A Call to Standardize Teratoma Assays Used to Define Human Pluripotent Cell Lines

SummaryThe teratoma assay is the gold standard for documenting pluripotency of human stem cells. However, reports of new human ESC and iPSC lines vary widely in both methods and analysis of teratoma data. We call for consensus standards to be established to make this assay worthy of its “golden” status

In Vitro Recapitulation of Developmental Transitions in Human Neural Stem Cells.

During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Recently, stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells and the fetal human hindbrain. Here, we explore whether these cell populations, similar to their in vivo counterparts, can give rise to neural stem (NS) cells with RG-like properties and whether region-specific NS cells can be generated from NES cells with different regional identities. In vivo RG cells are thought to form from NES cells with the onset of neurogenesis. Therefore, we cultured NES cells temporarily in differentiating conditions. Upon reinitiation of growth factor treatment, cells were found to enter a developmental stage reflecting major characteristics of RG-like NS cells. These NES cell-derived NS cells exhibited a very similar morphology and marker expression as primary NS cells generated from human fetal tissue, indicating that conversion of NES cells into NS cells recapitulates the developmental progression of early NES cells into RG cells observed in vivo. Importantly, NS cells generated from NES cells with different regional identities exhibited stable region-specific transcription factor expression and generated neurons appropriate for their positional identity. Stem Cells 2019;37:1429-1440

Applications of CRISPR/Cas9 for Selective Sequencing and Clinical Diagnostics

In this chapter, we will discuss the applications of CRISPR/Cas9 in the context of clinical diagnostics. We will provide an overview of existing methods and their use cases in the diagnostic field. Special attention will be given to selective sequencing approaches using third-generation sequencing and PAM-site requirements. As target sequences in an AT-rich environment cannot easily be accessed by the commercially available SpCas9 due to rarity of NGG PAM-sites, new enzymes such as ScCas9 with PAM-site requirements of NNG will be highlighted. Original research on CRISPR/Cas9 systems to determine molecular glioma markers by enriching regions of interest will be discussed in the context of potential future applications in clinical diagnostics

Creep in reactive colloidal gels: A nanomechanical study of cement hydrates

From soft polymeric gels to hardened cement paste, amorphous solids under constant load exhibit a pronounced time-dependent deformation called creep. The microscopic mechanism of such a phenomenon is poorly understood in amorphous materials and constitutes an even greater challenge in densely packed and chemically reactive granular systems. Both features are prominently present in hydrating cement pastes composed of calcium silicate hydrate (C-S-H) nanoparticles, whose packing density increases as a function of time, while cement hydration is taking place. Performing nanoindentation tests and porosity measurements on a large collection of samples at various stages of hydration, we show that the creep response of hydrating cement paste is mainly controlled by the interparticle distance and results from slippage between (C-S-H) nanoparticles. Our findings provide a unique insight into the microscopic mechanism underpinning the creep response in aging granular materials, thus paving the way for the design of concrete with improved creep resistance

Study protocol of the COMPARE-Interaction study: the impact of maternal comorbid depression and anxiety disorders in the peripartum period on child development

Introduction: To date, there are only few studies that compare the consequences of peripartum maternal depressive disorders (PD) versus depressive with comorbid anxiety disorders (PDCA) for infant and child development. As comorbidity is associated with greater impairment and symptom severity related to the primary diagnosis, comorbidity in mothers might raise their offspring’s risk of developing internalising or externalising disorders even more than has been noted in conjunction with PD alone. Methods and analysis: This study aims to analyse the impact of parental psychopathology, particularly peripartum depression in mothers with and without comorbid anxiety disorders according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) on child cognitive and socioemotional development. Maternal/paternal psychopathology, mother–infant/father–infant interaction and child development are assessed at four measurement points over the first 2 years (T1: 3–4 months postpartum, T2: 12 months postpartum, T3: 18 months postpartum and T4: 24 months postpartum). The mediating role of mother–infant/father–infant interaction and infant stress reactivity in the relationship between PD/PDCA and infant cognitive and socioemotional development will be analysed. In the ongoing study, 174 families (n=58 mothers with PD, n=58 mothers with PDCA and n=58 healthy controls) will be recruited in inpatient and outpatient centres as well as maternity hospitals in Munich and Heidelberg. Ethics and dissemination: This study is implemented in accordance with the current guidelines of the World Medical Association (revised Declaration of Helsinki) and the General Data Protection Regulation of the European Union. The study procedures were approved by the independent ethics committees of the Department of Psychology, Ludwig-Maximilians-University Munich (74_Reck_b) and of the Medical Faculty, University Heidelberg (S-446/2017). Participation is voluntary. A signed written informed consent form must be obtained from each study subject prior to any study-specific procedure. Participants can withdraw from the study at any point in time without giving a reason or being subjected to any future disadvantages. In case of withdrawal from the study, the subject’s data and material will be kept unless the participant asks for data removal. Results will be published and disseminated to further the discussion on the effects of maternal PD and PDCA on parent–infant interaction, infant stress reactivity and child development. Furthermore, study results will be presented at international congresses and expert conferences

Machine Learning Using a Single-Lead ECG to Identify Patients With Atrial Fibrillation-Induced Heart Failure

AIMS: Atrial fibrillation (AF) and heart failure often co-exist. Early identification of AF patients at risk for AF-induced heart failure (AF-HF) is desirable to reduce both morbidity and mortality as well as health care costs. We aimed to leverage the characteristics of beat-to-beat-patterns in AF to prospectively discriminate AF patients with and without AF-HF. METHODS: A dataset of 10,234 5-min length RR-interval time series derived from 26 AF-HF patients and 26 control patients was extracted from single-lead Holter-ECGs. A total of 14 features were extracted, and the most informative features were selected. Then, a decision tree classifier with 5-fold cross-validation was trained, validated, and tested on the dataset randomly split. The derived algorithm was then tested on 2,261 5-min segments from six AF-HF and six control patients and validated for various time segments. RESULTS: The algorithm based on the spectral entropy of the RR-intervals, the mean value of the relative RR-interval, and the root mean square of successive differences of the relative RR-interval yielded an accuracy of 73.5%, specificity of 91.4%, sensitivity of 64.7%, and PPV of 87.0% to correctly stratify segments to AF-HF. Considering the majority vote of the segments of each patient, 10/12 patients (83.33%) were correctly classified. CONCLUSION: Beat-to-beat-analysis using a machine learning classifier identifies patients with AF-induced heart failure with clinically relevant diagnostic properties. Application of this algorithm in routine care may improve early identification of patients at risk for AF-induced cardiomyopathy and improve the yield of targeted clinical follow-up

Sensory-neuropathy-causing mutations in ATL3 cause aberrant ER membrane tethering

The endoplasmic reticulum (ER) is a complex network of sheets and tubules that is continuously remodeled. The relevance of this membrane dynamics is underscored by the fact that mutations in atlastins (ATLs), the ER fusion proteins in mammals, cause neurodegeneration. How defects in this process disrupt neuronal homeostasis is unclear. Using electron microscopy (EM) volume reconstruction of transfected cells, neurons, and patient fibroblasts, we show that hereditary sensory and autonomic neuropathy (HSAN)-causing ATL3 mutants promote aberrant ER tethering hallmarked by bundles of laterally attached ER tubules. In vitro, these mutants cause excessive liposome tethering, recapitulating the results in cells. Moreover, ATL3 variants retain their dimerization-dependent GTPase activity but are unable to promote membrane fusion, suggesting a defect in an intermediate step of the ATL3 functional cycle. Our data show that the effects of ATL3 mutations on ER network organization go beyond a loss of fusion and shed light on neuropathies caused by atlastin defects

Neural stem cells genetically-modified to express neprilysin reduce pathology in Alzheimer transgenic models

INTRODUCTION: Short-term neural stem cell (NSC) transplantation improves cognition in Alzheimer’s disease (AD) transgenic mice by enhancing endogenous synaptic connectivity. However, this approach has no effect on the underlying beta-amyloid (Aβ) and neurofibrillary tangle pathology. Long term efficacy of cell based approaches may therefore require combinatorial approaches. METHODS: To begin to examine this question we genetically-modified NSCs to stably express and secrete the Aβ-degrading enzyme, neprilysin (sNEP). Next, we studied the effects of sNEP expression in vitro by quantifying Aβ-degrading activity, NSC multipotency markers, and Aβ-induced toxicity. To determine whether sNEP-expressing NSCs can also modulate AD-pathogenesis in vivo, control-modified and sNEP-NSCs were transplanted unilaterally into the hippocampus of two independent and well characterized transgenic models of AD: 3xTg-AD and Thy1-APP mice. After three months, stem cell engraftment, neprilysin expression, and AD pathology were examined. RESULTS: Our findings reveal that stem cell-mediated delivery of NEP provides marked and significant reductions in Aβ pathology and increases synaptic density in both 3xTg-AD and Thy1-APP transgenic mice. Remarkably, Aβ plaque loads are reduced not only in the hippocampus and subiculum adjacent to engrafted NSCs, but also within the amygdala and medial septum, areas that receive afferent projections from the engrafted region. CONCLUSIONS: Taken together, our data suggest that genetically-modified NSCs could provide a powerful combinatorial approach to not only enhance synaptic plasticity but to also target and modify underlying Alzheimer’s disease pathology

Efficacy of repeated intrathecal triamcinolone acetonide application in progressive multiple sclerosis patients with spinal symptoms

BACKGROUND: There are controversial results on the efficacy of the abandoned, intrathecal predominant methylprednisolone application in multiple sclerosis (MS) in contrast to the proven effectiveness in intractable postherpetic neuralgia. METHODS: We performed an analysis of the efficacy of the application of 40 mg of the sustained release steroid triamcinolone acetonide (TCA). We intrathecally injected in sterile saline dissolved TCA six times within three weeks on a regular basis every third day in 161 hospitalized primary and predominant secondary progressive MS patients with spinal symptoms. The MS patients did not experience an acute onset of exacerbation or recent distinct increased progression of symptoms. We simultaneously scored the MS patients with the EDSS and the Barthel index, estimated the walking distance and measured somatosensory evoked potentials. Additionally the MS patients received a standardized rehabilitation treatment. RESULTS: EDSS score and Barthel index improved, walking distance increased, latencies of somatosensory evoked potentials of the median and tibial nerves shortened in all MS patients with serial evaluation (p < 0.0001 for all variables). Side effects were rare, five patients stopped TCA application due to onset of a post lumbar puncture syndrome. CONCLUSIONS: Repeated intrathecal TCA application improves spinal symptoms, walking distance and SSEP latencies in progressive MS patients in this uncontrolled study. Future trials should evaluate the long-term benefit of this invasive treatment