NANOS3 downregulation in Down syndrome hiPSCs during primordial germ cell-like cell differentiation.

Human infertility is a complex disorder at the genetic, molecular, cellular, organ, and hormonal levels. New developing technology based on the generation of human primordial germ cell-like cells (hPGCLCs) from induced pluripotent stem cells (hiPSCs) might improve understanding of early germ cell development (specification, migration, gametogenesis, and epigenetic reconstitutions), as well as offering a solution for infertility and hereditary disorders. In this study, we differentiated hiPSCs with trisomy 21 into hPGCLCs. In vitro-derived germ cells from hiPSCs with Down syndrome (DS) express hPGCLC core circuitry, EOMES, SOX17, and PRDM14 at relatively low levels. TFAP2C and PRDM1 were expressed and remained elevated, whereas NANOS3 and NANOG were downregulated in BMP4-induced hiPSCs with DS. The low level of NANOG and NANOS3 expression might negatively influence hPGCLC generation in DS hiPSCs. We suggest that DS hPGCLCs could be a suitable model for studying human early germ cell development, the epigenetic and molecular mechanisms of PGC specification and formation, as well as related infertility disorders, such as azoospermia and teratozoospermia.

Immortalization of Human Keratinocytes Using the Catalytic Subunit of Telomerase.

A new stable line of human keratinocytes was obtained. The cells have altered morphology, both abnormal chromosomal composition and expression of keratinocyte markers, do not show contact inhibition, could be cultured in various media and have limited stratification ability in vitro. Upon transplantation into nude mice the cells have tumorigenic properties.

Mesenchymal Stem Cell Therapy-Is the Vessel Half Full or Half Empty?

The urgency of the search and introduction into medical practice of the method for the therapy of severe forms of pneumonia COVID-19 is due to the lack of effective treatment methods that can destroy the pathogen. Expectations of a good clinical effect from the application of mesenchymal stem cells (MSCs) are not groundless: there is a scientific justification in using MSCs for the treatment of inflammatory diseases and of the proven mechanisms of their action. Along with this, there are very little reliable data about the mechanism of MSCs' action when they are systemically administrated to a human or on the distribution of cells in the body and the long-term consequences of such administration. Data from model experiments are contradictory both concerning the specific action of MSCs and their safety. If clinical studies show an acceptable risk/benefit ratio for the application of MSCs, countries in which such studies have been conducted can expect their introduction into medical practice. In Russia, it is necessary to initiate experimental verification of the specific action of MSCs and the risks of their use in COVID-19 conditions in a sufficient quantity, and, in parallel, to create a mechanism for accelerated but justified admission of biomedical cell products into practice.

[Nutriome as the direction of the "main blow": determination of physiological needs in macro- and micronutrients, minor biologically active substances].

One of the essential parts of fundamental research in Nutrition Science is the determination of the physiological requirements of humans for energy and food substances. Research that has been carried out in this area over the past 90 years, consistently develops and improves the norms of physiological requirements for energy and nutrients for various groups of the population of the Russian Federation. In the 50 years of the last century in this research field, determining the values of daily intake for macronutrients (proteins, lipids and carbohydrates), was in the first place. Then the Era of micronutrients (vitamins, minerals, trace elements) was started, and, finally, now there is the Era of minor food biologically active substances. More and more facts are accumulating about their leading role in regulating metabolism. They can be recognized as endogenous regulators, the primary vital components involved in the formation of human health. In recent years, the new definition of Nutriome is introduced into Nutrition Science. It is considered as a set of essential nutritional factors to maintain a dynamic equilibrium between human being and the environment, aimed to ensure viability, the preservation and reproduction of the species, keeping the adaptive capacity, the system of antioxidant defence, apoptosis, metabolism, and immune system function. The Nutriome is a formula for optimal nutrition, which is continually being improved and supplemented. Knowledge of this formula is the key to forming an optimal diet for a person, and, therefore, to save their health. It is evident that at the population level, the Nutriome has its characteristics, its structure for each age period of human life. The need to develop a formula for optimal nutrition and, consequently, updating nutrient-based dietary guidelines is induced by socio-economic and demographic changes in population, changes in anthropometric characteristics of children and adults, increasing prevalence of socially significant non-communicable diseases, developing studies of the significance of particular food substances and establishing the relationship between nutrition and health.

Detection of small numbers of iPSCs in different heterogeneous cell mixtures with highly sensitive droplet digital PCR.

Induced pluripotent stem cells (iPS cells) are a prospective resource for regenerative biomedicine. iPS cells can differentiate into any type of stem, progenitor and somatic cells to help replace structures within damaged organs or tissues. However, iPS cells themselves, can produce malignant tumors if they are injected into the body of an immunocompatible or immunodeficient recipient. Thus, it is necessary to detect any residual iPS cells content in biomedical cell products obtained from iPS cells and destined for transplantation. In this article we describe searches for iPS cells in heterogeneous cell mixtures, using two different methods-quantitative RT-PCR and droplet digital PCR (ddPCR). In experiments with various heterogeneous mixtures, including mixtures with neural stem cells, we found that the OCT4, TDGF1 and LIN28 genes are the best markers for such a search, and droplet digital PCR provides the greatest measurement accuracy, which is 0.002%. Thus, we have confirmed the advantage of using droplet digital PCR in the search for pluripotent stem cells in heterogeneous cell mixtures. We hope that this data can be useful for biosafety control in regenerative biomedicine.

Modern Technologies Deriving Human Primordial Germ Cells in vitro.

Primordial germ cells (PGCs) are a unique type of stem cells capable of giving rise to totipotent stem cells and ensuring the fertility of an organism and the transfer of its genome to the next generation. PGC research is an important perspective research field of developmental biology that handles many questions of embryogenesis and holds promise for treatments of infertility in the future. Considering ethical concerns related to human embryos, the main research approach in understanding the biology of human PGCs is in vitro studies. In this review, we consider the historical perspective of human PGC studies in vitro, the main existing models, and further outlooks and applications in medicine and science.

[The significance of fibroblasts in experimental modeling of proliferative vitreoretinopathy]. / Rol' fibroblastov v modelirovanii proliferativnoi vitreoretinopatii.

AIM: to investigate the role of heterogeneous fibroblasts in the development of epiretinal membrane in eyes with modeled proliferative vitreoretinopathy. MATERIAL AND METHODS: The material for investigation were 6 eyes of 3 Chinchilla rabbits. Suspended fibroblasts (fibroblasts of the human skin - 200000 cells in 0.1 ml) were injected into the vitreous cavity via the pars plana. The animals were followed up for 1 month and then made out of the experiment. The eyes were enucleated and fixed in 10% neutral buffered formalin for routine histological examination. Microscopy was performed on the Leica system. RESULTS: The main clinical and morphological criteria for a rabbit model of PVR induced by intravitreal injection of heterogenic fibroblasts have been established: epiretinal membrane formation, changes in intraocular structures (the retinal pigment epithelium and retina), and inflammation (due to transplantation immunity). Particularities of the epiretinal membrane development and the role of different intraocular structures have been described. CONCLUSION: The experimental fibroblastic model of PVR reproduces the final, fibrous, stage of PVR, which is significant for efficacy evaluation of antiproliferative drugs.

Effect of Acute Emotional Stress on Proteomic Profile of Selected Brain Areas and Lysosomal Proteolysis in Rats with Different Behavioral Activity.

We compared proteome profiles of selected brain areas (cortex, amygdala, hippocampus, and reticular formation) and measured cathepsins B and D activity in liver lysosomal fraction in rats with different behavioral activity under conditions of emotional stress. In passive rats, the expression of some proteins in various brain regions was changed and baseline cathepsin B activity was higher than in active animals. Taken together, the results attest to differences in the adaptive response formation in rats, depending on behavioral features.

Compensatory function of submandibular gland in mice with streptozotocin diabetes under conditions of transplantation.

Transplantation of the submandibular gland under the renal capsule of mice with streptozotocin-induced diabetes mellitus stimulated the compensatory function of the recipient submandibular gland. An increase in insulin I and insulin II gene expression in the submandibular gland after transplantation was demonstrated by PCR. More intensive production and extrusion of these proteins in the apical and basal directions in granular compartment cells of the submandibular gland was confirmed by electron microscopy. All these changes led to a reduction of blood glucose levels in diabetic animals as soon as 2-2.5 weeks after transplantation.

Peculiarities of rat serum proteome profile in metabolic stress.

We identified individual proteins characterizing starvation-induced metabolic stress. Sustained changes at the level of proteome caused by metabolic stress were demonstrated. Intensified synthesis of S6 kinase ribosomal protein, immunoglobulin lambda light chains, and inter-alpha-trypsin inhibitor was observed in the experimental groups.

Type 2 Diabetes Mellitus: Pathogenic Features and Experimental Models in Rodents.

Type 2 diabetes mellitus (T2DM) is the most common endocrine disorder (90%) in the world; it has numerous clinical, immunological, and genetic differences from type 1 diabetes mellitus. The pathogenesis of T2DM is complex and not fully clear. To date, animal models remain the main tool by which to study the pathophysiology and therapy of T2DM. Rodents are considered the best choice among animal models, because they are characterized by a small size, short induction period, easy diabetes induction, and economic efficiency. This review summarizes data on experimental models of T2DM that are currently used, evaluates their advantages and disadvantages vis-a-vis research, and describes in detail the factors that should be taken into account when using these models. Selection of a suitable model for tackling a particular issue is not always trivial; it affects study results and their interpretation.

Effects of lipoic acid on proliferation and apoptosis of liver cells in rats with metabolic stress.

Lipoic acid stimulated expression of heat shock proteins 25, 70, and 90 in liver cells of Wistar rats with metabolic stress (5 days of food deprivation followed by complete resumption of nutrition). Lipoic acid in a dose of 25 mg/kg reduced proliferation of hepatic lymphocytes during fasting, while after resumption of feeding it stimulated hepatocyte proliferation due to differentiated regulation of the expression of cyclin D1 and Rb protein in these cell populations.

Effects of coenzyme Q10 on rat liver cells under conditions of metabolic stress.

Under conditions of metabolic stress induced in Wistar rats by 5-day starvation with subsequent refeeding, supplementation with coenzyme Q10 in doses of 10 and 100 µg/kg of body weight resulted in significant increase in liver weight after the experiment. Percent ratio of liver cell populations was changed, which was detected by flow cytometry. In addition, specific effects of low dose of coenzyme Q10 (10 mg/kg body weight) on hepatocytes was observed, which manifested in increased number of mitoses and percentage of S-phase cells, enhanced expression of D1 and Rb-protein expression, and reduced percent of apoptotic hepatocytes. Adaptive effects of coenzyme Q10 are associated with enhanced expression of Hsp25, Hsp70, and Hsp90 in hepatocytes during metabolic stress.

[Monoclonal antibody therapies for rapidly progressive and highly active multiple sclerosis in the era of the COVID-19 pandemic]. / Terapiya monoklonal'nymi antitelami bystroprogressiruyushchego i vysokoaktivnogo rasseyannogo skleroza v epokhu pandemii COVID-19.

As the COVID-19 pandemic continues, reducing the risk of infection for immunocompromised patients remains an important issue. Patients with aggressive multiple sclerosis (MS) require immunosuppressive therapy in order to control the overactive autoimmune response. Preliminary international and national trials demonstrate that older age, higher disability status and progressive MS are generally associated with a more severe clinical course of COVID-19. However, uncertainty remains about the effect of disease-modifying therapies on the COVID-19 clinical presentation. In this article, we pay special attention to monoclonal antibodies used for immune reconstitution therapy, which results in significant changes to the T-cell and/or B-cell repertoire. Based on the published data from registries in different countries, we attempted to estimate the benefits and risks of these therapies in a complicated epidemiological setting.

[Myelin oligodendrocyte glycoprotein immunoglobulin G-associated encephalomyelitis]. / Entsefalomielity, assotsiirovannye s antitelami k mielin-oligodendrotsitarnomu glikoproteinu.

The article discusses the role of myelin oligodendrocyte glycoprotein immunoglobulin G (MOG-IgG) in demyelinating diseases of the central nervous system. Clinical phenotypes of demyelinating syndromes associated with MOG-IgG that are currently included into neuromyelitis optica spectrum disorders (NMOSD) are described. However, it has been shown that encephalomyelitis associated with MOG-IgG (MOG-EM) has certain clinical, radiological, immunological and histopathological features that make it possible to single out these syndromes into a separate nosological form. We provide International recommendations that establish indications for testing MOG-IgG using cell-based assay. We discuss epidemiological issues and classification challenges of the disease. Various approaches to treatment and prevention of relapses of MOG-EM are analyzed.

Effect of metabolic stress on the expression of mTOR kinase in mouse liver cells.

The time course of changes in mTOR kinase protein and Akt and HSP70 molecular chaperone in hepatic cells was studied in a model system of metabolic stress (48-h food deprivation with subsequent restoration of normal volume of nutrition) on C57Bl/6 mice. The fluorescence intensity of mTOR protein stained with antibodies decreased in all liver cells during fasting. The expression of mTOR protein in different populations of liver cells was heterogeneous. The percentage of cells with initially high expression of mTOR protein decreased during 48-h food deprivation, while the percentage of cells with low mTOR expression increased. The levels of mTOR, Akt, and HSP70 returned to normal only on day 4 after normalization of nutrition.

[Neuromyelitis optica and neuromyelitis optica spectrum disorders]. / Optikoneiromielit i zabolevaniia spektra optikoneiromielita.

The review is devoted to up-to-date data on epidemiology, aspects of the pathogenesis of neuromyelitis optica (NMO) and neuromyelitis optica spectrum disorders (NMOSD). The authors consider a role of myelin oligodendrocyte glycoprotein immunoglobulin G (MOG-IgG) in the syndromes phenotypically similar to NMO and NMOSD. Special attention is drawn to the methods of MOG-IgG antibodies detection and indications for testing. The approaches and management for treatment and prevention of NMO relapses, risks of complications during pregnancy and immediately after delivery, as well as methods for their prevention and treatment, are described.

Pathogenesis of Type 1 Diabetes Mellitus and Rodent Experimental Models.

The global prevalence of diabetes mellitus and its severe complications is on the rise. The study of the pathogenesis of the onset and the progression of complications related to the disease, as well as the search for new therapeutic agents and methods of treatment, remains relevant. Experimental models are extremely important in the study of diabetes. This survey contains a synthesis of the most commonly used experimental animal models described in scientific literature. The mechanisms of the streptozotocin model are also analyzed and discussed, as it is considered as the most adequate and easily reproducible diabetes model. A review of the significant advantages and disadvantages of the described models has also been conducted.

New genes for accurate normalization of qRT-PCR results in study of iPS and iPS-derived cells.

iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed, iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro, phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation, validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data, typically by relating the obtained levels of gene expression to the levels of expression of a "house keeping gene", a gene whose expression is presumed not to change during manipulation of the cells. In the literature, typically only one such reference gene is used and its stability of expression during cell manipulation is not demonstrated. We are not aware of any study systematically looking at the expression of such genes in human iPSC or during their differentiation into neurons. Here we compare the expression of 16 reference genes in iPSC, neural stem cells (NSC) and neurons derived from iPSC. The applications GeNorm and NormFinder were used to identify the most suitable reference genes. We showed that ACTb, C1orf43, PSMB4, GAPDH and HMBS have the most stable expression. The use of these reference genes allows an accurate normalization of qRT-PCR results in all the cell types discussed above. We hope that this report will help to enable the performance of proper qRT-PCR results normalization in studies with iPSC-derived cells and in disease-modeling reports.