Multicomponent drug Neurexan mitigates acute stress-induced insomnia in rats.

The aim of this study was to determine whether the multicomponent drug Neurexan could mitigate acute insomnia after exposure to a psychosocial stressor. We administered Neurexan orally to rats and examined stress-induced insomnia using the male rat dirty cage exchange method. The neurocircuitry and electrophysiological correlates of the model are characterised, and it represents various human insomnia conditions. Male rats were randomly assigned in a crossover design to six treatment groups and electroencephalography (EEG) electrodes attached. Three groups were exposed to a cage inhabited by another male rat for a week and the other three groups received a clean cage. Prior to cage change, rats were given either no drug, vehicle control or Neurexan. Non-rapid eye movement (NREM) sleep, REM sleep, and waking were assessed manually via EEG recordings. Group means were compared for sleep latency and for the 2 h after cage change for: time in each state, state-specific episode duration/frequency, in addition to NREM delta, gamma and REM theta EEG spectral power. Rats administered Neurexan fell asleep faster than vehicle-treated rats and spent less time awake with shorter, albeit more waking episodes and increased NREM episodes after dirty cage exposure. Neurexan-treated rats given dirty cages were not statistically different on any outcomes from Neurexan-treated rats given clean cages, thereby mitigating the stressor. In the EEG power spectra analysed, changes between treatment groups were not detected. This research confirms that Neurexan treatment has somnogenic effects and ameliorates psychological stressor-induced acute insomnia.

The Natural Combination Medicine Traumeel (Tr14) Improves Resolution of Inflammation by Promoting the Biosynthesis of Specialized Pro-Resolving Mediators.

The resolution of inflammation is an integral part of the acute inflammatory response and eventually leads to the return to homeostasis. It is supported by specialized pro-resolving mediators (SPMs) that act as immunoresolvents via specific G-protein-coupled receptors. In contrast to classical non-steroidal anti-inflammatory drugs (NSAIDs) that suppress the formation of pro-inflammatory lipid mediators such as prostaglandins, novel pharmacotherapeutic concepts propose to foster the biosynthesis of beneficial SPMs. Here, we demonstrate that the natural combination medicine Traumeel (Tr14) improves resolution of inflammation by promoting SPM formation. Tr14 enhanced the biosynthesis of 12-/15-lipoxygenase (LOX) products and of SPMs in zymosan-induced mouse peritonitis as well as in human monocyte-derived macrophages challenged with Staphylococcus aureus. Importantly, in the peritonitis model, Tr14 supported the recruitment of innate leukocytes and the efferocytotic capacity of macrophages, and positively influenced the inflammation resolution index. Taken together, we suggest that based on these properties Tr14 may possess therapeutic potential as an enhancer for the resolution of inflammatory processes.

Reduction of Matrix Metallopeptidase 13 and Promotion of Chondrogenesis by Zeel T in Primary Human Osteoarthritic Chondrocytes.

Objectives: Zeel T (Ze14) is a multicomponent medicinal product. Initial preclinical data suggested a preventive effect on cartilage degradation. Clinical observational studies demonstrated that Ze14 reduced symptoms of osteoarthritis (OA), including stiffness and pain. This study aimed to explore these effects further to better understand the mode of action of Ze14 on human OA chondrocytes in vitro. Methods: Primary chondrocytes were obtained from the knees of 19 OA patients and cultured either as monolayers or in alginate beads. The cultures were treated with 20% or 10% (v/v) Ze14 or placebo. For RNA-seq, reads were generated with Illumina NextSeq5000 sequencer and aligned to the human reference genome (UCSC hg19). Differential expression analysis between Ze14 and placebo was performed in R using the DESeq2 package. Protein quantification by ELISA was performed on selected genes from the culture medium and/or the cellular fractions of primary human OA chondrocyte cultures. Results: In monolayer cultures, Ze14 20% (v/v) significantly modified the expression of 13 genes in OA chondrocytes by at least 10% with an adjusted p-value < 0.05: EGR1, FOS, NR4A1, DUSP1, ZFP36, ZFP36L1, NFKBIZ, and CCN1 were upregulated and ATF7IP, TXNIP, DEPP1, CLEC3A, and MMP13 were downregulated after 24 h Ze14 treatment. Ze14 significantly increased (mean 2.3-fold after 24 h, p = 0.0444 and 72 h, p = 0.0239) the CCN1 protein production in human OA chondrocytes. After 72 h, Ze14 significantly increased type II collagen pro-peptide production by mean 27% (p = 0.0147). For both time points CCN1 production by OA chondrocytes was correlated with aggrecan (r = 0.66, p = 0.0004) and type II collagen pro-peptide (r = 0.64, p = 0.0008) production. In alginate beads cultures, pro-MMP-13 was decreased by Ze14 from day 7-14 (from -16 to -25%, p < 0.05) and from day 17-21 (-22%, p = 0.0331) in comparison to controls. Conclusion: Ze14 significantly modified the expression of DUSP1, DEPP1, ZFP36/ZFP36L1, and CLEC3A, which may reduce MMP13 expression and activation. Protein analysis confirmed that Ze14 significantly reduced the production of pro-MMP-13. As MMP-13 is involved in type II collagen degradation, Ze14 may limit cartilage degradation. Ze14 also promoted extracellular matrix formation arguably through CCN1 production, a growth factor well correlated with type II collagen and aggrecan production.

Machine learning-assisted neurotoxicity prediction in human midbrain organoids.

INTRODUCTION: Brain organoids are highly complex multi-cellular tissue proxies, which have recently risen as novel tools to study neurodegenerative diseases such as Parkinson's disease (PD). However, with increasing complexity of the system, usage of quantitative tools becomes challenging. OBJECTIVES: The primary objective of this study was to develop a neurotoxin-induced PD organoid model and to assess the neurotoxic effect on dopaminergic neurons using microscopy-based phenotyping in a high-content fashion. METHODS: We describe a pipeline for a machine learning-based analytical method, allowing for detailed image-based cell profiling and toxicity prediction in brain organoids treated with the neurotoxic compound 6-hydroxydopamine (6-OHDA). RESULTS: We quantified features such as dopaminergic neuron count and neuronal complexity and built a machine learning classifier with the data to optimize data processing strategies and to discriminate between different treatment conditions. We validated the approach with high content imaging data from PD patient derived midbrain organoids. CONCLUSIONS: The here described model is a valuable tool for advanced in vitro PD modeling and to test putative neurotoxic compounds.

Impaired serine metabolism complements LRRK2-G2019S pathogenicity in PD patients.

Parkinson's disease (PD) is a multifactorial disorder with complex etiology. The most prevalent PD associated mutation, LRRK2-G2019S is linked to familial and sporadic cases. Based on the multitude of genetic predispositions in PD and the incomplete penetrance of LRRK2-G2019S, we hypothesize that modifiers in the patients' genetic background act as susceptibility factors for developing PD. To assess LRRK2-G2019S modifiers, we used human induced pluripotent stem cell-derived neuroepithelial stem cells (NESCs). Isogenic controls distinguish between LRRK2-G2019S dependent and independent cellular phenotypes. LRRK2-G2019S patient and healthy mutagenized lines showed altered NESC self-renewal and viability, as well as impaired serine metabolism. In patient cells, phenotypes were only partly LRRK2-G2019S dependent, suggesting a significant contribution of the genetic background. In this context we identified the gene serine racemase (SRR) as a novel patient-specific, developmental, genetic modifier contributing to the aberrant phenotypes. Its enzymatic product, d-serine, rescued altered cellular phenotypes. Susceptibility factors in the genetic background, such as SRR, could be new targets for early PD diagnosis and treatment.

Modeling Parkinson's disease in midbrain-like organoids.

Modeling Parkinson's disease (PD) using advanced experimental in vitro models is a powerful tool to study disease mechanisms and to elucidate unexplored aspects of this neurodegenerative disorder. Here, we demonstrate that three-dimensional (3D) differentiation of expandable midbrain floor plate neural progenitor cells (mfNPCs) leads to organoids that resemble key features of the human midbrain. These organoids are composed of midbrain dopaminergic neurons (mDANs), which produce and secrete dopamine. Midbrain-specific organoids derived from PD patients carrying the LRRK2-G2019S mutation recapitulate disease-relevant phenotypes. Automated high-content image analysis shows a decrease in the number and complexity of mDANs in LRRK2-G2019S compared to control organoids. The floor plate marker FOXA2, required for mDAN generation, increases in PD patient-derived midbrain organoids, suggesting a neurodevelopmental defect in mDANs expressing LRRK2-G2019S. Thus, we provide a robust method to reproducibly generate 3D human midbrain organoids containing mDANs to investigate PD-relevant patho-mechanisms.

Derivation of Human Midbrain-Specific Organoids from Neuroepithelial Stem Cells.

Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson's disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.

Hypoxia facilitates neurogenic dural plasma protein extravasation in mice: a novel animal model for migraine pathophysiology.

Migraine animal models generally mimic the onset of attacks and acute treatment processes. A guinea pig model used the application of meta-chlorophenylpiperazine (mCPP) to trigger immediate dural plasma protein extravasation (PPE) mediated by 5-HT2B receptors. This model has predictive value for antimigraine drugs but cannot explain the delayed onset of efficacy of 5-HT2B receptor antagonists when clinically used for migraine prophylaxis. We found that mCPP failed to induce dural PPE in mice. Considering the role 5-HT2B receptors play in hypoxia-induced pulmonary vessel muscularization, we were encouraged to keep mice under hypoxic conditions and tested whether this treatment will render them susceptible to mCPP-induced dural PPE. Following four-week of hypoxia, PPE, associated with increased transendothelial transport, was induced by mCPP. The effect was blocked by sumatriptan. Chronic application of 5-HT2B receptor or nitric oxide synthase blockers during hypoxia prevented the development of susceptibility. Here we present a migraine model that distinguishes between a migraine-like state (hypoxic mice) and normal, normoxic mice and mimics processes that are related to chronic activation of 5-HT2B receptors under hypoxia. It seems striking, that chronic endogenous activation of 5-HT2B receptors is crucial for the sensitization since 5-HT2B receptor antagonists have strong, albeit delayed migraine prophylactic efficacy.

Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.

Origin-dependent neural cell identities in differentiated human iPSCs in vitro and after transplantation into the mouse brain.

The differentiation capability of induced pluripotent stem cells (iPSCs) toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs), dermal fibroblasts, or cord blood CD34(+) hematopoietic progenitor cells. Neural progenitor cells (NPCs) and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.

Induced neural stem cells achieve long-term survival and functional integration in the adult mouse brain.

Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications.

Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling.

Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.

A systemic transcriptome analysis reveals the regulation of neural stem cell maintenance by an E2F1-miRNA feedback loop.

Stem cell fate decisions are controlled by a molecular network in which transcription factors and miRNAs are of key importance. To systemically investigate their impact on neural stem cell (NSC) maintenance and neuronal commitment, we performed a high-throughput mRNA and miRNA profiling and isolated functional interaction networks of involved mechanisms. Thereby, we identified an E2F1-miRNA feedback loop as important regulator of NSC fate decisions. Although E2F1 supports NSC proliferation and represses transcription of miRNAs from the miR-17∼92 and miR-106a∼363 clusters, these miRNAs are transiently up-regulated at early stages of neuronal differentiation. In these early committed cells, increased miRNAs expression levels directly repress E2F1 mRNA levels and inhibit cellular proliferation. In mice, we demonstrated that these miRNAs are expressed in the neurogenic areas and that E2F1 inhibition represses NSC proliferation. The here presented data suggest a novel interaction mechanism between E2F1 and miR-17∼92 / miR-106a∼363 miRNAs in controlling NSC proliferation and neuronal differentiation.

Induced pluripotent stem cells generated from adult bone marrow-derived cells of the nonhuman primate (Callithrix jacchus) using a novel quad-cistronic and excisable lentiviral vector.

Regenerative medicine is in need of solid, large animal models as a link between rodents and humans to evaluate the functionality, immunogenicity, and clinical safety of stem cell-derived cell types. The common marmoset (Callithrix jacchus) is an excellent large animal model, genetically close to humans and readily used worldwide in clinical research. Until now, only two groups showed the generation of induced pluripotent stem cells (iPSCs) from the common marmoset using integrating retroviral vectors. Therefore, we reprogrammed bone marrow-derived mesenchymal cells (MSCs) of adult marmosets in the presence of TAV, SB431542, PD0325901, and ascorbic acid via a novel, excisable lentiviral spleen focus-forming virus (SFFV)-driven quad-cistronic vector system (OCT3/4, KLF4, SOX2, C-MYC). Endogenous pluripotency markers like OCT3/4, KLF4, SOX2, C-MYC, LIN28, NANOG, and strong alkaline phosphatase signals were detected. Exogenous genes were silenced and additionally the cassette was removed with a retroviral Gag precursor system. The cell line could be cultured in absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) and could be successfully differentiated into embryoid bodies and teratomas with presence of all three germ layers. Directed differentiation generated neural progenitors, megakaryocytes, adipocytes, chondrocytes, and osteogenic cells. Thus, all criteria for fully reprogrammed bone marrow-MSCs of a nonhuman primate with a genetically sophisticated construct could be demonstrated. These cells will be a promising tool for future autologous transplantations.

Anti-inflammatory treatment induced regenerative oligodendrogenesis in parkinsonian mice.

INTRODUCTION: The adult mammalian brain retains niches for neural stem cells (NSCs), which can generate glial and neuronal components of the brain tissue. However, it is barely established how chronic neuroinflammation, as it occurs in neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, affects adult neurogenesis and, therefore, modulates the brain's potential for self-regeneration. METHODS: Neural stem cell culture techniques, intraventricular tumor necrosis factor (TNF)-α infusion and the 6-hydroxydopamine mouse model were used to investigate the influence of neuroinflammation on adult neurogenesis in the Parkinson's disease background. Microscopic methods and behavioral tests were used to analyze samples. RESULTS: Here, we demonstrate that differences in the chronicity of TNF-α application to cultured NSCs result in opposed effects on their proliferation. However, chronic TNF-α treatment, mimicking Parkinson's disease associated neuroinflammation, shows detrimental effects on neural progenitor cell activity. Inversely, pharmacological inhibition of neuroinflammation in a 6-hydroxydopamine mouse model led to increased neural progenitor cell proliferation in the subventricular zone and neuroblast migration into the lesioned striatum. Four months after surgery, we measured improved Parkinson's disease-associated behavior, which was correlated with long-term anti-inflammatory treatment. But surprisingly, instead of newly generated striatal neurons, oligodendrogenesis in the striatum of treated mice was enhanced. CONCLUSIONS: We conclude that anti-inflammatory treatment, in a 6-hydroxydopamine mouse model for Parkinson's disease, leads to activation of adult neural stem cells. These adult neural stem cells generate striatal oligodendrocytes. The higher numbers of newborn oligodendrocytes possibly contribute to axonal stability and function in this mouse model of Parkinson's disease and thereby attenuate dysfunctions of basalganglian motor-control.

Direct reprogramming of fibroblasts into neural stem cells by defined factors.

Recent studies have shown that defined sets of transcription factors can directly reprogram differentiated somatic cells to a different differentiated cell type without passing through a pluripotent state, but the restricted proliferative and lineage potential of the resulting cells limits the scope of their potential applications. Here we show that a combination of transcription factors (Brn4/Pou3f4, Sox2, Klf4, c-Myc, plus E47/Tcf3) induces mouse fibroblasts to directly acquire a neural stem cell identity-which we term as induced neural stem cells (iNSCs). Direct reprogramming of fibroblasts into iNSCs is a gradual process in which the donor transcriptional program is silenced over time. iNSCs exhibit cell morphology, gene expression, epigenetic features, differentiation potential, and self-renewing capacity, as well as in vitro and in vivo functionality similar to those of wild-type NSCs. We conclude that differentiated cells can be reprogrammed directly into specific somatic stem cell types by defined sets of specific transcription factors.

JAM-C is an apical surface marker for neural stem cells.

Junctional adhesion molecule-C (JAM-C) is an adhesive cell surface protein expressed in various cell types. JAM-C localizes to the apically localized tight junctions (TJs) between contacting endothelial and epithelial cells, where it contributes to cell-cell adhesions. Just as those epithelial cells, also neural stem cells are highly polarized along their apical-basal axis. The defining feature of all stem cells, including neural stem cells (NSCs) is their ability to self renew. This self-renewal depends on the tight control of symmetric and asymmetric cell divisions. In NSCs, the decision whether a division is symmetric or asymmetric largely depends on the distribution of the apical membrane and cell fate determinants on the basal pole of the cell. In this study we demonstrate that JAM-C is expressed on neural progenitor cells and neural stem cells in the embryonic as well as the adult mouse brain. Furthermore, we demonstrate that in vivo JAM-C shows enrichment at the apical surface and therefore is asymmetrically distributed during cell divisions. These results define JAM-C as a novel surface marker for neural stem cells.