comprehensive data set based on varying hESC differentiation conditions in a systematic manner and recording high-resolution differentiation time-courses analyzed by genome-wide expression profiling (GEO accession number GSE67154). As a baseline, hESCs were differentiated into cardiomyocytes under optimal conditions. Moreover, in additional time-series, individual signaling factors were withdrawn from the initial stimulation cocktail to reveal their specific roles via comparison to the standard condition. Hence, this data set presents a rich resource for hypothesis generation in studying human cardiac induction, as we reveal numbers of known as well as uncharacterized genes prominently marking distinct intermediate stages in the process. These data will also be useful for identifying putative cardiac master regulators in the human system as well as for characterizing expandable cardiac stem cells.