Search

The prospects for the human embryos genome editing cause intense debates both in the scientific community and in general public. While the main attention of scientists is focused on the safety, effectiveness and clinical feasibility of the inherited genome editing, the public pays attention to the bioethical aspects of the issue - the prospects of a baby design, the development of new forms of social inequality and intervention in human evolution. The authors conducted an interdisciplinary analysis of medical genetics and bioethical issues of human embryo genome editing, revealed the possibilities and limitations of genome editing technology, and considered the specifics of ethical discussions. The conceptualization of the main approaches of natural and social sciences in a general theoretical framework made it possible not only to take into account the complex nature of the issues, but also to create the prerequisites for its further productive discussion.

MYB is a key regulator of definitive hematopoiesis and it is dispensable for the development of primitive hematopoietic cells in vertebrates. To delineate definitive versus primitive hematopoiesis during differentiation of human embryonic stem cells, we have introduced reporters into the MYB locus and inactivated the gene by bi-allelic targeting. To recapitulate the early developmental events more adequately, the mutant and wild type human embryonic stem cell lines were differentiated in defined culture conditions without the addition of hematopoietic cytokines. The differentiation of the reporter cell lines demonstrated that MYB is specifically expressed throughout emerging hematopoietic cell populations. Here we show that the disruption of the MYB gene leads to severe defects in the development and proliferation of primitive hematopoietic progenitors while the emergence of primitive blood cells is not affected. We also provide evidence that MYB is essential for neutrophil and T cell development and the upregulation of innate immunity genes during hematopoietic differentiation. Our results suggest that the endothelial origin of primitive blood cells is direct and does not include the intermediate step of primitive hematopoietic progenitors.

The adeno-associated virus (AAV) is one of the most potent vectors in gene therapy. The experimental profile of this vector shows its efficiency and accepted safety, which explains its increased usage by scientists for the research and treatment of a wide range of diseases. These studies require using functional, pure, and high titers of vector particles. In fact, the current knowledge of AAV structure and genome helps improve the scalable production of AAV vectors. In this review, we summarize the latest studies on the optimization of scalable AAV production through modifying the AAV genome or biological processes inside the cell.

The adrenal glands are important endocrine organs that play a major role in the stress response. Some adrenal glands abnormalities are treated with hormone replacement therapy, which does not address physiological requirements. Modern technologies make it possible to develop gene therapy drugs that can completely cure diseases caused by mutations in specific genes. Congenital adrenal hyperplasia (CAH) is an example of such a potentially treatable monogenic disease. CAH is an autosomal recessive inherited disease with an overall incidence of 1:9500–1:20,000 newborns. To date, there are several promising drugs for CAH gene therapy. At the same time, it remains unclear how new approaches can be tested, as there are no models for this disease. The present review focuses on modern models for inherited adrenal gland insufficiency and their detailed characterization. In addition, the advantages and disadvantages of various pathological models are discussed, and ways of further development are suggested.

Tissue autofluorescence of fixed tissue sections is a major concern of fluorescence microscopy. The adrenal cortex emits intense intrinsic fluorescence that interferes with signals from fluorescent labels, resulting in poor-quality images and complicating data analysis. We used confocal scanning laser microscopy imaging and lambda scanning to characterize the mouse adrenal cortex autofluorescence. We evaluated the efficacy of tissue treatment methods in reducing the intensity of the observed autofluorescence, such as trypan blue, copper sulfate, ammonia/ethanol, Sudan Black B, TrueVIEWTM Autofluorescence Quenching Kit, MaxBlockTM Autofluorescence Reducing Reagent Kit, and TrueBlackTM Lipofuscin Autofluorescence Quencher. Quantitative analysis demonstrated autofluorescence reduction by 12–95%, depending on the tissue treatment method and excitation wavelength. TrueBlackTM Lipofuscin Autofluorescence Quencher and MaxBlockTM Autofluorescence Reducing Reagent Kit were the most effective treatments, reducing the autofluorescence intensity by 89–93% and 90–95%, respectively. The treatment with TrueBlackTM Lipofuscin Autofluorescence Quencher preserved the specific fluorescence signals and tissue integrity, allowing reliable detection of fluorescent labels in the adrenal cortex tissue. This study demonstrates a feasible, easy-to-perform, and cost-effective method to quench tissue autofluorescence and improve the signal-to-noise ratio in adrenal tissue sections for fluorescence microscopy.

Despite measures taken world-wide, the coronavirus disease 2019 (COVID-19) pandemic continues. Because efficient antiviral drugs are not yet widely available, vaccination is the best option to control the infection rate. Although this option is obvious in the case of COVID-19–naive individuals, it is still unclear when individuals who have recovered from a previous SARS-CoV-2 infection should be vaccinated and whether the vaccination raises immune responses against the coronavirus and its novel variants. In this study, we collected peripheral blood from 84 healthy human donors of different COVID-19 status who were vaccinated with the Sputnik Light vaccine and measured the dynamics of the Ab and T cell responses, as well as the virus-neutralizing activity (VNA) in serum, against two SARS-CoV-2 variants, B.1.1.1 and B.1.617.2. We showed that vaccination of individuals previously exposed to the virus considerably boosts the existing immune response. In these individuals, receptor-binding domain (RBD)–specific IgG titers and VNA in serum were already elevated on the 7th day after vaccination, whereas COVID-19–naive individuals developed the Ab response and VNA mainly 21 d postvaccination. Additionally, we found a strong correlation between RBD-specific IgG titers and VNA in serum, and according to these data vaccination may be recommended when the RBD-specific IgG titers drop to 142.7 binding Ab units/ml or below. In summary, the results of the study demonstrate that vaccination is beneficial for both COVID-19–naive and recovered individuals, especially since it raises serum VNA against the B.1.617.2 variant, one of the five SARS-CoV-2 variants of concern.

Despite the measures taken worldwide, COVID-19 pandemic still progresses. While efficient antiviral drugs are not yet widely available, vaccination is the best option to control the infection rate. Although this option is obvious in case of COVID-19–naïve individuals, it is still unclear when individuals who have recovered from a previous SARS-CoV-2 infection should be vaccinated and whether the vaccination raises immune responses against the coronavirus and its novel variants. Here we measured the dynamics of the antibody and T-cell responses, as well as virus neutralizing activity (VNA) in serum against two SARS-CoV-2 variants, B.1.1.1 and B.1.617.2, among 84 individuals with different COVID-19 status who were vaccinated with Sputnik Light vaccine. We showed that vaccination of individuals previously exposed to the virus considerably boosts the existing immune response. In these individuals, RBD-specific IgG titers and VNA in serum were already elevated on the 7th day after vaccination, while COVID-19–naïve individuals developed the antibody response and VNA mainly 21 days post–vaccination. Additionally, we found a strong correlation between RBD-specific IgG titers and VNA in serum, and according to these data vaccination may be recommended if the RBD-specific IgG titers drop to 142.7 BAU/mL or below. In summary, the results of the study demonstrate that vaccination is beneficial both for COVID-19–naïve and recovered individuals, especially since it raises serum VNA against the B.1.617.2 variant – one of four the SARS-CoV-2 variants of concern.

Background During the ongoing coronavirus disease 2019 (COVID-19) pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An important unanswered question is what levels of T-cell and antibody responses are sufficient to protect from the infection. Methods In 5340 Moscow residents, we evaluated anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin M (IgM)/immunoglobulin G (IgG) titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using interferon gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay. Additionally, we evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFN-γ and interleukin 2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T-cell responses, using the Kaplan–Meier estimator method, for up to 300 days postinclusion. Results We showed that T-cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, whereas the T-cell response by itself granted only intermediate protection. Conclusions We found that the contribution of the virus-specific antibodies to protection against SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized healthcare and public anti–COVID-19 policies. Clinical Trials Registration. NCT04898140.

Background Coronavirus disease COVID-19 has spread worldwide extremely rapidly. Although many individuals have been infected and have cleared the virus, developing virus-specific antibodies and effector/memory T cells, an important question still to be answered is what levels of T cell and antibody responses are sufficient to protect from the infection. Methods In 5,340 Moscow residents, we evaluated the anti-SARS-CoV-2 IgM/IgG titers and the frequencies of the T cells specific to the nucleocapsid, membrane, and spike proteins of SARS-CoV-2, using IFNγ ELISpot, and we also evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFNγ and IL2 followed by flow cytometry. Furthermore, we analyzed the post-inclusion COVID-19 rates as a function of the assessed antibody and T cell responses using the Kaplan-Meyer estimator method. Results We showed that T cell and antibody responses are closely interconnected and commonly are induced concurrently. Individuals positive for both antibody and T cell immunities demonstrated the highest levels of protectivity against the SARS-CoV-2 infection, indistinguishably from individuals with antibody response only. Meanwhile, individuals with T cell response only demonstrated slightly higher protectivity than individuals without both types of immunity, as measured from N-protein–specific or CD4+IL2+ T cells. However, these individuals were characterized by higher IgG titers than individuals without any immunity, although the titers were below the seropositivity cut-off. Conclusions The results of the study indicated the advantage of serology testing over the analysis of T cell responses for the prediction of SARS-CoV-2 infection rates on a populational level.

The National Medical Research Center for Endocrinology (NMRCE) received the right to implement the development program of the World-class Research Centre “The National Center for personalized medicine of endocrine diseases” (NMCPMED). The objective of the NMCPMED will be not only the creation of a system of personalized treatment, but also the training of new specialists for medicine. Fundamental researches, carried out on the basis of the already existing institutes and laboratories of the NMRCE will be expanded by creating new laboratories of the NCPMED created de novo in accordance with the approved project. This article introduces the reader to the most important laboratories that would be created in NCPMED. These are laboratories of general, molecular and population genetics, bioinformatics, pharmacogenomics, microbiota, genome editing, mathematical and digital technologies, non-invasive technologies for the diagnosis of endocrinopathies, cellular technologies, artificial intelligence and a fundamentally new laboratory of metabolic visualization and radioteranostics. The authors hope that readers of one of the main journals for endocrinologists in our country will actively participate in the implementation of NMRCE, as both young and experienced talented researchers will have a chance to be a part of the Centre. To realize the ambitious implementation plans for the achievements of the Centre, it is necessary to radically change the worldview of the doctors in our country, to train them in a new way, and to expand the structure of the Center’s team by increasing the number of specialists in medical genetics, transcriptomics, biostatistics and bioinformatics, working at the intersection of experimental and clinical endocrinology, and ensuring the transit of innovative technologies into clinical practice. New laboratories of the World-Class Research Center, will become the place of routine work of a new generation of doctors, who possess not only the basics of clinical work, but also the skills of fundamental researches that will allow them to significantly improve the methods of diagnosis and treatment.

Objective To analyze the clinical presentations of patients with endogenous Cushing’s syndrome (CS) affected by Coronavirus disease-19 (COVID-19). Materials and methods Patients who were referred to our clinic with active CS from 31st March to 15th May 2020 were screened for COVID-19 using real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Late-night serum cortisol (64–327 nmol/L), late-night salivary cortisol (LNSC) (0.5–9.4 nmol/L), or 24-h urinary free cortisol (24 hUFC) (100–379 nmol/24 h) were measured by electrochemiluminescence immunoassay. Results Among 22 patients with active CS we found three cases affected by COVID-19. Nonspecific inflammation markers were within the reference range or slightly elevated in these patients. A 71-year-old woman with newly diagnosed CS (late-night serum cortisol >1750 nmol/L, LNSC 908.6 nmol/L) developed dyspnea as an only symptom and died from bilateral polysegmantal hemorrhagic pneumonia 7 days later. A 38-year-old woman with a 5-year medical history of active Cushing’s disease (CD) (late-night serum cortisol 581.3 nmol/L, 24 hUFC 959.7 nmol/24-h) suffered from dyspnea, cough, fever (39.3 °C) and chest pain. Oxygen therapy, antibiotics and symptomatic treatments lead to full recovery 24 days later. A 66-year-old woman with a 4-year medical history of mild CD (late-night serum cortisol 603.4 nmol/L, LNSC 10.03 nmol/L) tested positive for COVID-19 in routine screening and remained asymptomatic. Conclusions The outcome of COVID-19 in patients with CS depends on the severity of hypercortisolism. Thus, severe hypercortisolism is a warning sign that CS affected by COVID-19 could require emergency care despite a lack of clinical presentations and low inflammation biomarkers.

We have developed a deep learning-based computer algorithm to recognize and predict retinal differentiation in stem cell-derived organoids based on bright-field imaging. The three-dimensional “organoid” approach for the differentiation of pluripotent stem cells (PSC) into retinal and other neural tissues has become a major in vitro strategy to recapitulate development. We decided to develop a universal, robust, and non-invasive method to assess retinal differentiation that would not require chemical probes or reporter gene expression. We hypothesized that basic-contrast bright-field (BF) images contain sufficient information on tissue specification, and it is possible to extract this data using convolutional neural networks (CNNs). Retina-specific Rx-green fluorescent protein mouse embryonic reporter stem cells have been used for all of the differentiation experiments in this work. The BF images of organoids have been taken on day 5 and fluorescent on day 9. To train the CNN, we utilized a transfer learning approach: ImageNet pre-trained ResNet50v2, VGG19, Xception, and DenseNet121 CNNs had been trained on labeled BF images of the organoids, divided into two categories (retina and non-retina), based on the fluorescent reporter gene expression. The best-performing classifier with ResNet50v2 architecture showed a receiver operating characteristic-area under the curve score of 0.91 on a test dataset. A comparison of the best-performing CNN with the human-based classifier showed that the CNN algorithm performs better than the expert in predicting organoid fate (84% vs. 67 ± 6% of correct predictions, respectively), confirming our original hypothesis. Overall, we have demonstrated that the computer algorithm can successfully recognize and predict retinal differentiation in organoids before the onset of reporter gene expression. This is the first demonstration of CNN’s ability to classify stem cell-derived tissue in vitro.

Avian influenza is one of the largest known threats to domestic poultry. Influenza outbreaks on poultry farms typically lead to the complete slaughter of the entire domestic bird population, causing severe economic losses worldwide. Moreover, there are highly pathogenic avian influenza (HPAI) strains that are able to infect the swine or human population in addition to their primary avian host and, as such, have the potential of being a global zoonotic and pandemic threat. Migratory birds, especially waterfowl, are a natural reservoir of the avian influenza virus; they carry and exchange different virus strains along their migration routes, leading to antigenic drift and antigenic shift, which results in the emergence of novel HPAI viruses. This requires monitoring over time and in different locations to allow for the upkeep of relevant knowledge on avian influenza virus evolution and the prevention of novel epizootic and epidemic outbreaks. In this review, we assess the role of migratory birds in the spread and introduction of influenza strains on a global level, based on recent data. Our analysis sheds light on the details of viral dissemination linked to avian migration, the viral exchange between migratory waterfowl and domestic poultry, virus ecology in general, and viral evolution as a process tightly linked to bird migration. We also provide insight into methods used to detect and quantify avian influenza in the wild. This review may be beneficial for the influenza research community and may pave the way to novel strategies of avian influenza and HPAI zoonosis outbreak monitoring and prevention.

MYB is a key regulator of definitive hematopoiesis that plays a critical role in the maintenance and multilineage differentiation of hematopoietic stem cells (HSCs). In vertebrate developmental models, MYB is thought to be dispensable for primitive hematopoiesis. To explore the role of MYB in human hematopoietic development, we have subjected human pluripotent stem cells (hPSCs) to mono- and bi-allelic gene targeting followed by hematopoietic differentiation in defined culture conditions. Here we show that MYB plays a central role in the development of human primitive blood cells. MYB expression was hematopoietic-specific and its induction coincided with emergence of the earliest primitive blood cells. Bi-allelic inactivation of MYB most severely affected the primitive erythroid progenitors of greater proliferative capacity and multilineage hematopoietic progenitors. The initial phase of the hematopoietic differentiation was not affected by the bi-allelic gene deficiency, but maturation of the primitive myeloid cells was found to be MYB-depended. Rescuing MYB expression in MYB-null cells shows that the gene is required for both development and proliferation of primitive clonogenic progenitors. In addition, our findings suggest that human primitive hematopoiesis emerge in several developmental cohorts.

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 and H7N9 viruses have a potential pandemic threat for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialylation. Tissue specific expression of the sialidases could protect domestic birds from infection.

Avian primordial germ cells (PGCs) have unique migration capacity towards the gonads via the bloodstream. Therefore, PGCs invitro genome modification is a dominant approach for poultry genetic modification. The aim of this study was to improve cultivation conditions of PGCs in terms of proliferation activity and further analyse their migration abilities. We isolated PGCs from whole blood cells collected from the embryonic dorsal aorta (Hamburger-Hamilton (HH) stage 14-17) and cultured them without feeder cells in customized avian knockout Dulbecco's modified Eagle's medium basal medium supplemented with fibroblast growth factor 2, Activin A and B-27 supplement containing insulin. We analysed the number of cells during the month of culture. Proliferation of PGCs increased in the first 7d of culture when both BMP4 and Activin A growth factors were added to the medium (four out of seven samples). Kinetic of stemness and PGC-marker genes (Nanog, PouV, DAZL) were similar to expression in mature gonadal PGCs (gPGCs). Expression levels of marker genes were significantly greater in the freshly isolated PGCs compared to the gPGCs and decreased during cultivation. In order to confirm the migration activity of the cultivated PGCs, cells were labelled with lentivirus (ZsGreen) and injected into the embryo blood stream (HH 14-17). Gonads from the recipient embryos were retrieved (HH 20-26) and analysed. Due to the low level of fluorescence in mature cells, the presence of transgenic PGC in the gonads was detected by ZsGreen-specific quantitative PCR. We confirmed that the cultured cells maintained migration activity, and efficient engraftment was detected in 75% of embryos.

Removal of regulatory T cells precipitates tissue inflammation and a systemic autoimmune lympho- and myeloproliferative syndrome, even in germ-free mice., Suppression mediated by regulatory T cells (T reg cells) represents a unique, cell-extrinsic mechanism of in-trans negative regulation that restrains multiple types of immune cells. The loss of T reg cells leads to fatal, highly aggressive, and widespread immune-mediated lesions. This severe autoimmunity may be driven by commensal microbiota, the largest source of non-self ligands activating the innate and adaptive immune systems. Alternatively, T reg cells may primarily restrain T cells with a diverse self–major histocompatibility complex (MHC)–restricted T cell receptor repertoire independently of commensal microbiota. In this study, we demonstrate that in germ-free (GF) mice, ablation of the otherwise fully functional T reg cells resulted in a systemic autoimmune lympho- and myeloproliferative syndrome and tissue inflammation comparable with those in T reg cell–ablated conventional mice. Importantly, there were two exceptions: in GF mice deprived of T reg cells, the inflammation in the small intestine was delayed, whereas exocrine pancreatitis was markedly accelerated compared with T reg cell–ablated conventional mice. These findings suggest that the main function of T reg cells is restraint of self-MHC–restricted T cell responsiveness, which, regardless of the presence of commensal microbiota, poses a threat of autoimmunity.

Human embryonic stem cells (hESCs) are a promising model for studying mechanisms of regulation of early development and differentiation. OCT4, NANOG, OCT4-related genes and some others were recently described to be important in pluripotency maintenance. Lesser is known about molecular mechanisms involved in their regulation. Apart from genetic regulation of gene expression epigenetic events, particularly methylation, play an important role in early development. Using RT-PCR we studied the expression of pluripotency-related genes OCT4, NANOG, DPPA3 and DPPA5 during hESCs differentiation to embryoid bodies. Analysis of methylation profiles of promoter or putative regulatory regions of the indicated genes demonstrated that expression of the pluripotency-maintaining genes correlated with their methylation status, whereas methylation of DPPA3 and DPPA5 varied between cell lines. We propose that DNA methylation underlies the developmental stage-specific mechanisms of pluripotency-related genes expression and reactivation and may have an impact on differentiation potential of hESC lines.

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 virus is getting increasingly infective for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild aquatic birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase (hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialilation to prevent infection. Tissue specific expression of the sialidases could protect domestic birds from infection.

Primordial germ cells (PGC) are the precursors of male and female progenitor cells. The cells are considered a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the PGC from chick donor embryos, transformation of the cells, and injection into the dorsal aorta of recipient embryos. After injection, the PGC are involved in the process of embryo development and differentiate into male or female sex cells. The aim of the research was to optimize the individual stages of this technology to increase the efficiency of transgenesis. The PGC were extracted from embryo gonads at stage 26 to 27 (H&H) using the trypsinization process. The trypsin concentration and incubation time were determined experimentally. Treatment of chick embryos with a 0.05% trypsin solution for 5 min was optimal for obtaining culture of embryonic cells. Separation of the PGC from other types of embryonic cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population for further cultivation was established by transfer (twice) of the supernatant containing unattached cells after 1 h of cultivation in a new culture dish. The cell population is represented mainly by the PGC (81 ± 4%). Additional purification of the PGC from other cell types using magnetic-activated cell sorting (MACS) increased the proportion of these cells up to 93 ± 2%. The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the PGC. The efficiency of infection of PGC with lentiviral particles (TU/mL = 2.5 × 108) was 70 ± 3%. The transformed cells were injected into the dorsal aorta of recipient embryos on Day 2.5 (n = 80). Before injecting donor PGC, recipient embryos were treated with busulfan to remove the endogenous PGC. The optimal dose of busulfan was selected experimentally. A series of experiments introducing busulfan in concentrations from 50 to 250 μg into chick embryos at 24 h of incubation showed that the optimal dose was 100 μg/embryo. The efficacy of colonization of gonads with donor PGC was assessed on Day-10 embryos (n = 32) and 4-week-old hatched chickens (n = 12). Cells from gonads were studied using fluorescence microscopy, fluorescence-activated cell sorting (FACS) and qPCR. The presence of fluorescent cells in the gonads of recipients was established in both embryos and hatched chickens. The relative number of the recombinant DNA copies and the relative level of expression were confirmed by qPCR. The FACS analysis of sex cells isolated from gonads of recipients showed that the percentage of transformed germ cells reached 55.8% in females (n = 5) and 31.9% in males (n = 7). Thus, the effectiveness of poultry transgenesis can be enhanced by preparation of donor PGC for injection into embryo recipients and elimination of endogenous PGC in recipients. Both the purification of PGC from other cell types based on adhesive capacity as well as treatment of embryo recipients at 24 h incubation with busulfan (100 μg/embryo) increased the effectiveness of transgenesis. Study supported by the RSF within project No. 16-16-10059.

It is known that avian influenza penetrates into the host cell by binding with sialic acids, the terminal residues of oligosaccharides. Avian influenza A virus preferably recognises α(2,3)-linked sialic acid residues as a receptor for penetration whereas human influenza A virus preferably binds with α(2,6)-linked sialic acids. Prevention of transfer of sialic acids to sugar bond or removal of it could be a defensive strategy against viral infection. There are 6 known sialyltransferases (ST3Gal1-6) that transfer α(2,3)-linked sialic acid residues to sugar branches. Most avian influenza virus isolates bind strongly to a sugar chain containing Neu5Aca(2,3) residues. In our study, we have shown that knockout of sialyltransferases leads to inhibition of viral infection. To find the expressed sialyltransferases in respiratory and digestive tracts, we used RT-qPCR. Tissue samples were taken from 3 chickens of Haisex white cross. Expression of mRNA was measured by RT-qPCR in 3 repeats and serial dilutions. Data analysis was carried out using the 2−ΔΔCt method. The amount of total RNA was normalised using GAPDH mRNA. For CRISPR/Cas9 targeting sialyltransferases, 3 guide RNAs for each gene were designed. We confirmed knockout (KO) of ST3GAL1 and ST3GAL6 by T7E assay. To estimate sialylation level on the cell surface, we performed a lectin-binding assay. For the assay, cells were incubated with fluorescein isothiocyanate (FITC)-labelled Maackia amurensis lectins and then subjected to flow-cytometry analysis to quantify the percentage of α(2,3)-sialylated cells in DF1 knockout (KO) v. DF1 wild type (wt) cell line. To estimate resistance to viral infection, a hemagglutinin binding assay was done, using fluorescein isothiocyanate (FITC)-labelled HA1 from H5N1 (A/Vietnam/1203/2004). To quantify the percentage of agglutinated HA1 molecules, DF1 KO and DF1 wt cells were analysed by flow cytometry. We found that mainly ST3GAL4 and ST3GAL5 are expressed in the chicken intestine (3-fold and 20-fold less compared with GAPDH level, respectively; other STs were not detected), and mainly ST3GAL1 and ST3GAL6 are expressed in the chicken respiratory tract (5-fold and 1.2-fold more compared with GAPDH level respectively; other STs were not detected). The expression profile of α(2,3)-sialyltransferases in the DF1 chicken cell line showed the noticeable expression of ST3GAL1 and ST3GAL6 compared with others as has been shown for the respiratory tract (500- and 1000-fold less compared with GAPDH respectively; other STs were not detected). In this study, we adopted the CRISPR/Cas9 system to knock out ST3GAL1 and ST3GAL6 genes in the chicken DF1 cell line. We confirmed that knockout of the genes leads to extinction of α(2,3)-sialic residues from the cell surface (7% v. 100% for DF1 KO v. DF1 wt cell line). Finally, we showed that knockout of sialyltransferases in the DF1 cells increases resistance against influenza A infection (16% v. 100% for DF1 KO v. DF1 wt cell line). Thus, creation of transgenic poultry with tissue-specific knockout of the α(2,3) sialyltransferases might protect domestic birds against influenza virus and block possible transfer of avian flu to human population.

Recently it has been demonstrated that CD30 expression was rather specific for transformed than for normal human ES cells and therefore CD30 maybe suggested as a potential marker for human ES cells bearing chromosomal abnormalities. Using immunohistochemistry and RT-PCR analysis we examined ÐiD30 expression in 10 hESCs lines with normal and abberant karyotypes. All hESC lines expressed CD30 antigen and RNA in undifferentiated state whether cell line beared chromosomal abnormalities or not. In contrast to previous notions our data demonstrate that CD30 could be considered as marker of undifferentiated hESCs without respect to karyotype changes.

Human embryonic stem cells (hESCs) are to be considered as a valuable source for regenerative medicine because of their capacity to differentiate into all cell types. We have developed an efficient culture system to differentiate hECSs into endothelial cells without the formation of embryoid bodies Establishing appropriate culture conditions with a cocktail of growth factors allowed us to differentiate hESCs directly to endothelial primary culture with about 50% efficiency. CD31 immunomagnetic cell sorting was used to purify derived endothelium from the primary culture of hESCs. Isolated endothelial cells expressed immunological markers (vWF, CD105), specific genes (VE-cadherin, KDR, GATA-2, GATA-3, eNOS), and formed cord-like structures on collagen matrix and in Matrigel assay. During differentiation to endothelial lineage promoter regions of the genes involved in specific cell fate determination and homeostasis (GATA-2,-3, and eNOS) underwent intensive hypomethylation which correlated with the gene expression. Overall our data demonstrate that direct differentiation of hESCs leads to endothelial cells that acquire epigenetic patterning similar to the functional endothelial cells of the organism.