Reconstructing human brown fat developmental trajectory in vitro.

Brown adipocytes (BAs) represent a specialized cell type that is able to uncouple nutrient catabolism from ATP generation to dissipate energy as heat. In humans, the brown fat tissue is composed of discrete depots found throughout the neck and trunk region. BAs originate from a precursor common to skeletal muscle, but their developmental trajectory remains poorly understood. Here, we used single-cell RNA sequencing to characterize the development of interscapular brown fat in mice. Our analysis identified a transient stage of BA differentiation characterized by the expression of the transcription factor GATA6. We show that recapitulating the sequence of signaling cues identified in mice can lead to efficient differentiation of BAs in vitro from human pluripotent stem cells. These precursors can in turn be efficiently converted into functional BAs that can respond to signals mimicking adrenergic stimuli by increasing their metabolism, resulting in heat production.

Reconstruction and deconstruction of human somitogenesis in vitro.

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1-4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.

Social Justice Activism as Interpretation in a Loewaldian World.

At a time when many questions are arising about the nexus between psychoanalysis and social justice, the writings of Hans Loewald open an avenue for broadened conceptualizations of psychoanalytic activity and the role of interpretation within it. The pursuit of social justice, it is argued, is integral to psychoanalytic ethics, and the relation between activists and society can be formulated in Loewaldian terms. Using Loewald, and considering case examples from social justice informed advocacy, direct action, and protest speech in AIDS activism, social justice activism can be understood as a spontaneously emergent psychoanalytic interpretation delivered by activists to their social surround, effectively accomplishing multiple forms of therapeutic action. The therapeutic action includes a working through in two phases of the negative social transference, a concept proposed here to elaborate a mechanism for the transformation, through the interpretive aspects of activism, of psychic material directed toward marginalized subjects and those expressing marginalized subjective positions. Resistance to social justice activism is examined using the forms of resistance identified by Freud.

Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers.

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell-derived skeletal muscle in vitro.

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

Observations on Use of the N-Word in Psychoanalytic Conferences.

In a frequently repeated group phenomenon, a racial slur is spoken in psychoanalytic conferences, after which a range of defensive responses emerge to counter acknowledgment of the meanings of having done so. After a discussion of the literature relevant to the use of slurs in psychoanalytic professional settings, Freud's concept of Nachträglichkeit, or deferred action, is used to identify and explore these events as a series of discriminatory gestures that evoke racial trauma. The defensive responses that emerge to protect the use of these gestures indicate ties to the traumatic legacy of slavery and to white supremacy as it appears in contemporary psychoanalytic culture. "Gestures of the open hand" are proposed, and their profound reparative potential is discussed. The intimate link between epistemic justice and psychoanalytic endeavors is delineated.

Efficacy of a novel extract of fenugreek seeds in alleviating vasomotor symptoms and depression in perimenopausal women: A randomized, double-blinded, placebo-controlled study.

The present randomized, double-blinded, placebo-controlled study investigated the effect of a standardized fenugreek extract (FHE) on perimenopausal discomforts and its influence on hormonal balance and safety. Healthy women characterized with perimenopausal symptoms (n = 48), as assessed by MRS questionnaire, were randomized either to FHE (n = 24) or placebo (n = 24) and supplemented with 250 mg × 2/day for 42 days. Both inter and intra-group comparison revealed a significant improvement in somatic, psychological, and urogenital scores in FHE group, especially for hot flashes (25.9%), night sweats (26.5%), depression (31.8%), and insomnia (21.6%). Further hormone analysis revealed an enhancement in serum estradiol (18.9%), free testosterone (38.2%), and progesterone (19.9%) concentrations and a significant decrease in FSH (38.2%) and SHBG (21.1%) concentrations toward establishing a hormonal balance among FHE-group; without significant changes in other clinical safety parameters. Thus, FHE supplementation offered a significant reduction in vasomotor effects and depression in perimenopausal women, without any adverse effects PRACTICAL APPLICATIONS: Fenugreek is a popular kitchen spice and Ayurvedic medicine for a variety of health conditions including diabetes, hypercholesterolemia, hepatotoxicity, gastritis, and also for a variety of hormone-related health conditions such as sexual functions, lactation, osteoporosis, PCOS, and post/perimenopausal discomforts. Fenugreek is rich in alkaloids, steroidal saponins, flavonoids and 4-hydroxyisoleucine. The present randomized-controlled study investigated the plausible application of a standardized hydro-ethanolic extract of fenugreek seeds (FHE) having a unique 3:1 ratio for protodioscin to trigonelline in the management of perimenopausal discomforts. It was observed that FHE at a dosage of 250 mg × 2/day for 42 days significantly reduced the discomforts, especially vasomotor symptoms and depression, and helped to attain a hormonal balance without any adverse effects or deviations in clinical safety parameters. Thus, FHE could be a potential natural agent for the management of post and perimenopausal discomforts and has to be explored in future studies.

Effectiveness of fluoride varnish, diode laser, and their combination in treatment of dentin hypersensitivity: A randomized split-mouth clinical trial.

BACKGROUND: Dentin hypersensitivity is a common dental problem with no permanent cure and predictable prognosis. AIM: The aim of this study was to evaluate the effectiveness of fluoride varnish (sodium fluoride [NaF]), diode laser, and the combination of NaF and diode laser in the treatment of dentin hypersensitivity. SETTINGS AND DESIGN: This was a randomized split-mouth clinical trial. MATERIALS AND METHODS: Sixty patients aged 20-60 years suffering from dentin hypersensitivity to air-blast, cold, and tactile stimulation corresponding to 4 cm and above on the Visual Analog Scale (VAS) in three quadrants with at least two hypersensitive teeth per quadrant were selected. Hypersensitive teeth were allotted to Group 1 - 5% NaF varnish application alone, Group 2 - 810-nm gallium-aluminum-arsenide laser (GaAlAs) diode laser (0.5 W) irradiation alone, and Group 3 - NaF varnish application, followed by diode laser irradiation. VAS score was recorded at baseline, 1 week, 2 weeks, 1 month, 3 months, and 6 months. RESULTS: A statistically significant reduction in dentin hypersensitivity was observed in all the three groups, from the baseline to the 1st-, 3rd-, and 6th-month follow-ups (P < 0.05). Group 2 and Group 3 demonstrated a significantly higher reduction (P < 0.05) in dentin hypersensitivity for all the stimuli as opposed to Group 1 at all follow-up intervals. However, no statistically significant difference (P > 0.05) was present between Group 2 and Group 3 at all follow-ups. CONCLUSION: Diode laser is significantly more effective than fluoride varnish alone in the treatment of dentin hypersensitivity over a period of 6 months.

Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.

Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.

Effect of Nonsurgical Treatment on Salivary HGF Levels in Population with Periodontal Disease: A Quasi-experimental Study.

AIM: To assess the effect of nonsurgical treatment on salivary hepatocyte growth factor (sHGF) levels in a population with periodontal disease: a quasi-experimental study. METHODS: Eighty-one patients (aged 30-70 years) were divided into three groups based on the gingival index, probing depth, clinical attachment loss, and radiographic evidence of bone loss: healthy (group I), gingivitis (group II), and chronic periodontitis (group III). Saliva samples were collected from these groups at baseline. At 8 weeks, saliva samples were collected again from group II and group III after the patients went through nonsurgical periodontal treatment. The levels of HGF were estimated using enzyme-linked immunosorbent assay (ELISA). The clinical parameters and HGF levels among all groups were analyzed using a one-way analysis of variance (ANOVA) using SPSS 17 version. RESULTS: At baseline, the highest mean HGF concentration in saliva was observed for group III (3455.83 ± 1463.44 pg/mL), and the least in group I (469.43 ± 317.13 pg/mL). Following nonsurgical periodontal treatment, the mean HGF concentration decreased significantly in group III and group II (p < 0.05). A significant positive correlation between clinical parameters and HGF levels was also seen (p < 0.05). CONCLUSION: HGF concentration showed a positive correlation with the progression of periodontal disease. CLINICAL SIGNIFICANCE: Following nonsurgical periodontal therapy, the levels of HGF decreased significantly, suggesting that HGF could be useful for monitoring the response to periodontal therapy. HOW TO CITE THIS ARTICLE: Alreja D, Rao JR, Kataria S, et al. Effect of Nonsurgical Treatment on Salivary HGF Levels in Population with Periodontal Disease: A Quasi-experimental Study. Euroasian J Hepato-Gastroenterol 2020;10(2):51-55.

SarcTrack.

RATIONALE: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues. OBJECTIVE: We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate. METHODS AND RESULTS: We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C ( MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment. CONCLUSIONS: SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.

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.

Wnt signaling positively regulates endothelial cell fate specification in the Fli1a-positive progenitor population via Lef1.

During vertebrate embryogenesis, vascular endothelial cells (ECs) and primitive erythrocytes become specified within close proximity in the posterior lateral plate mesoderm (LPM) from a common progenitor. However, the signaling cascades regulating the specification into either lineage remain largely elusive. Here, we analyze the contribution of ß-catenin dependent Wnt signaling to EC and erythrocyte specification during zebrafish embryogenesis. We generated novel ß-catenin dependent Wnt signaling reporters which, by using destabilized fluorophores (Venus-Pest, dGFP), specifically allow us to detect Wnt signaling responses in narrow time windows as well as in spatially restricted domains, defined by Cre recombinase expression (Tg(axin2BAC:Venus-Pest)mu288; Tg(14TCF:loxP-STOP-loxP-dGFP)mu202). We therefore can detect ß-catenin dependent Wnt signaling activity in a subset of the Fli1a-positive progenitor population. Additionally, we show that mesodermal Wnt3a-mediated signaling via the transcription factor Lef1 positively regulates EC specification (defined by kdrl expression) at the expense of primitive erythrocyte specification (defined by gata1 expression) in zebrafish embryos. Using mesoderm derived from human embryonic stem cells, we identified the same principle of Wnt signaling dependent EC specification in conjunction with auto-upregulation of LEF1. Our data indicate a novel role of ß-catenin dependent Wnt signaling in regulating EC specification during vasculogenesis.

Concise Review: Signaling Control of Early Fate Decisions Around the Human Pluripotent Stem Cell State.

Human embryonic stem cells (hESCs) present a fascinating and powerful system for generating specialized cell types of the human body. Culture and directed differentiation of these cells however requires an understanding of the pluripotent ground state and of how cell lineage decisions in this system are made. In this review, we highlight both these aspects in light of recent findings and technical progress. Hence, advances in culturing the human preimplantation embryo beyond the implantation barrier and in analyzing it at the single-cell level shed new light on the hESC tissue of origin. We argue that these findings have important implications for our view of hESC identity and we critically discuss recent efforts in converting these cells to a more primitive state. With an emphasis on the roles played by major signaling pathways, we furthermore attempt to infer key principles underlying cell fate control in hESCs from recently published work. This integrated model combines defined signaling pathway manipulation with the regulation of core hESC genes, to aid in controlling cell lineage allocation in a rational manner. Stem Cells 2017;35:277-283.

Stepwise Clearance of Repressive Roadblocks Drives Cardiac Induction in Human ESCs.

Cardiac induction requires stepwise integration of BMP and WNT pathway activity. Human embryonic stem cells (hESCs) are developmentally and clinically relevant for studying the poorly understood molecular mechanisms downstream of these cascades. We show that BMP and WNT signaling drive cardiac specification by removing sequential roadblocks that otherwise redirect hESC differentiation toward competing fates, rather than activating a cardiac program per se. First, BMP and WNT signals pattern mesendoderm through cooperative repression of SOX2, a potent mesoderm antagonist. BMP signaling promotes miRNA-877 maturation to induce SOX2 mRNA degradation, while WNT-driven EOMES induction transcriptionally represses SOX2. Following mesoderm formation, cardiac differentiation requires inhibition of WNT activity. We found that WNT inhibition serves to restrict expression of anti-cardiac regulators MSX1 and CDX2/1. Conversely, their simultaneous disruption partially abrogates the requirement for WNT inactivation. These results suggest that human cardiac induction depends on multi-stage repression of alternate lineages, with implications for deriving expandable cardiac stem cells.

Human pluripotent stem cell-derived cardiomyocytes: Genome-wide expression profiling of long-term in vitro maturation in comparison to human heart tissue.

Cardiomyocyte-like cells (CMs) derived from human pluripotent stem cells (hPSCs) present a valuable model for human disease modeling, studying early human development and, potentially, developing cell therapeutic approaches. However, the specification of early hPSC-derived CMs into defined cardiac subtypes such as atrial and ventricular cells is not well understood and, thus, poorly controlled. Moreover, the maturation status of hPSC-CMs is not well defined, yet it is known that these cells undergo at least some degree of maturation upon longer term in vitro culture. To gain insight into this process, and to assess their developmental status, we have recently generated a data set of hPSC-CMs monitoring global changes in gene expression upon long term maintenance in vitro, in comparison to human atrial and ventricular heart samples (GEO accession number GEO: GSE64189). These data present a rich resource for evaluating the maturation status of hPSC-CMs, for identifying suitable markers for subtype-specific gene expression, as well as for the generation of functional hypotheses. Here, we provide additional details and quality checks of this data set, and exemplify how it can be used to identify maturation-associated as well as cardiac subtype-specific markers.

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.

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.