SMAD2/3-SMYD2 and developmental transcription factors cooperate with cell cycle inhibitors to guide tissue formation.

Tissue formation and organ homeostasis is achieved by precise coordination of proliferation and differentiation of stem cells and progenitors. While deregulation of these processes can result in degenerative disease or cancer, their molecular interplays remain unclear. Here we show that the switch of human pluripotent stem cell (hPSC) self-renewal to differentiation is associated with the induction of distinct cyclin dependent kinase inhibitors (CDKIs). In hPSCs, Activin/Nodal/TGFß signalling maintains CDKIs in a poised state via SMAD2/3-NANOG-OCT4-EZH2-SNON transcriptional complex. Upon gradual differentiation, CDKIs are induced by successive transcriptional complexes between SMAD2/3-SMYD2 and developmental regulators such as EOMES, thereby lengthening the G1 phase. This, in turn, induces SMAD2/3 transcriptional activity by blocking its linker phosphorylation. Such SMAD2/3-CDKI positive feedback loops drive the exit from pluripotency and stepwise cell fate specification that could be harnessed for producing cells for therapeutic applications. Our study uncovers fundamental mechanisms how cell fate specification is interconnected to cell cycle dynamics and provides insight to autonomous circuitries governing tissue self-formation.

BRD9-SMAD2/3 Orchestrates Stemness and Tumorigenesis in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. The epigenetic mechanisms regulating CSCs are currently insufficiently understood, which hampers the development of novel strategies for eliminating CSCs. METHODS: By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodeling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signaling pathway. RESULTS: Inhibition and genetic ablation of BRD9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumors from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. CONCLUSIONS: Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

KMT2A associates with PHF5A-PHF14-HMG20A-RAI1 subcomplex in pancreatic cancer stem cells and epigenetically regulates their characteristics.

Pancreatic cancer (PC), one of the most aggressive and life-threatening human malignancies, is known for its resistance to cytotoxic therapies. This is increasingly ascribed to the subpopulation of undifferentiated cells, known as pancreatic cancer stem cells (PCSCs), which display greater evolutionary fitness than other tumor cells to evade the cytotoxic effects of chemotherapy. PCSCs are crucial for tumor relapse as they possess 'stem cell-like' features that are characterized by self-renewal and differentiation. However, the molecular mechanisms that maintain the unique characteristics of PCSCs are poorly understood. Here, we identify the histone methyltransferase KMT2A as a physical binding partner of an RNA polymerase-associated PHF5A-PHF14-HMG20A-RAI1 protein subcomplex and an epigenetic regulator of PCSC properties and functions. Targeting the protein subcomplex in PCSCs with a KMT2A-WDR5 inhibitor attenuates their self-renewal capacity, cell viability, and in vivo tumorigenicity.

PepFect14 mediates the delivery of mRNA into human primary keratinocytes and in vivo.

mRNA-based vaccines and candidate therapeutics have great potential in various medical fields. For the delivery of mRNA into target cells and tissues, lipid formulations are often employed. However, this approach could cause the activation of immune responses, making it unsuitable for the treatment of inflammatory conditions. Therefore, alternative delivery systems are highly demanded. In this study, we evaluated the transport efficiency and characteristics of cell-penetrating peptide PepFect14 (PF14) and mRNA nanoparticles in the presence of different additives. Our results show that all PF14-mRNA formulations entered cultured cells, while calcium chloride enhanced the transport and production of the encoded protein in HeLa and HaCaT cell lines, and polysorbate 80 did so in primary human keratinocytes. All formulations had similar physical properties and did not remarkably affect cell viability. By selectively blocking endocytosis pathways, we show that PF14-mRNA nanoparticles primarily entered HeLa cells via macropinocytosis and HaCaT cells via both macropinocytosis and clathrin-mediated endocytosis, while none of the blockers significantly affected the delivery into primary keratinocytes. Finally, subcutaneous injection of PF14-mRNA nanoparticles before inducing mouse irritant contact dermatitis resulted in the expression of a reporter protein without provoking harmful immune responses in the skin. Together, our findings suggest that PF14-mRNA nanoparticles have the potential for developing mRNA-based therapeutics for treating inflammatory skin conditions.

BRD9-SMAD2/3 orchestrates stemness and tumorigenesis in pancreatic ductal adenocarcinoma.

The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

Skin Colonization with S. aureus Can Lead to Increased NLRP1 Inflammasome Activation in Patients with Atopic Dermatitis.

The role of NLRP1 inflammasome activation and subsequent production of IL-1 family cytokines in the development of atopic dermatitis (AD) is not clearly understood. Staphylococcus aureus is known to be associated with increased mRNA levels of IL1 family cytokines in the skin and more severe AD. In this study, the altered expression of IL-1 family cytokines and inflammasome-related genes was confirmed, and a positive relationship between mRNA levels of inflammasome sensor NLRP1 and IL1B or IL18 was determined. Enhanced expression of the NLRP1 and PYCARD proteins and increased caspase-1 activity were detected in the skin of patients with AD. The genetic association of IL18R1 and IL18RAP with AD was confirmed, and the involvement of various immune cell types was predicted using published GWAS and expression quantitative trait loci datasets. In keratinocytes, the inoculation with S. aureus led to the increased secretion of IL-1ß and IL-18, whereas small interfering RNA silencing of NLRP1 inhibited the production of these cytokines. Our results suggest that skin colonization with S. aureus may cause the activation of the NLRP1 inflammasome in keratinocytes, which leads to the secretion of IL-1ß and IL-18 and thereby may contribute to the pathogenesis of AD, particularly in the presence of genetic variations in the IL-18 pathway.

Paradoxical attenuation of neuroinflammatory response upon LPS challenge in miR-146b deficient mice.

The miR-146 family consists of two microRNAs (miRNAs), miR-146a and miR-146b (miR-146a/b), both of which are known to suppress immune responses in a variety of conditions. Here, we studied how constitutive deficiency of miR-146b (Mir146b-/-) affects lipopolysaccharide (LPS)-induced neuroinflammation in mice. Our experiments demonstrated that miR-146b deficiency results in the attenuation of LPS-induced neuroinflammation, as it was evidenced by the reduction of sickness behavior, a decrease in the inflammatory status of microglia, and the loss of morphological signs of microglial activation in the hippocampus. Gene expression analysis revealed that LPS-induced upregulation of hippocampal pro-inflammatory cytokines is attenuated in Mir146b-/- mice, compared to wild-type (WT) mice. In addition, reduced expression of the NF-κB nuclear protein p65, reduced miR-146 family target TLR4 expression and relatively stronger upregulation of miR-146a was found in Mir146b-/- mice as compared to WT mice upon LPS challenge. Compensatory upregulation of miR-146a can explain the attenuation of the LPS-induced neuroinflammation. This was supported by experiments conducted with miR-146a/b deficient mice (Mir146a/b-/-), which demonstrated that additional deletion of the miR-146a led to the restoration of LPS-induced sickness behavior and proinflammatory cytokines. Our experiments also showed that the observed upregulation of miR-146a in Mir146b-/- mice is due to the overexpression of a miR-146a transcription inducer, interferon regulatory factor 7 (Irf7). Altogether, our results show the existence of crosstalk between miR-146a and mir-146b in the regulation of LPS-induced neuroinflammation.

Colon cancer cell differentiation by sodium butyrate modulates metabolic plasticity of Caco-2 cells via alteration of phosphotransfer network.

The ability of butyrate to promote differentiation of cancer cells has important implication for colorectal cancer (CRC) prevention and therapy. In this study, we examined the effect of sodium butyrate (NaBT) on the energy metabolism of colon adenocarcinoma Caco-2 cells coupled with their differentiation. NaBT increased the activity of alkaline phosphatase indicating differentiation of Caco-2 cells. Changes in the expression of pluripotency-associated markers OCT4, NANOG and SOX2 were characterized during the induced differentiation at mRNA level along with the measures that allowed distinguishing the expression of different transcript variants. The functional activity of mitochondria was studied by high-resolution respirometry. Glycolytic pathway and phosphotransfer network were analyzed using enzymatical assays. The treatment of Caco-2 cells with NaBT increased production of ATP by oxidative phosphorylation, enhanced mitochondrial spare respiratory capacity and caused rearrangement of the cellular phosphotransfer networks. The flexibility of phosphotransfer networks depended on the availability of glutamine, but not glucose in the cell growth medium. These changes were accompanied by suppressed cell proliferation and altered gene expression of the main pluripotency-associated transcription factors. This study supports the view that modulating cell metabolism through NaBT can be an effective strategy for treating CRC. Our data indicate a close relationship between the phosphotransfer performance and metabolic plasticity of CRC, which is associated with the cell differentiation state.

Impact of AHR Ligand TCDD on Human Embryonic Stem Cells and Early Differentiation.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, which mediates the effects of a variety of environmental stimuli in multiple tissues. Recent advances in AHR biology have underlined its importance in cells with high developmental potency, including pluripotent stem cells. Nonetheless, there is little data on AHR expression and its role during the initial stages of stem cell differentiation. The purpose of this study was to investigate the temporal pattern of AHR expression during directed differentiation of human embryonic stem cells (hESC) into neural progenitor, early mesoderm and definitive endoderm cells. Additionally, we investigated the effect of the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the gene expression profile in hESCs and differentiated cells by RNA-seq, accompanied by identification of AHR binding sites by ChIP-seq and epigenetic landscape analysis by ATAC-seq. We showed that AHR is differentially regulated in distinct lineages. We provided evidence that TCDD alters gene expression patterns in hESCs and during early differentiation. Additionally, we identified novel potential AHR target genes, which expand our understanding on the role of this protein in different cell types.

Utilising FGF2, IGF2 and FSH in serum-free protocol for long-term in vitro cultivation of primary human granulosa cells.

Human granulosa cells acquired as leftover from IVF treatment can be used to study infertility problems and are a valuable tool in the research of follicle maturation and ovulation. There is a need for more defined and long-term culture protocols for studying the response of granulosa cells upon treatment with selected hormones/chemicals. In the current study, we tested the effect of adding FGF2, IGF2 and FSH into defined basal medium in order to find culture conditions that would support proliferation of cumulus and mural granulosa cells along with the expression of common granulosa cell type markers such as FSHR, AMHR2, LHR and CYP19A1. We found that FGF2, IGF2 together with FSH helped to retain granulosa cell marker expression while supporting cell survival at least for two weeks of culture. The defined serum-free culture conditions for long-term culturing will be valuable in providing new standards in the research of human granulosa cells.

Target prediction and validation of microRNAs expressed from FSHR and aromatase genes in human ovarian granulosa cells.

MicroRNAs (miRNAs) are known post-transcriptional regulators of various biological processes including ovarian follicle development. We have previously identified miRNAs from human pre-ovulatory ovarian granulosa cells that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. The present study aims to identify the target genes regulated by these miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were transfected into KGN cell line and the gene expression changes were analyzed by Affymetrix microarray. Potential miRNA-regulated genes were further filtered by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. LIFR, PTEN, NEO1 and SP110 were confirmed as targets for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM19, PXDN and FMNL3 also passed all verification steps. Additionally, the expression pattern of the miRNAs was studied in human primary cumulus granulosa cell culture in relation to the expression of their host genes and FSH stimulation. Based on our findings we propose the involvement of hsa-miR-548ba in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions by regulating the expression of its identified targets.

MUC20 expression marks the receptive phase of the human endometrium.

RESEARCH QUESTION: How does mucin MUC20 expression change during the menstrual cycle in different cell types of human endometrium? DESIGN: Study involved examination of MUC20 expression in two previously published RNA-seq datasets in whole endometrial tissue (n = 10), sorted endometrial epithelial (n = 44) or stromal (n = 42) cell samples. RNA-Seq results were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in whole tissue (n = 10), sorted epithelial (n = 17) and stromal (n = 17) cell samples. MUC20 protein localization and expression were analysed in human endometrium by immunohistochemical analysis of intact endometrial tissue (n = 6) and also Western blot of cultured stromal and epithelial cells (n = 2). RESULTS: MUC20 is differentially expressed in the endometrium between the pre-receptive and receptive phases. We show that MUC20 is predominantly expressed by epithelial cells of the receptive endometrium, both at the mRNA (RNA-Seq, P = 0.005; qRT-PCR, P = 0.039) and protein levels (Western blot; immunohistochemistry, P = 0.029). CONCLUSION: Our results indicate MUC20 as a novel marker of mid-secretory endometrial biology. We propose a model of MUC20 function in the hepatocyte growth factor (HGF)-activated mesenchymal-epithelial transition (MET) receptor signalling specifically in the receptive phase. Further investigations should reveal the precise function of MUC20 in human endometrium and the possible connection between MUC20 and HGF-activated MET receptor signalling. MUC20 could potentially be included in the list of endometrial receptivity markers after further clinical validation.

Targeted gene silencing in human embryonic stem cells using cell-penetrating peptide PepFect 14.

BACKGROUND: Human embryonic stem (hES) cells serve as an invaluable tool for research and future medicine, but their transfection often leads to unwanted side effects as the method itself may induce differentiation. On the other hand, RNA interference (RNAi)-based targeted gene silencing is a quick, cost-effective, and easy-to-perform method to address questions regarding the function of genes, especially when hypomorphic knockdowns are needed. Therefore, effective transfection method with minimal side effects is essential for applying RNAi to hES cells. Here, we report a highly promising approach for targeted gene silencing in hES cells with siRNA complexed with cell-penetrating peptide PepFect 14 (PF14). This strategy provides researchers with efficient tool for unraveling the functions of genes or addressing the differentiation of pluripotent stem cells. METHODS: We present a method for delivery of siRNA into hES cells with cell-penetrating peptide PF14. Accordingly, hES cells were transfected in ROCK inhibitor containing medium for 24 h right after EDTA passaging as small cell clumps. Fluorescently labeled siRNA and siRNAs targeting OCT4 or beta-2-microglobulin (B2M) mRNA sequences were used to evaluate the efficiency of transfection and silencing. Analyses were performed at various time points by flow cytometry, RT-qPCR, and immunofluorescence microscopy. RESULTS: Effective downregulation of OCT4 in 70% of treated hES cells at protein level was achieved, along with 90% reduction at mRNA level in bulk population of cells. The applicability of this low-cost and easy-to-perform method was confirmed by inducing silencing of another target not associated with hES cell pluripotency (B2M). Furthermore, we discovered that downregulation of OCT4 induces neuroectodermal differentiation accompanied by reduced expression of B2M during early stage of this lineage. CONCLUSIONS: The results demonstrate PF14 as a promising tool for studying gene function and regulatory networks in hES cells by using RNAi.

2102Ep embryonal carcinoma cells have compromised respiration and shifted bioenergetic profile distinct from H9 human embryonic stem cells.

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells, it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system, aerobic glycolysis, and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells, while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass, increased proton leak, and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference, and highlight the importance of further research concerning energy metabolism of stem cells.

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, ß1, ß2 and γ1 chains, hESC express α2, α3, ß3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.

Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells.

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.

Effect of glucose content on thermally cross-linked fibrous gelatin scaffolds for tissue engineering.

Thermally cross-linked glucose-containing electrospun gelatin meshes were studied as possible cell substrate materials. FTIR analysis was used to study the effect of glucose on cross-linking reactions. It was found that the presence of glucose increases the extent of cross-linking of fibrous gelatin scaffolds, which in return determines scaffold properties and their usability in tissue engineering applications. Easy to handle fabric-like scaffolds were obtained from blends containing up to 15% glucose. Maximum extent of cross-linking was reached at nearly 20% glucose content. Cross-linking effectively resulted in decreased solubility and increased resistance to enzymatic degradation. Preliminary short-term cell culture experiments indicate that such thermally cross-linked gelatin-glucose scaffolds are suitable for tissue engineering applications.

SOX2 Is Regulated Differently from NANOG and OCT4 in Human Embryonic Stem Cells during Early Differentiation Initiated with Sodium Butyrate.

Transcription factors NANOG, OCT4, and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however, their expression profiles during early differentiation of hES cells are unclear. In this study, we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG, OCT4, and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells, characterized by specific expression patterns of NANOG, OCT4, and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time, indicating a gradual mode of developmental transition in these cells. Notably, distinct regulation of SOX2 during early differentiation events was detected, highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation.

Fibroblast growth on micro- and nanopatterned surfaces prepared by a novel sol-gel phase separation method.

Physical characteristics of the growth substrate including nano- and microstructure play crucial role in determining the behaviour of the cells in a given biological context. To test the effect of varying the supporting surface structure on cell growth we applied a novel sol-gel phase separation-based method to prepare micro- and nanopatterned surfaces with round surface structure features. Variation in the size of structural elements was achieved by solvent variation and adjustment of sol concentration. Growth characteristics and morphology of primary human dermal fibroblasts were found to be significantly modulated by the microstructure of the substrate. The increase in the size of the structural elements, lead to increased inhibition of cell growth, altered morphology (increased cytoplasmic volume), enlarged cell shape, decrease in the number of filopodia) and enhancement of cell senescence. These effects are likely mediated by the decreased contact between the cell membrane and the growth substrate. However, in the case of large surface structural elements other factors like changes in the 3D topology of the cell's cytoplasm might also play a role.

Nocodazole treatment decreases expression of pluripotency markers Nanog and Oct4 in human embryonic stem cells.

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4.