The cytoskeleton plays a crucial role in regulating cellular behavior, acting as both a structural framework and a mediator of mechanical and biochemical signals that influence cell fate. In the context of cellular reprogramming, modifications to the cytoskeleton can have profound effects on lineage commitment and differentiation efficiency. This review explores the impact of mechanical forces such as substrate stiffness, topography, extracellular fluid viscosity, and cell seeding density on cytoskeletal organization and mechanotransduction pathways, including Rho/ROCK and YAP/TAZ signaling. Additionally, we examine the influence of biochemical agents that modulate cytoskeletal dynamics, such as actin and microtubule polymerization inhibitors, and their effects on stem cell differentiation. By understanding how cytoskeletal remodeling governs cellular identity, this review highlights potential strategies for improving reprogramming efficiency and directing cell fate by manipulating mechanical and biochemical cues.

Objective: Histone modifications play a crucial role in shaping the epigenetic landscape of chromatin, influencing its structure and function. These histone modifications change throughout the cell cycle and during processes such as morphogenesis, differentiation, stress adaptation, and aging. Among these, histone H3 Lys9 trimethylation (H3K9me3) plays a critical role in gene silencing and cellular identity. Despite the importance of histone modifications in regulating gene expression, the dynamic visualization of these modifications in live cells remains a significant challenge. Here, we aim to develop a method to track H3K9me3 changes of induced pluripotent stem cells (iPSCs) during spontaneous differentiation into embryoid bodies (EBs) with the genetically encoded fluorescent sensor. Methods: We created a stable iPSC line, KUIFMSi004-A-1, expressing the MPP8-Green fluorescent sensor, which binds H3K9me3. This sensor combines two copies of the natural M-phase phosphoprotein 8 (MPP8) reader domain with the green fluorescent protein mNeonGreen. The iPSC line was induced to form EBs, and the distribution of H3K9me3 was monitored using live-cell fluorescence microscopy. Results and Discussion: The expression of the MPP8-Green sensor allowed real-time visualization of H3K9me3 modifications during spontaneous differentiation of iPSCs into EBs. We observed two distinct groups of cells with different patterns of H3K9me3 distribution: one with characteristic chromatin “dots” and another with diffuse sensor distribution. The sensor enabled tracking of epigenetic landscape changes during differentiation, providing insights into the dynamics of H3K9me3 during early embryoid body formation. Conclusions: Our study demonstrates the potential of using the MPP8-Green sensor to monitor the dynamic changes of H3K9me3 during iPSC differentiation. This method offers a novel approach for studying the temporal and spatial regulation of histone modifications in live cells, advancing our understanding of epigenetic regulation during development.

Background Large genetic effects from rare coding mutations (Singh et al. 2022; Liu et al. 2023) and polygenic burden from common variants (Trubetskoy et al. 2022) both contribute to the genetic liability of schizophrenia (SZ). However, the exact parameters of their interaction are currently under active investigation. For schizophrenia and other traits, it has been demonstrated that one might expect that in an individual with low polygenic risk, the chance of finding a new genetic factor with a large effect is higher (Bergen et al. 2019; Antaki et al. 2022). Here, we describe a case of an SZ patient with an extremely low SZ polygenic risk. We utilised expression data from his cortex samples to investigate whether some unique mutations in the patient's genome could be linked to gene expression features in his brain. Methods Polygenic risk scores (PRS) were calculated using data obtained from the Illumina GSA chip (v3) and imputed against the HRC 1.1 reference panel. SZ-PRS (PGC3) were defined with LDpred2 (auto). As a comparison sample, we employed an in-house sample predominantly consisting of individuals of Slavic descent from the Moscow region. Chromosomal abnormalities were checked with the SurePrint G3 Human CGH Microarray. Exome sequencing was performed with 2 × 150 PE reads, x70 coverage, and its results were analysed with the VEP annotation tool. Post-mortem human brain samples were obtained with informed consent from donors or their next of kin. Brain tissues were dissected to locate the Brodmann areas BA9, BA22p, and BA4. Using RNA isolated from the samples, we constructed cDNA libraries and sequenced them (2 × 150 PE, 20M reads per sample). The reads were aligned to the human genome with STAR and quantified with Salmon. Cell type fractions were inferred using a containerised version of CIBERSORTx with a detailed cortex cell composition reference from Siletti et al., 2023 data. The analysis for differentially expressed genes was performed with DESeq2, and further affected pathways were identified with GSEA. Results During the routine genotyping of the Skolkovo SZ brain collection, we found an individual with an SZ-PRS polygenic score well below the first percentile distribution for ancestry-matched SZ patients from the MHRC genetic data. The patient was diagnosed with a paranoid SZ subtype with a typical clinical presentation. We checked for large chromosomal anomalies in the patient's genome with aCGH, and none were found. Using exome sequencing, we isolated a set of potentially damaging mutations, notably in the LIMK1, LSR, MCTP1, NAPA, RAPGEF3, TAF15, and UNC13C genes. We obtained transcriptomics data for a set of samples from cortex tissues (total n = 33, including 11 for the patient). We used a digital cytometry approach to compensate for cell composition imbalances in the transcriptomes. Using public gene expression data, we assessed whether the found mutations have an impact on gene expression in the brain of the patient. Discussion Accounting for polygenic scores in the WES analysis promises to identify prospective rare disease-related genetic variants. Here, we use this idea in an attempt to investigate whether individual genetic factors are driving the disease of the schizophrenia patient with low SZ-PRS. The main limitation of the study is that it is a case report and transcriptomic analysis from an individual cannot be free from confounders so all results should be considered preliminary. The study was supported by RSF (grant 21-15-00124) and MoSHE (grant 075-15-2019-1789, cDNA libraries).

Introduction The main finding of a large-scale collaborative study (Rees et al. Nat Neurosci 2020;23(2) 179-184), which focused on de novo mutations in schizophrenia, was the discovery of an enrichment of these mutations in the SLC6A1 gene. This gene encodes the gamma-aminobutyric acid (GABA) transporter GAT1, thereby encouraging further research into novel schizophrenia targets within the GABA pathway. However, the gene was not highlighted in recent schizophrenia genetic studies, while typically pathogenic SLC6A1 mutations result in epilepsy, motor dysfunction, autistic spectrum disorder (ASD) and developmental delay. The absence of genetic replication for SLC6A1’s involvement in schizophrenia and the differing clinical spectrum for SLC6A1 mutations led us to study in depth one of the only three original probands from the Rees et al. 2020 study. Objectives In our comprehensive case study, we delved deep into the relationship between the SLC6A1 mutation and schizophrenia. Methods Our subject, a patient who first presented with acute mania symptoms at age 15 and was later diagnosed with schizophrenia, carried the SLC6A1 Arg211Cys mutation. Over a detailed 25-year follow-up, we conducted an array of assessments and tests, including cognitive testing, personality assessments, EEG, and 1H-MRS. Results Notably, we discovered abnormal GABA levels, potentially indicating a dysfunction in GABA reuptake, adding a new layer of complexity to our understanding. Further analysis revealed a significant correlation between the patient’s clinical picture and a polygenic background, rather than the SLC6A1 mutation. Despite having a high polygenic risk score for bipolar disorder, the dominant features of his condition were more representative of schizophrenia. Interestingly, neither the patient nor his father, who also showed a higher BP PRS, had a diagnosis of bipolar disorder. The pathogenic significance of the mutation warrants investigation in cells of neuronal origin. We generated induced pluripotent stem cells (iPSC) from the patient and his parents. This approach provides us with a platform for future investigations into the pathogenic significance of the mutation in neuronal cells. The Human Pluripotent Stem Cell Registry accession numbers of those cells are MHRCCGi001-A (patient), MHRCCGi005-A (mother) and MHRCCGi004-A (father). Conclusions In the presented case the clinical picture is rather explained by the polygenic background than by the SLC6A1 Arg211Cys mutation. The study is supported by Russian Science Foundation, grant 21-15-00124 (https://rscf.ru/project/21-15-00124) Disclosure of Interest None Declared

BACKGROUND:Mesenchymal stromal cells (MSCs) are recognized for their potential in regenerative medicine, attributed to their multipotent differentiation capabilities and immunomodulatory properties. Despite this potential, the classification and detailed characterization of MSCs, especially those derived from specific tissues like the pancreas, remains challenging leading to a proliferation of terminology in the literature. This study aims to address these challenges by providing a thorough characterization of human pancreatic islets-derived mesenchymal stromal cells (hPD-MSCs). METHODS:hPD-MSCs were isolated from donor islets using enzymatic digestion, immortalized through lentiviral transduction of human telomerase reverse transcriptase (hTERT). Cells were characterized by immunostaining, flow cytometry and multilineage differentiation potential into adipogenic and osteogenic lineages. Further a transcriptomic analysis was done to compare the gene expression profiles of hPD-MSCs with other mesenchymal cells. RESULTS:We show that hPD-MSCs express the classical MSC features, including morphological characteristics, surface markers expression (CD90, CD73, CD105, CD44, and CD106) and the ability to differentiate into both adipogenic and osteogenic lineages. Furthermore, transcriptomic analysis revealed distinct gene expression profiles, showing notable similarities between hPD-MSCs and pancreatic stellate cells (PSCs). The study also identified specific genes that distinguish hPD-MSCs from MSCs of other origins, including genes associated with pancreatic function (e.g., ISL1) and neural development (e.g., NPTX1, ZNF804A). A novel gene with an unknown function (ENSG00000286190) was also discovered. CONCLUSIONS:This study enhances the understanding of hPD-MSCs, demonstrating their unique characteristics and potential applications in therapeutic strategies. The identification of specific gene expression profiles differentiates hPD-MSCs from other mesenchymal cells and opens new avenues for research into their role in pancreatic function and neural development.

Direct pro-neural reprogramming is a conversion of differentiated somatic cells to neural cells without an intermediate pluripotency stage. It is usually achieved via ectopic expression (EE) of certain transcription factors (TFs) or other reprogramming factors (RFs). Determining the transcriptional changes (TCs) caused by particular RFs in a given cell line enables an informed approach to reprogramming initiation. Here, we characterized TCs in the human fibroblast cell line LF1 on the 5th day after EE of the single well-known pro-neural RFs NGN2, ASCL1, SOX2, and MSI1. As assessed by expression analysis of the bona fide neuronal markers nestin and beta-III tubulin, all four RFs initiated pro-neuronal phenotype conversion; analysis by RNA-seq revealed striking differences in the resulting TCs, although some pathways were overlapping. ASCL1 and SOX2 were not sufficient to induce significant pro-neural phenotype switches using our EE system. NGN2 induced TCs indicative of cell phenotype changes towards neural crest cells, neural stem cells, mature neurons, as well as radial glia, astrocytes, and oligodendrocyte precursors and their mature forms. MSI1 mainly induced a switch towards early stem-like cells, such as radial glia.

Gene transfection is a fundamental technique in the fields of biological research and therapeutic innovation. Due to their biocompatibility and membrane-mimetic properties, lipid vectors serve as essential tools in transfection. The successful delivery of genetic material into the cytoplasm is contingent upon the fusion of the vector and cellular membranes, which enables hydrophilic polynucleic acids to traverse the hydrophobic barriers of two intervening membranes. This review examines the critical role of membrane fusion in lipofection efficiency, with a particular focus on the molecular mechanisms that govern lipoplex-membrane interactions. This analysis will examine the key challenges inherent to the fusion process, from achieving initial membrane proximity to facilitating final content release through membrane remodeling. In contrast to viral vectors, which utilize specialized fusion proteins, lipid vectors necessitate a strategic formulation and environmental optimization to enhance their fusogenicity. This review discusses recent advances in vector design and fusion-promoting strategies, emphasizing their potential to improve gene delivery yield. It highlights the importance of understanding lipoplex-membrane fusion mechanisms for developing next-generation delivery systems and emphasizes the need for continued fundamental research to advance lipid-mediated transfection technology.

The rete testis (RT) is a region of the mammalian testis that plays an important role in testicular physiology. The RT epithelium consists of cells sharing some well-known gene markers with supporting Sertoli cells (SCs). However, little is known about the differences in gene expression between these two cell populations. Here, we used fluorescence-activated cell sorting (FACS) to obtain pure cultures of neonatal RT cells and SCs and identified differentially expressed genes (DEGs) between these cell types using RNA sequencing (RNA-seq). We then compared our data with the RNA-seq data of other studies that examined RT cells and SCs of mice of different ages and generated a list of DEGs permanently upregulated in RT cells throughout testis development and in culture, which included 86 genes, and a list of 79 DEGs permanently upregulated in SCs. The analysis of studies on DMRT1 function revealed that nearly half of the permanent DEGs could be regulated by this SC upregulated transcription factor. We suggest that useful cell lineage markers and candidate genes for the specification of both RT cells and SCs may be present among these permanent DEGs.

Introduction:Culturing of human neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSC) is a promising area of research, as these cells have the potential to treat a wide range of neurological, neurodegenerative and psychiatric diseases. However, the development of optimal protocols for the production and long-term culturing of NSCs remains a challenge. One of the most important aspects of this problem is to determine the stability of NSCs during long-term in vitro passaging. To address this problem, our study was aimed at investigating the spontaneous differentiation profile in different iPSC-derived human NSCs cultures during long-term cultivation using.Methods:Four different IPSC lines were used to generate NSC and spontaneously differentiated neural cultures using DUAL SMAD inhibition. These cells were analyzed at different passages using immunocytochemistry, qPCR, bulk transcriptomes and scRNA-seq.Results:We found that various NSC lines generate significantly different spectrums of differentiated neural cells, which can also change significantly during long-term cultivation in vitro.Discussion:Our results indicate that both internal (genetic and epigenetic) and external (conditions and duration of cultivation) factors influence the stability of NSCs. These results have important implications for the development of optimal NSCs culturing protocols and highlight the need to further investigate the factors influencing the stability of these cells in vitro.

Human immunodeficiency virus type 1 (HIV-1) remains a significant challenge for global public health as limited therapeutic options are available for HIV-infected individuals receiving combination antiretroviral therapy. Additionally, individuals with HIV-1/acquired immunodeficiency syndrome (AIDS) complications have a reduced life expectancy. In recent decades, gene and cell-based strategies have shown promise in achieving a functional cure for HIV-1 infection. The outcomes of therapies with patients in Berlin and London have led to moderate optimism for a highly effective HIV-1 treatment. This review categorizes current strategies for HIV-1 treatment into RNA- and antibody-based therapies, cell and genome editing approaches, and methods for eradicating latent reservoirs. These findings demonstrate how the use of various anti-HIV-1 agents enhances our understanding of HIV-1 infection and may provide important insights for potential HIV-1 treatment.

Clear cell renal cell carcinoma (ccRCC) accounts for 80–90% of kidney cancers worldwide. Small C-terminal domain phosphatases CTDSP1, CTDSP2, and CTDSPL (also known as SCP1, 2, 3) are involved in the regulation of several important pathways associated with carcinogenesis. In various cancer types, these phosphatases may demonstrate either antitumor or oncogenic activity. Tumor-suppressive activity of these phosphatases in kidney cancer has been shown previously, but in general case, the antitumor activity may be dependent on the choice of cell line. In the present work, transfection of the Caki-1 cell line (ccRCC morphologic phenotype) with expression constructs containing the coding regions of these genes resulted in inhibition of cell growth in vitro in the case of CTDSP1 (p < 0.001) and CTDSPL (p < 0.05) but not CTDSP2. The analysis of The Cancer Genome Atlas (TCGA) data showed differential expression of some of CTDSP genes and of their target, RB1. These results were confirmed by quantitative RT-PCR using an independent sample of primary ccRCC tumors (n = 52). We observed CTDSPL downregulation and found a positive correlation of expression for two gene pairs: CTDSP1 and CTDSP2 (rs = 0.76; p < 0.001) and CTDSPL and RB1 (rs = 0.38; p < 0.05). Survival analysis based on TCGA data demonstrated a strong association of lower expression of CTDSP1, CTDSP2, CTDSPL, and RB1 with poor survival of ccRCC patients (p < 0.001). In addition, according to TCGA, CTDSP1, CTDSP2, and RB1 were differently expressed in two subtypes of ccRCC—ccA and ccB, characterized by different survival rates. These results confirm that CTDSP1 and CTDSPL have tumor suppressor properties in ccRCC and reflect their association with the more aggressive ccRCC phenotype.

Trisomy is the presence of one extra copy of an entire chromosome or its part in a cell nucleus. In humans, autosomal trisomies are associated with severe developmental abnormalities leading to embryonic lethality, miscarriage or pronounced deviations of various organs and systems at birth. Trisomies are characterized by alterations in gene expression level, not exclusively on the trisomic chromosome, but throughout the genome. Here, we applied the high-throughput chromosome conformation capture technique (Hi-C) to study chromatin 3D structure in human chorion cells carrying either additional chromosome 13 (Patau syndrome) or chromosome 16 and in cultured fibroblasts with extra chromosome 18 (Edwards syndrome). The presence of extra chromosomes results in systematic changes of contact frequencies between small and large chromosomes. Analyzing the behavior of individual chromosomes, we found that a limited number of chromosomes change their contact patterns stochastically in trisomic cells and that it could be associated with lamina-associated domains (LAD) and gene content. For trisomy 13 and 18, but not for trisomy 16, the proportion of compacted loci on a chromosome is correlated with LAD content. We also found that regions of the genome that become more compact in trisomic cells are enriched in housekeeping genes, indicating a possible decrease in chromatin accessibility and transcription level of these genes. These results provide a framework for understanding the mechanisms of pan-genome transcription dysregulation in trisomies in the context of chromatin spatial organization.

Human immunodeficiency virus type 1 (HIV-1) remains a significant challenge for global public health as limited therapeutic options are available for HIV-infected individuals receiving combination antiretroviral therapy. Additionally, individuals with HIV-1/acquired immunodeficiency syndrome (AIDS) complications have a reduced life expectancy. In recent decades, gene and cell-based strategies have shown promise in achieving a functional cure for HIV-1 infection. The outcomes of therapies with patients in Berlin and London have led to moderate optimism for a highly effective HIV-1 treatment. This review categorizes current strategies for HIV-1 treatment into RNA- and antibody-based therapies, cell and genome editing approaches, and methods for eradicating latent reservoirs. These findings demonstrate how the use of various anti-HIV-1 agents enhances our understanding of HIV-1 infection and may provide important insights for potential HIV-1 treatment.

Introduction Schizophrenia is a severe mental disorder mainly caused by genetic risk factors. Many studies have demonstrated that both multiple genetic variants and rare mutations are associated with schizophrenia risk. The next step is to study the causal effect of the gene on the phenotype. Recently, a large family-based study identified de novo mutations, which may increase liability to schizophrenia (Rees et al 2020). In particular, a mutation in the GABA transporter (SLC6A1) gene (rs756927822 C/T) was identified in one patient from our subsample. Objectives Here, we present a case report of this patient and describe the procedure of derivation of induced pluripotent stem cells (iPSCs) from fibroblast cultures. Methods Clinical, psychometric and neuropsychological methods were used. iPSCs were derived from patients’ and both unaffected parents’ fibroblasts. Human fibroblasts, cultured in fibroblast medium, are infected with lentivirus vectors expressing the transcription factors Oct4, Sox2, c-Myc, and KLF4. All iPSCs were immunocytochemical stained for intracellular (Oct4, Sox2) and extracellular (SSEA4, Tra-1-81, Tra-1-60) pluripotency markers. An qPCR analysis for pluripotency markers (TDGF1, Sox2, Oct4, REX1, LIN28, NANOG, KLF4, GDF3, DPPA4, DNMT3) was performed. All four iPSC lines formed embryoid bodies before the differentiating into three germ layers. Differentiation was confirmed by immunostaining for mesoderm (aSMA), ectoderm (Nestin, Desmin) andendoderm (FoxA2, Pax6) markers. Results A 47-year-old male patient was presented to psychiatry at the age of 16. There was no personal or family history of psychiatric disorder, the premorbid functioning was normal, the patient had no somatic diseases, showed high performance in sport (mountain skiing). On his first admission, he was diagnosed with schizoaffective psychosis. The patient showed signs of mania and catatonia. Neuropsychological testing revealed a decrease of cognitive functioning (short-term and associative memory). The patient was followed up for more than 20 years. The diagnosis was changed for schizophrenia at the age of 43 years. There was a deterioration in cognitive function (the apparent decrease in performance on neurocognitive tests (attention, memory, executive functions) from the first examination (1997) till last one (2019). The patient refused or was not able to perform most of the tasks. During follow-up, the patient shows good adherence to treatment. Conclusions For this patient, obtained lines might be valuable for investigating the disease mechanisms and screening candidate drugs. Disclosure of Interest None Declared

Sertoli cells are key somatic cells in the testis that form seminiferous tubules and support spermatogenesis. The isolation of pure Sertoli cells is important for their study. However, it is a difficult effort because of the close association of Sertoli cells with peritubular myoid cells surrounding seminiferous tubules. Here, we propose a novel approach to the establishment of a pure Sertoli cell culture from immature mouse testes. It is based on the staining of testicular cells for platelet-derived growth factor receptor alpha (PDGFRA), followed by fluorescence-activated cell sorting and culturing of a PDGFRA-negative cell population. Cells positive for a Sertoli cell marker WT1 accounted for more than 96% of cells in cultures from 6 to 12 days postpartum (dpp) mice. The numbers of peritubular myoid cells identified by ACTA2 staining did not exceed 4%. Cells in the cultures were also positive for Sertoli cell proteins SOX9 and DMRT1. Amh and Hsd17b3 expression decreased and Ar and Gata1 expression increased in 12 dpp cultures compared to 6 dpp cultures, which suggests that cultured Sertoli cells at least partially retained their differentiation status. This method can be employed in various applications including the analysis of differential gene expression and functional studies.

Diabetes has been a worldwide healthcare problem for many years. Current methods of treating diabetes are still largely directed at symptoms, aiming to control the manifestations of the pathology. This creates an overall need to find alternative measures that can impact on the causes of the disease, reverse diabetes, or make it more manageable. Understanding the role of key players in the pathogenesis of diabetes and the related β-cell functions is of great importance in combating diabetes. PDX1 is a master regulator in pancreas organogenesis, the maturation and identity preservation of β-cells, and of their role in normal insulin function. Mutations in the PDX1 gene are correlated with many pancreatic dysfunctions, including pancreatic agenesis (homozygous mutation) and MODY4 (heterozygous mutation), while in other types of diabetes, PDX1 expression is reduced. Therefore, alternative approaches to treat diabetes largely depend on knowledge of PDX1 regulation, its interaction with other transcription factors, and its role in obtaining β-cells through differentiation and transdifferentiation protocols. In this article, we review the basic functions of PDX1 and its regulation by genetic and epigenetic factors. Lastly, we summarize different variations of the differentiation protocols used to obtain β-cells from alternative cell sources, using PDX1 alone or in combination with various transcription factors and modified culture conditions. This review shows the unique position of PDX1 as a potential target in the genetic and cellular treatment of diabetes.

Transactivation systems are a promising application based on the CRISPR/Cas9 system and allow targeted control of gene expression levels in cell culture. However, their performance has been reported to vary considerably depending on the cell type and the activator system. Three activator systems (dCas9-VP160, dCas9-SunTag, and dCas9-VPR) were compared for the efficiency of activating expression of OCT4, NANOG, PDX1, FOXA2, NKX2-2, and NKX6-1 in an immortalized human skin fibroblast line. The activation efficiency was found to depend on the activation system type; the extent of activation depended on the system run time.

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA-DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA-chromatin interactions. Here, we introduce RedChIP, a technique combining RNA-DNA proximity ligation and chromatin immunoprecipitation for identifying RNA-chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA-DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.