Epigenetic reprogramming of muscle progenitors: inspiration for clinical therapies

In the context of regenerative medicine, based on the potential of stem cells to restore diseased tissues, epigenetics is becoming a pivotal area of interest. Therapeutic interventions that promote tissue and organ regeneration have as primary objective the selective control of gene expression in adult stem cells. This requires a deep understanding of the epigenetic mechanisms controlling transcriptional programs in tissue progenitors. This review attempts to elucidate the principle epigenetic regulations responsible of stem cells differentiation. In particular we focus on the current understanding of the epigenetic networks that regulate differentiation of muscle progenitors by the concerted action of chromatin-modifying enzymes and noncoding RNAs. The novel exciting role of exosome-bound microRNA in mediating epigenetic information transfer is also discussed. Finally we show an overview of the epigenetic strategies and therapies that aim to potentiate muscle regeneration and counteract the progression of Duchenne Muscular Dystrophy (DMD)

The key role of micrornas in self-renewal and differentiation of embryonic stem cells

Naïve pluripotent embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent distinctive developmental stages, mimicking the pre-and the post-implantation events during the embryo development, respectively. The complex molecular mechanisms governing the transition from ESCs into EpiSCs are orchestrated by fluctuating levels of pluripotency transcription factors (Nanog, Oct4, etc.) and wide-ranging remodeling of the epigenetic landscape. Recent studies highlighted the pivotal role of microRNAs (miRNAs) in balancing the switch from self-renewal to differentiation of ESCs. Of note, evidence deriving from miRNA-based reprogramming strategies underscores the role of the non-coding RNAs in the induction and maintenance of the stemness properties. In this review, we revised recent studies concerning the functions mediated by miRNAs in ESCs, with the aim of giving a comprehensive view of the highly dynamic miRNA-mediated tuning, essential to guarantee cell cycle progression, pluripotency maintenance and the proper commitment of ESCs

The effect of microRNA-375 overexpression, an inhibitor of Helicobacter pylori-induced carcinogenesis, on lncRNA SOX2OT

Background: Helicobacter pylori is a major human pathogenic bacterium in gastric mucosa. Although the association between gastric cancer and H. pylori has been well-established, the molecular mechanisms underlying H. pylori-induced carcinogenesis are still under investigation. MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the posttranscriptional level. Recently, studies have revealed that miRNAs are involved in immune response and host cell response to bacteria. Also, microRNA-375 (miR-375) is a key regulator of epithelial properties that are necessary for securing epithelium-immune system crosstalk. It has been recently reported that miR-375 acts as an inhibitor of H. pylori-induced gastric carcinogenesis. There are few reports on miRNA-mediated targeting long noncoding RNAs (lncRNAs). Objectives: This study aimed to examine the possible effect of miR-375 as an inhibitor of H. pylori-induced carcinogenesis on the expression of lncRNA SOX2 overlapping transcript (SOX2OT) and SOX2, a master regulator of pluripotency of cancer stem cells. Materials and Methods: In a model cell line, NT-2 was transfected with the constructed expression vector pEGFP-C1 contained miR- 375. The RNA isolations and cDNA synthesis were performed after 48 hours of transformation. Expression of miR-375 and SOX2OT and SOX2 were quantified using real-time polymerase chain reaction and compared with control cells transfected with pEGFP-C1-Mock clone. Cell cycle modification was also compared after transfections using the flow cytometry analysis. Results: Following ectopic expression of miR-375, SOX2OT and SOX2 expression analysis revealed a significant decrease in their expression level (P < 0.05) in NT-2 cells compared to the control. Cell cycle analysis following ectopic expression of miR-375 in the NT-2 cells using propidium iodine staining revealed significant extension in sub-G1 cell cycle. Conclusions: This is the first report to show down-regulation of SOX2OT and SOX2 following induced expression of miR-375. This findingmaysuggest expression regulation potential between different classes of ncRNAs, for example between miR-375andSOX2OT. This data not only extends our understanding of possible ncRNA interactions in cancers but also may open novel investigation lines towards elucidation of molecular mechanisms controlling H. pylori inflammation and carcinogenesis. © 2016, Ahvaz Jundishapur University of Medical Sciences

Dissecting Somatic Cell Reprogramming by MicroRNAs and Small Molecules: A Dissertation

Somatic cells could be reprogrammed into an ES-like state called induced pluripotent stem cells (iPSCs) by expression of four transcriptional factors: Oct4, Sox2, Klf4 and cMyc. iPSCs have full potentials to generate cells of all lineages and have become a valuable tool to understand human development and disease pathogenesis. However, reprogramming process suffers from extremely low efficiency and the molecular mechanism remains poorly understood. This dissertation is focused on studying the role of small non-coding RNAs (microRNAs) and kinases during the reprogramming process in order to understand how it is regulated and why only a small percentage of cells could achieve fully reprogrammed state. We demonstrate that loss of microRNA biogenesis pathway abolished the potential of mouse embryonic fibroblasts (MEFs) to be reprogrammed and revealed that several clusters of mES-specific microRNAs were highly induced by four factors during early stage of reprogramming. Among them, miR-93 and 106b were further confirmed to enhance iPSC generation by promoting mesenchymal-to-epithelial transition (MET) and targeting key p53 and TGFβ pathway components: p21 and Tgfbr2, which are important barrier genes to the process. To expand our view of microRNAs function during reprogramming, a systematic approach was used to analyze microRNA expression profile in iPSC-enriched early cell population. From a list of candiate microRNAs, miR-135b was found to be most highly induced and promoted reprogramming. Subsequent analysis revealed that it targeted an extracellular matrix network by directly modulating key regulator Wisp1. By regulating several downstream ECM genes including Tgfbi, Nov, Dkk2 and Igfbp5, Wisp1 coordinated IGF, TGFβ and Wnt signaling pathways, all of which were strongly involved in the reprogramming process. Therefore, we have identified a microRNA-regulated network that modulates somatic cell reprogramming, involving both intracellular and extracellular networks. In addition to microRNAs, in order to identify new regulators and signaling pathways of reprogramming, we utilized small molecule kinase inhibitors. A collection of 244 kinase inhibitors were screened for both enhancers and inhibitors of the process. We identified that inhibition of several novel kinases including p38, IP3K and Aurora kinase could significantly enhance iPSC generation, the effects of which were also confirmed by RNAi of specific target genes. Further characterization revealed that inhibition of Aurora A kinase enhanced phosphorylation and inactivation of GSK3β, a process mediated by Akt kinase. All together, in this dissertation, we have identified novel role of both small non-coding RNAs and kinases in regulating the reprogramming of MEFs to iPSCs

microRNAs in cardiac regeneration and cardiovascular disease

microRNAs (miRNAs) are a class of small non-coding RNAs, which have been shown important to a wide range of biological process by post-transcriptionally regulating the expression of protein-coding genes. miRNAs have been demonstrated essential to normal cardiac development and function. Recently, numerous studies indicate miRNAs are involved in cardiac regeneration and cardiac disease, including cardiac hypertrophy, myocardial infarction and cardiac arrhythmia. These observations suggest miRNAs play important roles in cardiology. In this review, we summarize the recent progress of studying miRNAs in cardiac regeneration and cardiac disease. We also discuss the diagnostic and therapeutic potential of miRNAs in heart disease

Deciphering the stem cell machinery as a basis for understanding the molecular mechanism underlying reprogramming

Stem cells provide fascinating prospects for biomedical applications by combining the ability to renew themselves and to differentiate into specialized cell types. Since the first isolation of embryonic stem (ES) cells about 30 years ago, there has been a series of groundbreaking discoveries that have the potential to revolutionize modern life science. For a long time, embryos or germ cell-derived cells were thought to be the only source of pluripotency—a dogma that has been challenged during the last decade. Several findings revealed that cell differentiation from (stem) cells to mature cells is not in fact an irreversible process. The molecular mechanism underlying cellular reprogramming is poorly understood thus far. Identifying how pluripotency maintenance takes place in ES cells can help us to understand how pluripotency induction is regulated. Here, we review recent advances in the field of stem cell regulation focusing on key transcription factors and their functional interplay with non-coding RNAs

Small non-coding RNA landscape of extracellular vesicles from human stem cells

Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the ATMSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche.Peer reviewe

The non-coding genome in early human development-Recent advancements

Not that long ago, the human genome was discovered to be mainly non-coding, that is comprised of DNA se-quences that do not code for proteins. The initial paradigm that non-coding is also non-functional was soon overturned and today the work to uncover the functions of non-coding DNA and RNA in human early embryogenesis has commenced. Early human development is characterized by large-scale changes in genomic activity and the transcriptome that are partly driven by the coordinated activation and repression of repetitive DNA elements scattered across the genome. Here we provide examples of recent novel discoveries of non-coding DNA and RNA interactions and mechanisms that ensure accurate non-coding activity during human maternal-to -zygotic transition and lineage segregation. These include studies on small and long non-coding RNAs, trans-posable element regulation, and RNA tailing in human oocytes and early embryos. High-throughput approaches to dissect the non-coding regulatory networks governing early human development are a foundation for func-tional studies of specific genomic elements and molecules that has only begun and will provide a wider un-derstanding of early human embryogenesis and causes of infertility.Peer reviewe

Non-coding RNA analysis of iPSCs-derived hepatocyte-like cells

The liver is a crucial human organ with a complex architecture. Although the liver has great regeneration potential, deadly liver diseases are associated with irreversible hepatocytes damage. Currently, a liver transplant is the only treatment for liver failure. A shortage of donors forced extensive research for alternative treatments. The most promising hepatocyte source could be obtained from the differentiation of induced pluripotent stem cells (iPSCs). This technology can give us great amounts of pluripotent cells without ethical restrictions, which could be available in a variety of haplotypes to minimize the possibility of rejection. There are many reprogramming protocols available. However, there is still no standardised method to obtain clinical grade iPSCs. From those stem cells, it is possible to obtain hepatic-like cells (HLCs) by direct differentiation in vitro. HLCs express multiple hepatocyte-specific features, but their names signal that they still show fetal liver identity. A variety of hepatic differentiation protocols were described, although the process of hepatic differentiation must be improved in order to be translated into the clinic. Along with genes, microRNA (miRNA) is the well-known controller of cell fate. MiRNA is a type of non-coding RNA (ncRNA) which can influence gene transcription by inhibiting gene expression. In contrast to genes, many of the miRNA can affect up to thousands of genes simultaneously. Another group of ncRNA, which is a subject of potential differences are small nucleolar RNA (snoRNA). SnoRNA are involved in RNA chemical modifications by acting as a guide, mostly for ribosomal RNA (rRNA), but some of them have additional functions. In this study, a new iPSCs line was generated from skin fibroblasts using lipotransfection of episomal vectors. This method is free from exogene integration and shows low cytotoxicity. A pluripotency of generated cells was confirmed by morphological assessment, immunocytochemical staining, and spontaneous differentiation assay. To be sure that the genome of the cells was not changed, karyotype analysis was performed. Next, HLCs were derived from those iPSCs using a four-stage hepatic differentiation protocol. The obtained HLCs were characterised using, among others, a hepatic gene expression analysis. Cells after differentiation express mature and fetal hepatic markers, which is consistent with previous results. The attempt to improve differentiation using transient overexpression of master hepatic transcription factor – HNF4α, was not sufficient, as shown by gene expression analysis and whole slide scanning. Previous studies failed to point out the genetic inhibitors of hepatic maturation and non-coding RNA (ncRNA) profiles of iPSCs – derived HLCs were not investigated. In this study, the sequencing of ncRNA was performed in order to compare the expression profiles of HLCs on two stages of differentiation (Day 20 and 24) with mature hepatocytes. The obtained results indicate that HLCs express miRNA, which control hepatic differentiation and maintain their fetal liver character. In comparison to mature hepatocytes, differentially expressed miRNAs in HLCs control the pathways of fatty acid metabolism and synthesis, proteoglycan in cancer, the Hippo signaling pathway, ECM-receptor interaction and adherens junction. Some of those highly expressed miRNAs can potentially block maturation by inhibiting epithelial-mesenchymal transition (EMT) which has an impact that is essential during hepatic differentiation. However, this should be resolved in future research. In this work, differentially expressed snoRNA were also identified. A total of 68% of differentially expressed snoRNAs was C/D box class. This is interesting because this snoRNa class was previously indicated as capable to be processed by an miRNA processing pathway. Many of the differentially expressed snoRNAs belong to the imprinted loci, in which a different expression in a human were analysed before. In obteined dataset, copies of SNORD115 were upregulated in a liver, but not in HLCs, which is consistent with an earlier comparison of a liver and other endoderm organs. Additionally, an analysis of obtained sequencing data allowed for a discovery of 19 novel snoRNA genes. In summary, this work shows a new approach to the reprogramming of a fibroblast and investigates the involvement of miRNAs and snoRNAs in the dynamics of hepatic differentiation. This study has shed a light on the molecular and regulatory mechanisms that underlie the complex process of liver differentiation and will hopefully allow existing problems with the use of in vitro derived hepatocytes to be overcome. A dataset generated here can be the foundation for a hepatic-specialised rybosomes theory and enabled to discover novel snoRNA genes.:1. INTRODUCTION 11 1.1. PLURIPOTENT STEM CELLS 11 1.1.1. Pluripotency 11 1.1.2. IPSCs 13 1.1.3. Reprogramming methods 14 1.1.4. IPSCs as an alternative cell source for disease modelling and regenerative medicine 16 1.2. LIVER 18 1.2.1. Liver anatomy and function 18 1.2.2. Liver embryonal development 20 1.3. HEPATIC DIFFERENTIATION OF IPSCS IN VITRO 22 1.3.1. HLCs 22 1.3.2. Differentiation protocols into hepatocytes 24 1.4. NCRNA 25 1.4.1. MiRNA 26 1.4.2. SnoRNA 28 2. AIMS 31 3. MATERIALS 32 3.1. EQUIPMENT 32 3.2. SOFTWARE 32 3.3. ENZYMES, KITS AND TRANSFECTION REAGENTS 33 3.4. SOLUTIONS AND REAGENTS 33 3.5. CELL LINES 34 3.6. CELL CULTURE MEDIA AND CYTOKINES 34 3.7. PLASMIDS 35 3.8. PCR REAGENTS AND PRIMERS 35 3.8.1. PCR reagents 35 3.8.2. PCR primers 35 3.9. ANTIBODIES 36 4. METHODS 37 4.1. CELL BIOLOGY 37 4.1.1. Derivation and culture of primary human foreskin fibroblasts 37 4.1.2. Counting cells 37 4.1.3. Cryo-preservation of cells 37 4.1.4. Thawing of cryo-preserved cells 38 4.1.5. Cell reprogramming 38 4.1.6. Cultivation and expansion of iPSCs 39 4.2. IMMUNOCYTOCHEMISTRY 39 4.3. IN VITRO SPONTANEOUS DIFFERENTIATION 39 4.4. KARYOTYPE ANALYSIS 40 4.5. RNA ISOLATION 40 4.6. QUANTITATIVE PCR 40 4.7. PERIODIC ACID-SCHIFF (PAS) STAINING 41 4.8. INDOCYANINE GREEN UPTAKE AND RELEASE 41 4.9. PLASMID TRANSFECTION 42 4.10. HEPATIC DIFFERENTIATION 42 4.11. WHEAT GERM AGGLUTININ STAINING 42 4.12. VALIDATION OF HEPATIC DIFFERENTIATION EFFICIENCY 43 4.13. RNA ISOLATION AND SEQUENCING 43 4.14. BIOINFORMATIC ANALYSIS 44 4.14.1. Sequencing quality and mapping 44 4.14.2. Analysis of differential expressed ncRNAs 44 4.14.3. Target pathways prediction of differentially expressed miRNAs 44 4.14.4. Identification of novel ncRNAs candidates 45 5. RESULTS 46 5.1. GENERATION OF IPSCS USING EPISOMAL VECTORS 46 5.1.1. Cell transfection 46 5.1.2. Establishment of iPSCs line 48 5.2. PLURIPOTENCY CHARACTERISATION OF THE IPSCS 49 5.2.1. Pluripotency markers 49 5.2.2. Spontaneous differentiation assay 50 5.2.3. Karyotype 52 5.3. HEPATIC DIFFERENTIATION OF IPSCS AND HLCS CHARACTERISATION 53 5.3.1. iPSCs hepatic differentiation 53 5.3.2. Expression of hepatic markers 54 5.3.3. Hepatic gene expression in HLCs 56 5.3.4. Hepatic functions in HLCs 58 5.4. HNF4A OVEREXPRESSION DURING DIFFERENTIATION 59 5.4.1. Cell transfection during differentiation 59 5.4.2. Comparison of hepatic differentiation efficiency 60 5.4.3. Whole slide scanning 62 5.5. NON-CODING RNA ANALYSIS 64 5.5.1. Non-coding RNA sequencing quality 64 5.5.2. MicroRNA analysis 68 5.5.3. SnoRNA analysis 79 5.5.4. Short reads snoRNA analysis 84 5.5.5. New gene candidates 85 6. DISCUSSION 88 6.1. METHODICAL STRATEGY 88 6.2. CHARACTERISATION OF GENERATED IPSCS 89 6.3. HEPATIC DIFFERENTIATION OF IPSCS 89 6.3.1. Characterisation of HLCs 89 6.3.2. Protocol with HNF4a overexpression 90 6.3.3. Differentially expressed miRNA 90 6.3.4. Differentially expressed snoRNA 93 6.4. NOVEL SNORNA GENES 95 7. SUMMARY 96 8. REFERENCES 99 9. APPENDIX 118 ERKLÄRUNG ÜBER DIE EIGENSTÄNDIGE ABFASSUNG DER ARBEIT 122. ACKNOWLEDGEMENTS 12