Pulsating Extremely Low-Frequency Electromagnetic Fields Influence Differentiation of Mouse Neural Stem Cells towards Astrocyte-like Phenotypes: In Vitro Pilot Study.

Even though electromagnetic fields have been reported to assist endogenous neurogenesis, little is known about the exact mechanisms of their action. In this pilot study, we investigated the effects of pulsating extremely low-frequency electromagnetic fields on neural stem cell differentiation towards specific phenotypes, such as neurons and astrocytes. Neural stem cells isolated from the telencephalic wall of B6(Cg)-Tyrc-2J/J mouse embryos (E14.5) were randomly divided into three experimental groups and three controls. Electromagnetic field application setup included a solenoid placed within an incubator. Each of the experimental groups was exposed to 50Hz ELF-EMFs of varied strengths for 1 h. The expression of each marker (NES, GFAP, ß-3 tubulin) was then assessed by immunocytochemistry. The application of high-strength ELF-EMF significantly increased and low-strength ELF-EMF decreased the expression of GFAP. A similar pattern was observed for ß-3 tubulin, with high-strength ELF-EMFs significantly increasing the immunoreactivity of ß-3 tubulin and medium- and low-strength ELF-EMFs decreasing it. Changes in NES expression were observed for medium-strength ELF-EMFs, with a demonstrated significant upregulation. This suggests that, even though ELF-EMFs appear to inhibit or promote the differentiation of neural stem cells into neurons or astrocytes, this effect highly depends on the strength and frequency of the fields as well as the duration of their application. While numerous studies have demonstrated the capacity of EMFs to guide the differentiation of NSCs into neuron-like cells or ß-3 tubulin+ neurons, this is the first study to suggest that ELF-EMFs may also steer NSC differentiation towards astrocyte-like phenotypes.

Human Oral Mucosa Stem Cells Increase Survival of Neurons Affected by In Vitro Anoxia and Improve Recovery of Mice Affected by Stroke Through Time-limited Secretion of miR-514A-3p.

The success rate of regenerative medicine largely depends on the type of stem cells applied in such procedures. Consequently, to achieve the needed level for clinical standardization, we need to investigate the viability of accessible sources with sufficient quantity of cells. Since the oral region partly originates from the neural crest, which naturally develops in niche with decreased levels of oxygen, the main goal of this work was to test if human oral mucosa stem cells (hOMSC) might be used to treat neurons damaged by anoxia. Here we show that hOMSC are more resistant to anoxia than human induced pluripotent stem cells and that they secrete BDNF, GDNF, VEGF and NGF. When hOMSC were added to human neurons damaged by anoxia, they significantly improved their survival. This regenerative capability was at least partly achieved through miR-514A-3p and SHP-2 and it decreased in hOMSC exposed to neural cells for 14 or 28 days. In addition, the beneficial effect of hOMSC were also confirmed in mice affected by stroke. Hence, in this work we have confirmed that hOMSC, in a time-limited manner, improve the survival of anoxia-damaged neurons and significantly contribute to the recovery of experimental animals following stroke.

Effect of Fetal Bovine Serum or Basic Fibroblast Growth Factor on Cell Survival and the Proliferation of Neural Stem Cells: The Influence of Homocysteine Treatment.

In vitro cell culture is a routinely used method which is also applied for in vitro modeling of various neurological diseases. On the other hand, media used for cell culture are often not strictly standardized between laboratories, which hinders the comparison of the obtained results. Here, we compared the effects of homocysteine (Hcy), a molecule involved in neurodegeneration, on immature cells of the nervous system cultivated in basal medium or media supplemented by either fetal bovine serum or basic fibroblast growth factor. The number of cells in basal media supplemented with basic fibroblast growth factor (bFGF) was 2.5 times higher in comparison to the number of cells in basal media supplemented with fetal bovine serum (FBS). We also found that the neuron-specific ß-3-tubulin protein expression dose dependently decreased with increasing Hcy exposure. Interestingly, bFGF exerts a protective effect on ß-3-tubulin protein expression at a concentration of 1000 µM Hcy compared to FBS-treated neural stem cells on Day 7. Supplementation with bFGF increased SOX2 protein expression two-fold compared to FBS supplementation. GFAP protein expression increased five-fold on Day 3 in FBS-treated neural stem cells, whereas on Day 7, bFGF increased GFAP expression two-fold compared to FBS-treated neural stem cells. Here, we have clearly shown that the selection of culturing media significantly influences various cellular parameters, which, in turn, can lead to different conclusions in experiments based on in vitro models of pathological conditions.

Low-Molecular-Mass Fragments of Collagen Improve Parameters Related to Mass and Inflammation of the Adipose Tissue in the Obese Rat.

Research background: Despite clearly recognized links between increased body mass and increased risk for various pathological conditions, therapeutic options to treat obesity are still very limited. The aim of the present study is to explore the effect of low-molecular-mass collagen fragments obtained from the scales of Antarctic wild marine fish on rats' visceral and subcutaneous white adipose tissue in a high-calorie diet-induced obesity model. Experimental approach: The study was conducted on outbred rats, which were divided into 3 experimental groups: (i) control, consuming standard food (3.81 kcal/g), (ii) obese group, consuming a high-calorie diet (5.35 kcal/g), and (iii) obese group, consuming a high-calorie diet (5.35 kcal/g) with intragastric administration of low-molecular-mass collagen fragments (at a dose 1 g/kg of body mass during 6 weeks). Low-molecular-mass collagen fragments were obtained by a procedure that included collagen extraction from fish scales and enzymatic hydrolysis with pepsin. Apart from hematoxylin and eosin staining, fibrosis level was assessed by histochemical Van Gieson's trichrome picrofuchsin staining, and mast cells were analysed by toluidine blue O staining. Results and conclusions: Group treated with low-molecular-mass fragments of collagen exhibited decreased rate of mass gain, relative mass, area occupied by collagen fibre of both visceral and subcutaneous adipose tissue, and cross-sectional area of both visceral and subcutaneous adipocytes. Treatment with low-molecular-mass fragments of collagen reduced the infiltration of immune cells, number of mast cells and their redistribution back to the septa. This was also accompanied by a decreased number of the crown-like structures formed by the immune cells, which are markers of chronic inflammation that accompanies obesity. Novelty and scientific contribution: This is the first study that reports the anti-obesity effect of low-molecular-mass fragments produced as a result of controlled hydrolysis of collagen from the scales of Antarctic wild marine fish in the in vivo model. Another novelty of this work is the observation that the tested collagen fragments not only reduce the body mass, but also improve the morphological and inflammatory parameters (decrease in the number of crown-like structures, immune cell infiltration, fibrosis and mast cells). Altogether, our work suggests that low-molecular-mass collagen fragments are a promising candidate for amelioration of some comorbidities linked to obesity.

Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain.

A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements.

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.

How to face the aging world - lessons from dementia research.

A continuous rise in life expectancy has led to an increase in the number of senior citizens, now amounting to a fifth of the global population, and to a dramatic increase in the prevalence of diseases of the elderly. This review discusses the threat of dementia, a disease that imposes enormous financial burden on health systems and warrants efficient therapeutic solutions. What we learned from numerous failed clinical trials is that we have to immediately take into account two major elements: early detection of dementia, much before the onset of symptoms, and personalized (precision) medicine treatment approach. We also discuss some of the most promising therapeutic directions, including stem cells, exosomes, electromagnetic fields, and ozone.

Extracellular Vesicles as Innovative Tool for Diagnosis, Regeneration and Protection against Neurological Damage.

Extracellular vesicles (EVs) have recently attracted a great deal of interest as they may represent a new biosignaling paradigm. According to the mode of biogenesis, size and composition, two broad categories of EVs have been described, exosomes and microvesicles. EVs have been shown to carry cargoes of signaling proteins, RNA species, DNA and lipids. Once released, their content is selectively taken up by near or distant target cells, influencing their behavior. Exosomes are involved in cell-cell communication in a wide range of embryonic developmental processes and in fetal-maternal communication. In the present review, an outline of the role of EVs in neural development, regeneration and diseases is presented. EVs can act as regulators of normal homeostasis, but they can also promote either neuroinflammation/degeneration or tissue repair in pathological conditions, depending on their content. Since EV molecular cargo constitutes a representation of the origin cell status, EVs can be exploited in the diagnosis of several diseases. Due to their capability to cross the blood-brain barrier (BBB), EVs not only have been suggested for the diagnosis of central nervous system disorders by means of minimally invasive procedures, i.e., "liquid biopsies", but they are also considered attractive tools for targeted drug delivery across the BBB. From the therapeutic perspective, mesenchymal stem cells (MSCs) represent one of the most promising sources of EVs. In particular, the neuroprotective properties of MSCs derived from the dental pulp are here discussed.

Molecular mechanisms of microglia- and astrocyte-driven neurorestoration triggered by application of electromagnetic fields.

AIM: To propose potential mechanisms of action of electromagnetic fields (EMF) on astrocytes and microglia and to elucidate the role of heat shock proteins (HSP), adenosine triphosphate (ATP), calcium ions (Ca2+), and hypoxia-inducible factor 1α (HIF1α) in neurorestoration following the application of EMF. METHODS: We reviewed the existing studies within the public domain and cross-evaluated their results in order to conclude on the molecular mechanisms of microglia-astrocyte crosstalk at work during EMF treatment. RESULTS: The existing studies suggest that EMF induces the increase of HSP70 expression and inhibition of HIF1α, thus decreasing inflammation and allowing the microglia-astrocyte crosstalk to initiate the formation of a glial scar within the central nervous system. Furthermore, by potentially up-regulating A2A and A3 adenosine receptors, EMF increases cAMP accumulation from astrocytes and reduces the expression of inflammatory cytokines TNF α and IL-8, thus initiating neurorestoration. CONCLUSION: The microglia-astrocyte crosstalk during EMF treatment is crucial for the initiation of neurorestoration. Elucidating the exact mechanisms of EMF actions upon microglia and astrocytes, and its role in neurorestoration could be a key step in further research of the therapeutic potential of EMFs in various neurological disorders.

Necroptosis is one of the modalities of cell death accompanying ischemic brain stroke: from pathogenesis to therapeutic possibilities.

Due to very limited therapeutic options, ischemic brain injury is one of the leading causes of death and lifelong disability worldwide, which imposes enormous public health burden. One of the main events occurring with ischemic brain stroke is cell death. Necroptosis is a type of cell death described as a regulated necrosis characterized by cell membrane disruption mediated by phosphorylated mixed lineage kinase like protein (MLKL). It can be triggered by activation of death receptors (eg, FAS, TNFR1), which lead to receptor-interacting serine/threonine-protein kinase 3 (RIPK3) activation by RIPK1 in the absence of active caspase-8. Here, we review articles that have reported that necroptosis significantly contributes to negative events occurring with the ischemic brain stroke, and that its inhibition is protective both in vitro and in vivo. We also review articles describing positive effects obtained by reducing necroptosis, including the reduction of infarct volume and improved functional recovery in animal models. Since necroptosis is characterized by cell content leakage and subsequent inflammation, in addition to reducing cell death, inhibition of necroptosis in ischemic brain stroke also reduces some inflammatory cytokines. By comparing various approaches in inhibition of necroptosis, we analyze the achieved effects from the perspective of controlling necroptosis as a part of future therapeutic interventions in brain ischemia.

STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.

STAM2 (signal transducing adaptor molecule 2), a subunit of the ESCRT-0 complex, is an endosomal protein acting as a regulator of receptor signaling and trafficking. To analyze STAM2 in the nervous system, its gene expression and protein localization in the mouse brain were identified using three methods: mRNA in situ hybridization, immunohistochemistry, and via lacZ reporter in frame with Stam2 gene using the gene trap mouse line Stam2(Gt1Gaj). STAM2 intracellular localization was analyzed by subcellular fractionation and co-immunofluorescence using confocal microscopy. Stam2 was strongly expressed in the cerebral and cerebellar cortex, hippocampal formation, olfactory bulb, and medial habenula. The majority of STAM2-positive cells co-stained with the neuronal markers. In neurons STAM2 was found in the early endosomes and also in the nucleus. The other members of the ESCRT-0 complex co-localized with STAM2 in the cytoplasm, but they were not present in the nucleus. The newly identified neuron-specific nuclear localization of STAM2, together with its high expression in the brain indicated that STAM2 might have a specific function in the mouse nervous system.

Lusca: FIJI (ImageJ) based tool for automated morphological analysis of cellular and subcellular structures.

The human body consists of diverse subcellular, cellular and supracellular structures. Neurons possess varying-sized projections that interact with different cellular structures leading to the development of highly complex morphologies. Aiming to enhance image analysis of complex biological forms including neurons using available FIJI (ImageJ) plugins, Lusca, an advanced open-source tool, was developed. Lusca utilizes machine learning for image segmentation with intensity and size thresholds. It performs particle analysis to ascertain parameters such as area/volume, quantity, and intensity, in addition to skeletonization for determining length, branching, and width. Moreover, in conjunction with colocalization measurements, it provides an extensive set of 29 morphometric parameters for both 2D and 3D analysis. This is a significant enhancement compared to other scripts that offer only 5-15 parameters. Consequently, it ensures quicker and more precise quantification by effectively eliminating noise and discerning subtle details. With three times larger execution speed, fewer false positive and negative results, and the capacity to measure various parameters, Lusca surpasses other existing open-source solutions. Its implementation of machine learning-based segmentation facilitates versatile applications for different cell types and biological structures, including mitochondria, fibres, and vessels. Lusca's automated and precise measurement capability makes it an ideal choice for diverse biological image analyses.

Is it enough just to demonstrate that the advanced therapy medicinal products do work or we would prefer to keep walking on the Moon?

After several decades of continuous yet bumpy progress the advanced therapy medicinal products reached the stage when the first drugs with well documented efficacy started to be registered. However, in the disturbing chain of events, many of them were discontinued because of the lack of return on investment. By comparing this phenomenon to the fact that humans did not return to the Moon for already 50 years, primarily because of the lack of dedicated funds, this commentary proposes strategies how to avoid menace of the dead end threating to suffocate progress of the advanced medical therapies. While treatments for rare diseases can be defended by mixture of altruistic, inspiring and rational reasons, mostly covered by the fact that regardless of the price of the newly developed therapy, the total burden remains low, common diseases should be addressed in a different way. This needs to include precise modelling of the benefits which advanced therapy medicinal products bring for every condition, taking in account reduction of the costs of long, often life-long support of patients affected by such diseases. Without intention to steal romantic view on the scientific progress, powerful yet very expensive tools of advanced therapy medicinal products require urgent top-down decisions which include selection of priorities based on the financial modelling. Instead of spontaneous exploration in all directions, this commentary proposes an arranged marriage between scientific community and big investors sustained by combination of governmental requirements in the form of real time data sharing, reimbursement warranties according to demonstrated efficacy and clear recognition of the primary targets with accompanying pre-defined financial frameworks.

Mesenchymal stem cell therapy for neurological disorders: The light or the dark side of the force?

Neurological disorders are recognized as major causes of death and disability worldwide. Because of this, they represent one of the largest public health challenges. With awareness of the massive burden associated with these disorders, came the recognition that treatment options were disproportionately scarce and, oftentimes, ineffective. To address these problems, modern research is increasingly looking into novel, more effective methods to treat neurological patients; one of which is cell-based therapies. In this review, we present a critical analysis of the features, challenges, and prospects of one of the stem cell types that can be employed to treat numerous neurological disorders-mesenchymal stem cells (MSCs). Despite the fact that several studies have already established the safety of MSC-based treatment approaches, there are still some reservations within the field regarding their immunocompatibility, heterogeneity, stemness stability, and a range of adverse effects-one of which is their tumor-promoting ability. We additionally examine MSCs' mechanisms of action with respect to in vitro and in vivo research as well as detail the findings of past and ongoing clinical trials for Parkinson's and Alzheimer's disease, ischemic stroke, glioblastoma multiforme, and multiple sclerosis. Finally, this review discusses prospects for MSC-based therapeutics in the form of biomaterials, as well as the use of electromagnetic fields to enhance MSCs' proliferation and differentiation into neuronal cells.

Critical points for optimizing long-term culture and neural differentiation capacity of rodent and human neural stem cells to facilitate translation into clinical settings.

Despite several decades of research on the nature and functional properties of neural stem cells, which brought great advances in regenerative medicine, there is still a plethora of ambiguous protocols and interpretations linked to their applications. Here, we present a whole spectrum of protocol elements that should be standardized in order to obtain viable cell cultures and facilitate their translation into clinical settings. Additionally, this review also presents outstanding limitations and possible problems to be encountered when dealing with protocol optimization. Most importantly, we also outline the critical points that should be considered before starting any experiments utilizing neural stem cells or interpreting their results.

The Oxygen and Glucose Deprivation of Immature Cells of the Nervous System Exerts Distinct Effects on Mitochondria, Mitophagy, and Autophagy, Depending on the Cells' Differentiation Stage.

Perinatal brain damage, one of the most common causes of lifelong impairment, is predominantly caused by a lack of oxygen and glucose during early development. These conditions, in turn, affect cells of the nervous tissue through various stages of their maturation. To quantify the influence of these factors on cell differentiation and mitochondrial parameters, we exposed neural cell precursors to oxygen and glucose deprivation (OGD) during three stages of their differentiation: day 1, day 7, and day 14 (D1, D7, and D14, respectively). The obtained results show that OGD slows down cellular differentiation and causes cell death. Regardless of the level of cell maturity, the overall area of the mitochondria, their length, and the branching of their filaments decreased uniformly when exposed to OGD-related stress. Moreover, the cells in all stages of differentiation exhibited an increase in ROS production, hyperpolarization of the mitochondrial membrane, and autophagy. Interestingly, day 7 was the only stage in which a significant increase in mitochondrial fission, along with measurable instances of mitophagy, were detected. Taken together, the results of this study suggest that, apart from common reactions to a sudden lack of oxygen and glucose, cells in specific stages of neural differentiation can also exhibit increased preferences for mitochondrial fission and mitophagy. Such findings could play a role in guiding the future development of novel therapeutic approaches targeting perinatal brain damage during specific stages of nervous system development.