Oct4-mediated reprogramming has recently become a novel tool for the generation of various cell types from differentiated somatic cells. Although molecular mechanisms underlying this process are unknown, it is well documented that cells over-expressing Oct4 undergo transition from differentiated state into plastic state. This transition is associated with the acquisition of stem cells properties leading to epigenetically "open" state that is permissive to cell fate switch upon external stimuli. In order to contribute to our understanding of molecular mechanisms driving this process, we characterised human fibroblasts over-expressing Oct4 and performed comprehensive small-RNAseq analysis. Our analyses revealed new interesting aspects of Oct4-mediated cell plasticity induction. Cells over-expressing Oct4 lose their cell identity demonstrated by down-regulation of fibroblast-specific genes and up-regulation of epithelial genes. Interestingly, this process is associated with microRNA expression profile that is similar to microRNA profiles typically found in pluripotent stem cells. We also provide extensive network of microRNA families and clusters allowing us to precisely determine the miRNAome associated with the acquisition of Oct4-induced transient plastic state. Our data expands current knowledge of microRNA and their implications in cell fate alterations and contributing to understanding molecular mechanisms underlying it.

• MeSH
• Cell Line MeSH
• Embryo, Mammalian * MeSH
• Fibroblasts cytology   metabolism   MeSH
• Humans MeSH
• MicroRNAs * biosynthesis   genetics   MeSH
• Octamer Transcription Factor-3 * biosynthesis   genetics   MeSH
• Gene Expression Regulation * MeSH
• Cellular Reprogramming Techniques * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Aim: Human induced pluripotent stem cells (iPSCs) are inefficiently derived from somatic cells by overexpression of defined transcription factors. Overexpression of H2A histone variant macroH2A1.1, but not macroH2A1.2, leads to increased iPSC reprogramming by unclear mechanisms. Materials & methods: Cleavage under targets and tagmentation (CUT&Tag) allows robust epigenomic profiling of a low cell number. We performed an integrative CUT&Tag-RNA-Seq analysis of macroH2A1-dependent orchestration of iPSCs reprogramming using human endothelial cells. Results: We demonstrate wider genome occupancy, predicted transcription factors binding, and gene expression regulated by macroH2A1.1 during reprogramming, compared to macroH2A1.2. MacroH2A1.1, previously associated with neurodegenerative pathologies, specifically activated ectoderm/neural processes. Conclusion: CUT&Tag and RNA-Seq data integration is a powerful tool to investigate the epigenetic mechanisms occurring during cell reprogramming.

• MeSH
• Endothelial Cells metabolism   MeSH
• Histones * metabolism   MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Humans MeSH
• Cellular Reprogramming genetics   MeSH
• RNA-Seq MeSH
• Transcription Factors genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.

• MeSH
• Biomarkers MeSH
• Cell Differentiation genetics   MeSH
• Embryoid Bodies cytology   MeSH
• Fibroblasts cytology   metabolism   MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   MeSH
• Karyotype MeSH
• Stem Cells cytology   metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Natal Teeth cytology   MeSH
• Cellular Reprogramming * MeSH
• Transcriptome MeSH
• Gene Expression Regulation, Developmental MeSH
• Dental Pulp cytology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Reprogramming of non-endocrine pancreatic cells into insulin-producing cells represents a promising therapeutic approach for the restoration of endogenous insulin production in diabetic patients. In this paper, we report that human organoid cells derived from the pancreatic tissue can be reprogrammed into the insulin-producing cells (IPCs) by the combination of in vitro transcribed modified mRNA encoding transcription factor neurogenin 3 and small molecules modulating the epigenetic state and signalling pathways. Upon the reprogramming, IPCs formed 4.6 ± 1.2 % of the total cells and expressed typical markers (insulin, glucokinase, ABCC8, KCNJ11, SLC2A2, SLC30A8) and transcription factors (PDX1, NEUROD1, MAFA, NKX2.2, NKX6.1, PAX4, PAX6) needed for the proper function of pancreatic β-cells. Additionally, we have revealed a positive effect of ALK5 inhibitor RepSox on the overall reprogramming efficiency. However, the reprogrammed IPCs possessed only a partial insulin-secretory capacity, as they were not able to respond to the changes in the extracellular glucose concentration by increasing insulin secretion. Based on the achieved results we conclude that due to the incomplete reprogramming, the IPCs have immature character and only partial properties of native human β-cells.

• MeSH
• AC133 Antigen metabolism   MeSH
• Insulin-Secreting Cells cytology   drug effects   MeSH
• Adult MeSH
• Transcription, Genetic drug effects   MeSH
• Insulin biosynthesis   MeSH
• Small Molecule Libraries pharmacology   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Organoids cytology   MeSH
• Cellular Reprogramming drug effects   genetics   MeSH
• Cell Proliferation MeSH
• Nerve Tissue Proteins genetics   metabolism   MeSH
• Basic Helix-Loop-Helix Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

The possibility of replacing the originally discovered and widely used DNA reprogramming transcription factors is stimulating enormous effort to identify more effective compounds that would not alter the genetic information. Here, we describe the generation of induced pluripotent stem cells (iPSc) from head-derived primary culture of mouse embryonic cells using small chemical inhibitors of the MEK and TGF-beta pathways without delivery of exogenous transcription factors. These iPSc express standard pluripotency markers and retain their potential to differentiate into cells of all germ layers. Our data indicate that head-derived embryonic neural cells might have the reprogramming potential while neither the same primary cells cultivated over five passages in vitro nor a cell population derived from adult brain possesses this capacity. Our results reveal the potential for small molecules to functionally replace routinely used transcription factors and lift the veil on molecular regulation controlling pluripotency. The conditions described here could provide a platform upon which other genome non integrative and safer reprogramming processes could be developed. This work also shows novel potential for developing embryonic neural cells.

• MeSH
• Antigens, Differentiation biosynthesis   MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   MeSH
• MAP Kinase Signaling System * MeSH
• Mice MeSH
• Cellular Reprogramming * MeSH
• Transforming Growth Factor beta metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). METHODS: RNA sequencing, quantitative polymerase chain reaction,(13)C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U-(13)C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD(+) ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. RESULTS: We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD(+) ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated proliferation, and suppressed macrophage numbers and remodeling in the distal pulmonary vasculature. CONCLUSIONS: CtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target.

• MeSH
• Adventitia metabolism   pathology   MeSH
• Alcohol Oxidoreductases genetics   metabolism   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Familial Primary Pulmonary Hypertension genetics   metabolism   pathology   MeSH
• Phenotype MeSH
• Fibroblasts metabolism   pathology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Mice MeSH
• Hypertension, Pulmonary metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

This study investigated the effects of bone morphogenetic protein 6 (BMP6) supplementation in the medium during in vitro maturation (IVM) on the developmental potential of oocytes and in the subsequent development of cloned yak embryos. Cumulus-oocyte complexes (COCs) were aspirated from the antral follicles of yak ovaries and cultured with different concentrations of recombinant human BMP6 in oocyte maturation medium. Following maturation, the metaphase II (MII) oocytes were used for somatic cell nuclear transfer (SCNT), and these were cultured in vitro. The development of blastocysts and cell numbers were detected on day 8. The apoptosis and histone modifications of yak cloned blastocysts were evaluated by detecting the expression of relevant genes and proteins (Bax, Bcl-2, H3K9ac, H3K18ac, and H3K9me3) using relative quantitative RT-PCR or immunofluorescence. The presence of 100 ng/mL BMP6 significantly enhanced the oocyte maturation ratios (66.12 ± 2.04% vs. 73.11 ± 1.38%), cleavage rates (69.40 ± 1.03% vs. 78.16 ± 0.93%), and blastocyst formation rates (20.63 ± 1.32% vs. 28.16 ± 1.67%) of cloned yak embryos. The total blastocysts (85.24 ± 3.12 vs. 103.36 ± 5.28), inner cell mass (ICM) cell numbers (19.59 ± 2.17 vs. 32.20 ± 2.61), and ratio of ICM to trophectoderm (TE) (22.93 ± 1.43% vs. 31.21 ± 1.62%) were also enhanced (p < 0.05). The ratio of the Bax to the Bcl-2 gene was lowest in the SCNT + BMP6 groups (p < 0.05). The H3K9ac and H3K18ac levels were increased in SCNT + BMP6 groups (p < 0.05), whereas the H3K9me3 level was decreased; the differences in blastocysts were not significant (p > 0.05). These study results demonstrate that addition of oocyte maturation medium with recombinant BMP6 enhances yak oocyte developmental potential and the subsequent developmental competence of SCNT embryos, and provides evidence that BMP6 is an important determinant of mammalian oocyte developmental reprogramming.

• MeSH
• Apoptosis MeSH
• Blastocyst cytology   MeSH
• Embryo, Mammalian cytology   MeSH
• Fertilization in Vitro MeSH
• Microscopy, Fluorescence MeSH
• Cloning, Organism methods   MeSH
• Bone Morphogenetic Protein 6 pharmacology   MeSH
• Humans MeSH
• Oocytes cytology   MeSH
• Ovary pathology   MeSH
• Cellular Reprogramming MeSH
• Recombinant Proteins metabolism   MeSH
• Cattle MeSH
• Gene Expression Profiling MeSH
• Nuclear Transfer Techniques MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Cattle MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Transplantace inzulín produkující tkáně představuje optimální možnost léčby diabetu závislého na inzulínu. Vzhledem k nedostatku lidských orgánových dárců je však tento způsob léčby málo dostupný. Alternativním zdrojem dostupným pro všechny pacienty by mohly být inzulín produkující buňky získané řízenou přeměnou neendokrinních buněk pankreatu. Doposud publikované práce potvrdily, že je možné neendokrinní pankreatické buňky transdiferencovat na inzulín produkující buňky, které jsou po transplantaci schopny vyléčit diabetes. Zásadním problémem dosavadních postupů je však nízká efektivita přeměny na inzulín produkující buňky, nebo nutnost využít postupy genové terapie, které sebou nesou rizika nádorového zvratu. Nedostatky těchto postupů hodláme vyřešit s pomocí nové metody založené na použití modifikovaných molekul mRNA. V předkládaném projektu hodláme ke stimulaci transdiferenciace pankreatických neendokrinních buněk na inzulín produkující beta-buňky využít modifikované molekuly mRNA, které umožní expresi klíčových transkripčních faktorů stimulujících diferenciaci beta-buněk.; Transplantation of the whole pancreas or isolated Langerhans islets are currently the only available therapeutic options that allow to cure insulin-dependent diabetes. However, both of these options are significantly limited due to the lack of available organ donors. Insulin-producing cells (IPCs) derived from non-endocrine pancreatic cells (NEPCs) represent very promising alternative source that could be available for the treatment of all diabetic patients. Published data have confirmed that IPCs derived from NEPCs are able to cure diabetes. However, only a small percentage of pancreatic cells is able to transdifferentiate into IPCs under the standard conditions. Therefore a more efficient but also unsafe method that uses viral vectors has to be applied in order to improve efficiency of that process. New approach of cell reprogramming that is based on the application of synthetic modified mRNA could solve all these issues. Therefore we propose to use synthetic modified mRNAs encoding key transcription factors of beta-cell differentiation for transdifferentiation of NEPCs into IPCs.

• MeSH
• Insulin-Secreting Cells transplantation   MeSH
• Diabetes Mellitus surgery   MeSH
• Insulin therapeutic use   MeSH
• RNA, Messenger therapeutic use   MeSH
• Cellular Reprogramming MeSH
• Cell Transdifferentiation MeSH
• Transcription Factors MeSH
• Cell Transplantation methods   veterinary   MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• diabetologie
• transplantologie
• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications.

• MeSH
• Cell Differentiation genetics   MeSH
• Cell Line MeSH
• Fibroblasts cytology   metabolism   MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Human Embryonic Stem Cells cytology   metabolism   MeSH
• DNA Methylation * MeSH
• Cellular Reprogramming genetics   MeSH
• Cluster Analysis MeSH
• Gene Expression Profiling MeSH
• Gene Expression Regulation, Developmental MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

RNA silencing is a complex of mechanisms that regulate gene expression through small RNA molecules. The microRNA (miRNA) pathway is the most common of these in mammals. Genome-encoded miRNAs suppress translation in a sequence-specific manner and facilitate shifts in gene expression during developmental transitions. Here, we discuss the role of miRNAs in oocyte-to-zygote transition and in the control of pluripotency. Existing data suggest a common principle involving miRNAs in defining pluripotent and differentiated cells. RNA silencing pathways also rapidly evolve, resulting in many unique features of RNA silencing in different taxonomic groups. This is exemplified in the mouse model of oocyte-to-zygote transition, in which the endogenous RNA interference pathway has acquired a novel role in regulating protein-coding genes, while the miRNA pathway has become transiently suppressed.

• MeSH
• Phylogeny MeSH
• Humans MeSH
• RNA, Small Interfering genetics   metabolism   MeSH
• MicroRNAs classification   genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Oocytes cytology   physiology   MeSH
• Pluripotent Stem Cells cytology   physiology   MeSH
• RNA Interference MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Animals MeSH
• Zygote cytology   physiology   MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH