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.

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.

[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.

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.

[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.

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.

[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.

[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.

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.

Stem Cells in the Treatment of Insulin-Dependent Diabetes Mellitus.

Diabetes affects over 350 million people worldwide, with the figure projected to rise to nearly 500 million over the next 20 years, according to the World Health Organization. Insulin-dependent diabetes mellitus (type 1 diabetes) is an endocrine disorder caused by an autoimmune reaction that destroys insulin-producing ß-cells in the pancreas, which leads to insulin deficiency. Administration of exogenous insulin remains at the moment the treatment mainstay. This approach helps to regulate blood glucose levels and significantly increases the life expectancy of patients. However, type 1 diabetes is accompanied by long-term complications associated with the systemic nature of the disease and metabolic abnormalities having a profound impact on health. Of greater impact would be a therapeutic approach which would overcome these limitations by better control of blood glucose levels and prevention of acute and chronic complications. The current efforts in the field of regenerative medicine are aimed at finding such an approach. In this review, we discuss the time-honored technique of donor islets of Langerhans transplantation. We also focus on the use of pluripotent stem and committed cells and cellular reprogramming. The molecular mechanisms of pancreatic differentiation are highlighted. Much attention is devoted to the methods of grafts delivery and to the materials used during its creation.

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.

[STEM CELL NICHES AND REGENERATIVE MEDICINE].

The review is devoted to modern state of science in the area of stem cell niches. Fundamental characteristics of niche including extracellular matrix are considered. Key principles of niche functioning are demonstrated by the example of hematopoietic and epithelial cells. Special section discusses the use of stem cells and their microenvironment in regenerative medicine.

Reconstruction of rabbit urethral epithelium with skin keratinocytes.

We have investigated the living skin equivalent (LSE) as an alternative source of plastic material for closing full-thickness epithelial-stromal urethral injuries. The possibility of transdifferentiation of epidermal keratinocytes, a component of 3D tissue constructs, was investigated in vivo in a model of the recovery of urethral injuries in laboratory rabbits. Autologous grafting of LSE in de-epithelialized urethra showed that skin keratinocytes placed in a specific in vivo microenvironment can be incorporated into the damaged area and function as urothelium. The use of EGFP transfected keratinocytes allowed us to identify transplanted cells. The reconstructed urethral tubes did not develop strictures or fistulas at the site of the grafted LSE. Immunohistochemical studies of neo-urothelium revealed EGFP-positive cells expressing the urothelial markers K7 and UP3.

Valproic Acid Increases the Hepatic Differentiation Potential of Salivary Gland Cells.

The studies of cell plasticity and differentiation abilities are important problems in modern cellular biology. The use of histone deacetylase inhibitor - valproic acid is a promising approach to increasing the differentiation efficiency of various cell types. In this paper we investigate the ability of mouse submandibular salivary gland cells to differentiate into the hepatic direction and the effect of valproic acid on the efficiency of this differentiation. It was shown that the gene expression levels of hepatocyte markers (Aat, Afp, G6p, Pepck, Tat, Cyp3a13) and liver-enriched transcription factors (Hnf-3α, Hnf-3ß, Hnf-4α, Hnf-6) were increased after differentiation in salivary gland cells. Valproic acid increases the specificity of hepatic differentiation, reducing the expression levels of the ductal (Krt19, Hhex1, Cyp7a1) and acinar (Ptf1a) markers. After valproic acid exposure, the efficiency of hepatic differentiation also increases, as evidenced by the increase in the gene expression level of Alb and Tdo, and increase in urea production by differentiated cells. No change was found in DNA methylation of the promoter regions of the genes; however, valproic acid treatment and subsequent hepatic differentiation largely affected the histone H3 methylation of liver-enriched genes. Thus, mouse submandibular salivary gland cells are capable of effective differentiation in the hepatic direction. Valproic acid increases the specificity and efficiency of the hepatic differentiation of these cells.

Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells.

Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.

The role of integrins in the development and homeostasis of the epidermis and skin appendages.

Integrins play a critical role in the regulation of adhesion, migration, proliferation, and differentiation of cells. Because of the variety of the functions they play in the cell, they are necessary for the formation and maintenance of tissue structure integrity. The trove of data accumulated by researchers suggests that integrins participate in the morphogenesis of the epidermis and its appendages. The development of mice with tissue-specific integrin genes knockout and determination of the genetic basis for a number of skin diseases in humans showed the significance of integrins in the biology, physiology, and morphogenesis of the epidermis and hair follicles. This review discusses the data on the role of different classes of integrin receptors in the biology of epidermal cells, as well as the development of the epidermis and hair follicles.