Epigenetic regulation of Mash1 expression

Mash1 is a proneural gene important for specifying the neural fate. The Mash1 locus undergoes specific epigenetic changes in ES cells following neural induction. These include the loss of repressive H3K27 trimethylation and acquisition of H3K9 acetylation at the promoter, switch to an early replication timing and repositioning of the locus away from the nuclear periphery. Here I examine the relationship between nuclear localization and gene expression during neural differentiation and the role of the neuronal repressor REST in silencing Mash1 expression in ES cells. Following neural induction of ES cells, I observed that relocation of the Mash1 locus occurs from day 4-6 whereas overt expression begins at day 6. Mash1 expression was unaffected by REST removal in ES cells as well as the locus localization at the nuclear periphery. In contrast bona fide REST target genes were upregulated in REST -/- cells. Interestingly, among REST targets, loci that were more derepressed upon REST removal showed an interior location (Sthatmin, Synaptophysin), while those more resistant to REST withdrawal, showed a peripheral location (BDNF, Calbidin, Complexin). To ask whether the insulator protein CTCF together with the cohesin complex might be involved in regulating Mash1 in ES cells, I performed ChIP analysis of CTCF and cohesin binding across the Mash1 locus in ES cells and used RNAi to deplete CTCF and cohesin expression. A slight increase in the transcription of Mash1 was seen in cells upon Rad21 knock down, although it was not possible to exclude this was a consequence of delayed cell cycle progression. Finally ES cell lines that carried a Mash1 transgene were created as a tool to look at whether activation of Mash1 can affect the epigenetic properties of neighbouring genes

A systematic review of the asymmetric inheritance of cellular organelles in eukaryotes : A critique of basic science validity and imprecision

We performed a systematic review to identify all original publications describing the asymmetric inheritance of cellular organelles in normal animal eukaryotic cells and to critique the validity and imprecision of the evidence. Searches were performed in Embase, MEDLINE and Pubmed up to November 2015. Screening of titles, abstracts and full papers was performed by two independent reviewers. Data extraction and validity were performed by one reviewer and checked by a second reviewer. Study quality was assessed using the SYRCLE risk of bias tool, for animal studies and by developing validity tools for the experimental model, organelle markers and imprecision. A narrative data synthesis was performed. We identified 31 studies (34 publications) of the asymmetric inheritance of organelles after mitotic or meiotic division. Studies for the asymmetric inheritance of centrosomes (n = 9); endosomes (n = 6), P granules (n = 4), the midbody (n = 3), mitochondria (n = 3), proteosomes (n = 2), spectrosomes (n = 2), cilia (n = 2) and endoplasmic reticulum (n = 2) were identified. Asymmetry was defined and quantified by variable methods. Assessment of the statistical reliability of the results indicated only two studies (7%) were judged to have low concern, the majority of studies (77%) were 'unclear' and five (16%) were judged to have 'high concerns'; the main reasons were low technical repeats (<10). Assessment of model validity indicated that the majority of studies (61%) were judged to be valid, ten studies (32%) were unclear and two studies (7%) were judged to have 'high concerns'; both described 'stem cells' without providing experimental evidence to confirm this (pluripotency and self-renewal). Assessment of marker validity indicated that no studies had low concern, most studies were unclear (96.5%), indicating there were insufficient details to judge if the markers were appropriate. One study had high concern for marker validity due to the contradictory results of two markers for the same organelle. For most studies the validity and imprecision of results could not be confirmed. In particular, data were limited due to a lack of reporting of interassay variability, sample size calculations, controls and functional validation of organelle markers. An evaluation of 16 systematic reviews containing cell assays found that only 50% reported adherence to PRISMA or ARRIVE reporting guidelines and 38% reported a formal risk of bias assessment. 44% of the reviews did not consider how relevant or valid the models were to the research question. 75% reviews did not consider how valid the markers were. 69% of reviews did not consider the impact of the statistical reliability of the results. Future systematic reviews in basic or preclinical research should ensure the rigorous reporting of the statistical reliability of the results in addition to the validity of the methods. Increased awareness of the importance of reporting guidelines and validation tools is needed for the scientific community

Development of a novel in vitro 3D model to identify cancer genes via insertional mutagenesis

This thesis was submitted for the degree of Doctorate of Philosophy and awarded by Brunel University LondonThe aim of the presented research project was to develop a personalised in vitro based human model to test the genotoxicity of gene therapy viral vectors. For this purpose, human induced pluripotent stem cells (hiPSCs) and human induced pluripotent stem cell-derived 3D heps were used in combination with a number of lentiviral and adeno-associated viral vectors and molecular analysis was performed to detect insertion sites (IS) to assess the predictive power of 3D heps model to predict insertional mutagenesis. In this study, two previously designed plasmid were used, one of which contained the U3 region of the LTR from the wild type (pHV) and considered as “Unsafe” vector, and the other was a self-inactivating (SIN) lentiviral vector with no U3 region of the LTR (pHR) and used as a “Safe” vector. Initially, high-titre production of both lentiviral vectors was achieved in the HEK293 cell line. The titre was calculated using flow cytometry analysis. The recombinant AAV serotype-2 vectors were kindly provided by our collaborators in Australia. These vectors constructed with “Clean ITR” with strong and weak promoters, driving the green fluorescent protein (GFP) expression. In parallel, P106i, as an integration-free hiPSCs, was expanded and fully characterised, using flow cytometry, immunostaining and qPCR analysis. Expression of pluripotency cell surface markers such as SSEA4, TRA-1-81 and TRA-1-60 was detected in more than 90% of the cells using flow cytometry. The expression of POU domain class 5 transcription factor 1 (POU5F1 or OCT4) and SRY-Box Transcription Factor 2 (SOX2) as two major transcription factors regulating pluripotency were also confirmed at gene and protein levels by quantitative polymerase chain reaction (qPCR) and immunostaining, respectively. The high level of pluripotency markers confirmed the pluripotent state and the identity of P106i, which was used in this study. Then, a recently published protocol to generate phenotypically stable 3D heps from human embryonic stem cells (hESCs) were amended and optimised to generate 3D heps from hiPSC efficiently. This protocol addresses issues surrounding previous 3D protocols, such as scalability and long-term in vitro phenotypic stability. Notably, hiPSC-derived 3D heps displayed liver functions for an extended period. Standard characterisation tests were performed on day 20 of differentiation using a range of molecular and cell biology techniques, including immunofluorescence analysis of liver-specific markers such as HNF4-alpha and secretion of serum albumin (ALB) and alpha-fetoprotein (AFP) using ELISA assay. The result revealed a high level of ALB and a low level of AFP in hiPSC-derived 3D heps compared to conventional 2D hepatocyte-like cells. Following characterisation, hiPSCs and 3D heps were transduced with recombinant AAV serotype-2 vectors (at MOI 1E5) and lentiviral vectors (at MOI 20). Modifications were made to enhance transduction efficiency in 3D heps. The outer layer of the 3D heps exhibited the highest level of GFP expression, which transduced with rAVV serotype-2 vectors. In 3D heps transduced with pHR and pHV lentiviral vectors, the same pattern was observed; however, a higher level of GFP expression was observed in cells transduced with the pHR lentiviral vector. The result of successful transduction was confirmed by PCR analysis. hiPSCs were also transduced with pHR and pHV lentiviral vectors at two different time points, and the result was confirmed via PCR analysis. In order to see the effect of gene expression in the proximity of viral insertion at the individual cell level, single-cell cloning (SCC) was performed on hiPSCs which were transduced with pHR and pHV lentiviral vectors. The results showed positive GFP expression in hiPSCs, confirming transduction. The DNA and RNA of the single-cell clones, hiPSCs and 3D heps were collected and sent for downstream analysis of the insertion site (IS). Upon viral integration, it is crucial to detect IS in the setting of clonal dominance. For bioinformatic analysis, lentiviral IS was retrieved by EPTS/LM-PCR and CIS were detected in P106i and 3D heps samples. This result revealed an overall decrease in IS overtime in transduced cells for both lentiviral vectors by comparison of the days 3 and 30 time points. The number of identified IS in 3D heps transduced with pHR and pHV lentiviral was low compared to P106i cells. There was also an apparent reduction in IS in P106i cells transduced with pHR and pHV lentiviral vectors over time, suggesting possible cell death during propagation and culture expansion. Following EPTS/LM-PCR, identification of cancer-related genes IS, and CIS were performed in P106i cells and 3D heps. The result indicates that in P106i, the number of proto-oncogenes was higher in samples transduced with pHV lentiviral vectors, suggesting the possible effect of the full LTR vector on the number of cancer-related genes. In addition, polyclonality of pHV and pHR was observed in analysed single-cell clones of P106i. However, in clone G transduced with pHV lentiviral vector, the gene LINC01249 exhibited 93.4% of the viral sequence count. In addition, despite the polyclonality of the samples, the qPCR result revealed that the genes near the IS have increased in level of relative gene expression. In conclusion, the 3D heps provide a valuable in vitro tool to assess genotoxicity associated with viral vectors

New insights into probabilistic pattern formation of embryonic stem cells using agent-based modelling

Embryonic stem cells (ESCs) hold great potential for developing future therapies for a wide range of diseases. However, the mechanisms of pattern formation during embryonic development remain poorly understood. ESCs in culture self-organise to form spatial patterns of gene expression upon geometrical confinement indicating that patterning is an emergent phenomenon that results from the many interactions between the cells. Here, we applied an agent-based modelling approach to identify biologically plausible rules acting at the mesoscale within stem cell collectives that may explain spontaneous patterning. We tested different models involving differential motile behaviours including exploring effects due to neighbour interactions. We introduced a new metric, the stem cell aggregate pattern distance (SCAPD), to assess the deviation between the probabilistic experimental pattern formation (used as ground truth) and the probabilistic simulated outcome. We demonstrated our models can produce broadly realistic pattern formation (when compared to experimental data) with a quantified level of uncertainty. The best of our models improve fitness, evaluated by SCAPD, by 70% and 77% over the random models for a discoidal or an ellipsoidal stem cell confinement, respectively. Collectively, our findings provide compelling arguments that a parsimonious mechanism that involves differential motility is sufficient to explain the spontaneous patterning of the cells upon confinement. Furthermore, our work also defines a region of the parameter space that is compatible with patterning, which assists future studies in the field of cell engineering. We envisage that the novel approaches explored within this work will be applicable to many biological systems and will contribute towards facilitating progress by reducing the need for extensive and costly experiments

Injectable polypeptide hydrogels via methionine modification for neural stem cell delivery.

Injectable hydrogels with tunable physiochemical and biological properties are potential tools for improving neural stem/progenitor cell (NSPC) transplantation to treat central nervous system (CNS) injury and disease. Here, we developed injectable diblock copolypeptide hydrogels (DCH) for NSPC transplantation that contain hydrophilic segments of modified l-methionine (Met). Multiple Met-based DCH were fabricated by post-polymerization modification of Met to various functional derivatives, and incorporation of different amino acid comonomers into hydrophilic segments. Met-based DCH assembled into self-healing hydrogels with concentration and composition dependent mechanical properties. Mechanical properties of non-ionic Met-sulfoxide formulations (DCHMO) were stable across diverse aqueous media while cationic formulations showed salt ion dependent stiffness reduction. Murine NSPC survival in DCHMO was equivalent to that of standard culture conditions, and sulfoxide functionality imparted cell non-fouling character. Within serum rich environments in&nbsp;vitro, DCHMO was superior at preserving NSPC stemness and multipotency compared to cell adhesive materials. NSPC in DCHMO injected into uninjured forebrain remained local and, after 4 weeks, exhibited an immature astroglial phenotype that integrated with host neural tissue and acted as cellular substrates that supported growth of host-derived axons. These findings demonstrate that Met-based DCH are suitable vehicles for further study of NSPC transplantation in CNS injury and disease models

Molecular mapping of nuclear organization in the mouse preimplantation embryo

Upon fertilization the two parental genomes are extensively reprogrammed to give rise to a totipotent state. In the mammalian embryo, this epigenetic reprogramming involves an extensive three dimensional (3D) rearrangement of nuclear organization which only recently have been started to be investigated on a genome-wide scale. The positioning of loci relative to the nuclear periphery has been shown to change during differentiation, potentially regulating gene expression and chromatin. Therefore, it is of question whether the nuclear reorganization in early embryonic cells correlates with or even regulates genome function and embryo development. During my PhD work, we have created maps of lamina associated domains (LADs) from mouse preimplantation embryos and oocytes at the single cell level. LADs are genomic regions that reside at the nuclear periphery and represent a lowly transcribed, gene-poor fraction of the genome originally identified in somatic cells. We have found that LADs are absent in oocytes but become established already in zygotes and are dynamically rearranged during the 2- and 8-cell stages with little heterogeneity between individual cells. We obtained LAD data from hybrid embryos to distinguish the parental genomes by single nucleotide polymorphisms (SNPs) in sequencing. Our analysis unravelled differences in genome organization between the two parental alleles that likely reflect their different germline history. Moreover, we find that LAD formation precedes the maturation of topologically associated domains (TADs) in a DNA replication independent manner. Additionally, we observed that only the X chromosome contacts the lamina in oocytes, potentially through an interaction with the Lamin B Receptor (LBR) protein. Eventually, we identified an epigenetic asymmetry of H3K4 methylation on LADs between the paternal and maternal genomes in zygotes. We found that the experimental reduction of the H3K4me3 histone mark by the overexpression of the lysine demethylase Kdm5b results in a loss of LAD structure, specifically in the paternal zygotic genome. In conclusion, we have uncovered a novel mechanism of allele specific LAD formation through histone methylation. Additionally, this work provides genome wide information on mouse preimplantation nuclear organization contributing a resource for further epigenetic studies of early embryos

Nucleobase, Nucleoside, And Neighboring Nucleotides: Intrinsic Preferences For Tet Enzyme-Mediated Oxidation Of 5-Methylcytosine

The Ten-eleven-translocation (TET) family of enzymes can oxidize the fifth base of DNA, 5-methylcytosine (mC) sequentially, to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). The biochemical preference of TET enzymes for these substrates, in the canonical cytosine guanine dinucleotides (CpG), mimics the order in which they are generated and is reflected in levels of these oxidized modifications (oxmCs) detected in various genomes. Other than this exception, there is conflicting or limited data concerning intrinsic substrate preferences of TET, particularly with regards to different nucleic acid structures, sequence contexts, and extent to which TET mediates oxmCs in clustered proximity to one another. Thus, in this thesis, I present our efforts to determine intrinsic substrate preferences of TET enzymes, and in doing so expand upon our understanding of mechanisms driving these relative activities and the functional significance of observed levels of oxmCs in vivo. After a review of the field, in Chapter 2, I present our work comparing TET activity on different DNA and RNA structures in vitro. We found that TET is relatively promiscuous on a variety of DNA/RNA structures but prefers DNA, a specificity that is dictated by nucleic acid identity of the target base, as well helical conformation of the substrate. In Chapter 3, I newly expose the relative tolerance of TET activity on hmC with a non-G at the +1 base, although mCpG is still largely preferred. This tolerance for hmC oxidation by TET and fC and caC excision by TDG, regardless of the +1 base, supports a model explaining hmCpH depletion relative to mCpH and hmCpG in some genomes. In Chapter 4, I narrate our efforts to quantify clusters of oxmCs using modification-specific sequencing methods and observe that TET is intrinsically capable of clusters of at least fC and caC. Also, we explore the possibility that these clusters are mediated by either strand processivity of TET or underlying sequence context preferences. Finally, I propose two kinetics-based experiments to test our hypotheses regarding mechanisms driving these substrate preferences, along with ways to exploit our knowledge of TET enzymes for creation of more efficient and specific epigenetic editing tools

In Vivo Bioluminescent Imaging (BLI): Noninvasive Visualization and Interrogation of Biological Processes in Living Animals

In vivo bioluminescent imaging (BLI) is increasingly being utilized as a method for modern biological research. This process, which involves the noninvasive interrogation of living animals using light emitted from luciferase-expressing bioreporter cells, has been applied to study a wide range of biomolecular functions such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis, cancer treatment, and disease progression. This article will review the various bioreporter/biosensor integrations of BLI and discuss how BLI is being applied towards a new visual understanding of biological processes within the living organism