Role of miR-1305 in regulating pluripotency, cell cycle and apoptosis in human embryonic stem cells

PhD ThesisHuman embryonic stem cells and human induced pluripotent stem cells are defined as pluripotent in view of their ability to maintain self-renewal and differentiation to cells of all three germ layers. So far the mechanism underlying the cell cycle regulation, self-renewal and pluripotency of human pluripotent stem cells are not fully understood. In this study, we first screened for candidate miRNAs which might play important roles in regulating pluripotency and cell cycle by using a microarray based approach. miR-1305 was chosen as a target, as its expression profile changed during human embryonic stem cell differentiation and cell cycle. We also revealed the role of miR-1305 in regulating differentiation in human embryonic stem cells as well as cell cycle and apoptosis in human embryonic and induced pluripotent stem cells. Our results provide evidence that overexpression of miR-1305 induces significant human embryonic stem cell differentiation and downregulation of miR-1305 maintains human embryonic stem cell pluripotency. Furthermore, POLR3G was identified as a downstream target by which miR-1305 regulates human embryonic stem cell differentiation. Together our data corroborate previous findings indicating an intrinsic link between miRNA and maintenance of pluripotency in human embryonic stem cells

Deriving an in vitro source of canine corneal stromal cells for future studies of corneal disease and therapeutic applications

The cornea is the transparent tissue located at the front of the eye, that transmits and refracts light onto the retina. Despite great advances in corneal stem cell biology in human and laboratory animal research, no information is available in dogs. Corneal pathology, as corneal crystalline dystrophy has a prevalence of up to 15% and has been described in eight different canine breeds. Cholesterol and phospholipids are deposited in the stroma, similar to Schnyder’s dystrophy (SCD) in humans. Chronic corneal fibrosis is one of the leading causes of visual impairment in veterinary ophthalmology. Similar to the situation in human ophthalmology, there is a shortage of corneal donor tissue. Therefore, the overall aim was first to investigate whether corneal stromal stem cells exist in the canine cornea., The second aim was to determine the potential of deriving an in vitro source of corneal stroma cells from corneal stromal stem cells, adipose derived mesenchymal stromal cells (adMSC) and canine induced pluripotent stem cells (ciPSC), to provide a resource for studies investigating the pathogenesis of inherited stromal dystrophies, and for the development of novel cell-based therapies for dogs. First, a canine corneal stromal cell (CSC) population was characterised that demonstrated mesenchymal stromal cell properties, they differentiated into keratocyte-like cells (KDCs) in vitro and appeared to be immune privileged. Second, canine adMSC were differentiated into KDCs, but expressed high levels of a myofibroblastic marker, similar to those found in fibrotic tissue. Third, a modified protocol was established whereby ciPSCs were induced into neural crest (stem) cell lineages and then into KDCs. This led to the successful expression of some keratocyte associated markers in absence of a myofibroblastic expression. Taken together, a novel cell population was characterised in the canine corneal stroma. The differentiation protocols of adMSC and ciPSC led to preliminary results and built a basic foundation for future studies

Characterization of neural progenitor/stem cells derived from human embryonic stem cells

Human embryonic stem cells (hESCs) are able to proliferate indefinitely without losing their ability to differentiate into multiple cell types of all three germ layers. Due to these fascinating properties, hESCs have promise as a robust cell source for regenerative medicine and as an in vitro model for the study of human development. In my PhD study, I have investigated the neural differentiation process of hESCs using our established protocol, identified characteristics associated with each stage of the differentiation and explored possible signalling pathways underlying these dynamic changes. It was found that neural differentiation of hESCs could be divided into 5 stages according to their morphology, marker expression and differentiation potencies: hESCs, neural initiation, neural epithelium/rosette, neuronal progenitor cells and neural progenitor/stem cells (NPSCs) and 4 of these stages have been studied in more detail. At the neural initiation, hESCs firstly lose TRA-1-81 expression but retain SSEA4 expression. This transient cell population shows several similar properties to the primitive ectoderm. After neural-tube like structure/neural rosette formation, neural progenitor cells appear as typical bipolar structures and exhibit several properties of radial glial cells, including gene expression and pro-neuronal differentiation. The neural progenitor cells are able to grow in culture for a long time in the presence of growth factors bFGF and EGF. However, they gradually lose their bipolar morphology to triangular cell type and become pro-glial upon further differentiation. In addition, the state of neural progenitor and stem cells can be distinguished by their differential response to canonical Notch effector, C protein-binding factor 1. It was also found that delta like1 homolog (DLK-1) is temporally upregulated upon initial neural differentiation, but becomes undetectable after the neural progenitor stage. Overexpression of DLK-1 in NPSCs enhances neuronal differentiation in the presence of serum by blocking BMP and Notch pathways. These results show that neural differentiation of hESCs is a dynamic process in which cells go through sequential changes, and the events are reminiscent of the in vivo neurodevelopment process. Moreover, I have characterized stably transfected nestin-GFP reporter hESC lines and found that the cell lines maintained the features of hESCs and the expression of GFP is restricted to the neural lineage after differentiation. Therefore, these reporter lines will be useful for the study of factors that regulate neural differentiation and for the enrichment of neural progenitors from other lineages. Taken together, this study has demonstrated that hESCs are a good in vitro model to study the mechanisms and pathways that are involved in neural differentiation. The availability of hESCs allows us to explore previously inaccessible processes that occur during human embryogenesis, such as gastrulation and neurogenesis

The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition.

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development

Src Family Tyrosine Kinase Signaling in Mouse and Human Embryonic Stem Cells

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo and are characterized by self-renewal and pluripotency. Previous work has implicated the Src family of protein-tyrosine kinases (SFKs) in the self-renewal and differentiation of mouse ES (mES) cells. These kinases display dynamic expression and activity changes during ES cell differentiation, suggesting distinct functions in the control of developmental fate. To test the hypothesis that c-Src and its closest phylogenetic relative, c-Yes, act in biological opposition to one another, I first showed that enforced expression of active c-Yes blocked ES cell differentiation to embryoid bodies by maintaining pluripotency gene expression. To determine the interplay of c-Src and c-Yes in mES cell fate determination, I employed a chemical genetics approach to generate c-Src and c-Yes mutants that are resistant to A-419259, a potent pyrrolopyrimidine inhibitor of the Src kinase family. This method allowed us to investigate individual kinase function in the presence of A-419259. I found that c-Src activity alone induces mES cell differentiation to the ectoderm and endoderm, while c-Yes inhibits this process. These studies show that even closely related kinases such as c-Src and c-Yes have unique and opposing functions in the same cell type. While Src kinase signaling has been investigated in mES cells, the role of this kinase family in human ES (hES) cells is largely unknown. Using quantitative real-time RT-PCR, I determined the relative expression profile of individual SFK members in undifferentiated hES cells vs. embryoid bodies derived from them. Like mES cells, hES cells express multiple SFK members with dynamic transcription changes during EB differentiation, indicating that individual members may play non-redundant roles. To assess the role of SFK activity in hES cells, I treated hES cell cultures with SFK inhibitors. SFK inhibition maintained hES cell colony morphology and expression of the pluripotency marker Tra-1-60 in differentiation medium. These observations support a role for Src family kinase signaling in the regulation of hES fate, and suggest that some parallels may exist in mouse and human ES cells for this intracellular signaling network

Novel approaches to the isolation of farm animal embryonic stem cells

The establishment of stable immortal ES cell lines using embryos as a source of isolation in domesticated farm animals, in particular for pigs, which are closer to humans than other ungulates, has not been reported; hence this information could contribute to the improvement of regenerative medicine in humans, biotechnology and agriculture. Therefore, the discovery of effective protocols to derive and maintain ES cells and the induction of purified somatic cells from ES cells in pigs is of importance. The objectives of this study were to produce pES-like cells and direct differentiation of the ES-like cells obtained by improving the culture conditions. In vivo-derived porcine blastocysts at day 6-8 were classified into two groups distinguished by the exhibition of ICMs and epiblasts of the embryos. In each group, intact blastocysts and isolated ICMs or epiblasts were designed to culture in either KO4bh or DM40bh medium on mitotically inactivated MEFs under the humidified air of 5%CO2 at 39°C until the primary outgrowth of ES-like cells was observed. Two morphologically distinct pES-like cells, pESA-like and pESB-like cells were isolated from the epiblasts, whereas no cell lines were generated from ICMs. pESA-like cells were observed as individual small round cells containing one or multiple nucleoli along with a high ratio of nucleus to cytoplasm, while pESB-like cells formed dome-like colonies. The pESA-like cells were stained both negative and positive with the alkaline phosphatase enzyme, while pESB-like cells were all stained positive. With immunofluorescence staining of OCT-4 and nanog, the nuclei of pESB-like cells appeared not to be stained positive with these two antibodies, while the designed self-renewing genes such as OCT-4, nanog, SOX-2, REX-1 and DPPA-3 were detectable as similar to mES cells. Regarding the pluripotent abilities of pESB-like cells, they could be induced to form neuronal-like, neuronal supporting-like, smooth muscle-like and hepatic-like cells in a variety of desirable differentiation media under the feeder-free culture system. The cytoplasmic contents of certain induced mature cells were stained positive with nestin, α-smooth muscle actin and α-fetoprotein in association with the expression of differentiated genes specific to each germ layer such as nestin, α-smooth muscle actin, smooth muscle myosin heavy chain, α-cardiac actin, transthyretin, α-fetoprotein, albumin and HGF1β. In conclusion, pESB-like cells obtained in this study may possibly have the potential to be authentic ES cells isolated from early epiblast origin as mES cells

Dissecting Pluripotency and Mammalian Embryonic Development via Droplet Microfluidics

Pluripotency, the ability of a cell to differentiate towards any type of somatic cell is a transient feature of the developing embryo. In vitro, pluripotency can be captured in the form of embryonic stem cells (ESCs). In this study, I developed a microfluidic-based system to encapsulate ESCs into agarose microgels, three-dimensional scaffolds that are in terms of their mechanical and biochemical properties fundamentally different from conventional tissue culture in plastic dishes. Subsequently, I investigated how these microenvironmental changes influence pluripotency. Interestingly, microgel culture of ESCs was not just accompanied by drastic changes in morphology, but also a promotion in naïve pluripotency. RNA-sequencing of microgel cultured ESCs elucidated global transcriptional changes of which many affected members of the pluripotency network. I then identified plakoglobin, a homologue of b-catenin, as one of the strongest upregulated proteins upon microgel encapsulation. However, molecular functions of plakoglobin in embryonic stem cells remain largely elusive. To investigate plakoglobin’s potential role during naïve pluripotency, I created several ESC lines that constitutively expressed plakoglobin at varying levels. Cells expressing high amounts of plakoglobin, portrayed a distinct naïve phenotype with homogeneous transcription factor expression even under serum-based conditions. Single cell RNA-seq and the formation of blastocyst chimaeras were then used to confirm the re-establishment of the complete naïve network. In contrast, plakoglobin is absent or greatly reduced during primed pluripotency in epiblast-derived stem cells and conventional primate pluripotent stem cells. A finding that was further confirmed in the corresponding pre- and post-implantation embryo and naïve and primed marmoset and human pluripotent stem cells. Remarkably, forced expression of plakoglobin during primed pluripotency, unlike b-catenin, leads to stabilisation of the pluripotency network rather than differentiation. Finally, after having extensively elucidated plakoglobin’s role within the continuum of pluripotency I used the microgel system to co-encapsulate ESCs with extraembryonic endoderm (XEN) cells. Co-encapsulation of these cell types led to the formation of self-organising aggregates in which the XEN cells surrounded an inner core of ES cells. These aggregates, termed EX-structures, exhibited deposition of a basal lamina, acquired apical-basal polarity, and initiated lumen formation with subsequent lineage-specific differentiation. Taken together, I have developed a cross-species compatible, compartmentalised system, for the suspension-culture of microgel-encapsulated embryonic stem cells that is generated in microfluidic devices. This interdisciplinary approach led to the identification of plakoglobin as a hitherto unknown, evolutionary conserved, regulator of naïve pluripotency. Furthermore, I have shown that co-culture of ES and XEN in microgels can mimic spatiotemporal events reminiscent to the peri-implantation embryo

Exploring the Frontiers of Female Fertility to Unlock the Full Potential of Assisted Reproduction

In females, reproduction is a key facet of women’s health, livestock production, and conservation of endangered species. The ovary is the gonad of the female and is a central component of female reproductive physiology as it houses the germ cells (i.e., oocytes) within follicles and produces hormones to coordinate folliculogenesis. The ovary is a target organ for developing novel assisted reproductive technologies (ART) such as safeguarding fertility via cryopreserving ovarian tissue containing follicles and developing strategies to grow early staged preantral follicles in vitro for oocyte production. Moreover, a better understanding of cellular and molecular events constituting early gonad development would be widely valuable to further advance ART that capitalizes on components of the ovary. In this work, we describe three aims using the bovine as a model and collectively expand our current knowledge in 1) ovarian tissue cryopreservation (OTC), 2) in vitro bovine preantral follicle development, and 3) in vitro differentiation of bovine embryonic stem cells (bESCs) into progenitors of bipotential gonad-like cells. In Aim 1, we found that, overall, slow freezing outperformed vitrification when bovine ovarian fragments were thawed and cultured as indicated by higher rates of normal follicle morphology, lower rates of apoptosis in stromal cells, and proper expression of the gap junction protein connexin 37. In addition to the use of OTC and culturing ovarian tissue fragments to rescue folliculogenesis, in Aim 2 we explored the use of bioengineered and proteolytically degradable (poly)ethylene glycol (PEG) hydrogels for in vitro culture of isolated bovine preantral follicles. We also tested co-encapsulation of mesoderm-like cells (MeLCs) from bESCs or native bovine ovarian cells (BOCs) with follicles to determine their propensity to become theca-like cells and improve folliculogenesis in vitro. After customizing a PEG hydrogel to complement bovine preantral follicle extracellular matrix (ECM) enzyme expression, we found that smaller follicles grew better in PEG hydrogels compared to two-dimensional control over ten days. However, MeLCs did not survive PEG hydrogel encapsulation process. Although BOCs could survive in PEG hydrogels throughout the culture period, BOCs did not better support follicle survival and development when co-encapsulated compared to PEG-only control. Moreover, the culture conditions did not maintain expression of selected genes essential for theca cell differentiation. Finally, in Aim 3, we tested the ability of bESCs to be driven towards the intermediate mesoderm (IM), early coelomic epithelium (eCE), and steroidogenic state during in vitro culture using several protocols. Our data indicated that inclusion of the steroidogenic factor-1 (SF1) agonist, RJW100, did not upregulate steroidogenic genes during the differentiation. Similarly, the cytokines basic fibroblast growth factor (bFGF) and bone morphogenic protein-4 (BMP4) were dispensable for inducing IM and eCE gene expression, and WNT activation was a predominant driver of eCE and IM gene expression, high concentrations of BMP4 led to upregulation of lateral plate mesoderm gene expression, and paraxial mesoderm gene expression remained unchanged across all regimens. Collectively, this dissertation work sheds light on several facets of ovarian biology including a better understanding of the impacts of OTC, in vitro preantral folliculogenesis, and use of embryonic stem cells to recapitulate bipotential gonad somatic cell differentiation. Nevertheless, additional research is required to elucidate mechanisms that are pivotal to both early folliculogenesis and early embryonic gonad formation in the bovine model

The support of undifferentiated human embryonic stem cell lines by different matrices

The future of human embryonic stem cell (hESC) research with regards to their applicability in a therapeutic setting, relies on the development and standardisation of consistent and robust methods to demonstrate their defining characteristics; their pluripotent ability to form all three germ layers and their capacity for self-renewal. Although much research has been carried out to investigate new methods of culturing hESCs, many of these studies have not robustly concluded the impact of prolonged culture on genetic and genomic stability nor have they examined in any comparative detail the impact of the culture conditions such as differences in feeders used or the media composition in which the stem cells are cultured in. The aim of this thesis therefore was to investigate and evaluate methods for improving the uniform and robust culture and characterisation of hESCs over prolonged periods in culture. Four hESC lines ( RH5, HUES9, SHEF1 and NCL5) were chosen on the basis that they had not previously been well characterised and therefore could potentially benefit the wider stem cell community by increasing diversity, rather than continue to use the already small subset of well publicised lines. The RH5, HUES9, SHEF1 and NCL5 cells were subjected to long term passaging using recombinant enzyme TrypLE™ Express, on human feeders, mouse feeders and feeder free matrix Matrigel in combination with defined media mTeSR1, for uniform scale up. Changes in characteristic stem cell surface markers were compared using two techniques; flow cytometry and quantitative in situ fluorescence microscopy. Genomic stability was assessed by real time PCR. Chromosomal integrity was monitored using array genomic hybridisation (aCGH). Array genomic hybridisation analysis of cells cultured for 20 passages by enzymatic passaging revealed changes in copy number variations in all the stem cell lines. Aberrations on chromosomes 12, 17 and 20, appeared most commonly as a result of long term culture. Although no significant differences were seen between hESCs cultured on mouse and human feeders, cultures on Matrigel showed fewer detected chromosomal aberrations. Expression of cell surface stemness markers SSEA3, SSEA4, TRA1-60 and TRA1-81 were maintained by hESC cultured on all matrices and confirmed by the use of flow cytometry and high throughput quantitative immunofluorescence imaging using the TissueFaxs™ cell analysis microscopy system. In depth imaging revealed subtle but important differences in the way in which hESCs attach and proliferate on different matrices. Genetic profiling of each of the stem cell lines using Taqman Low density array cards to assess the expression of 96 genes by Real Time PCR, demonstrated the continued expression of stemness genes 21 at late passage, and low level expression of differentiation genes, inherent to particular stem cell lines. Although both mouse and human feeders and Matrigel support the undifferentiated growth of hESCs, subtle differences from the hESCs were seen as a result of their use, most obviously, changes in morphology and how they proliferate. This was further explored in the stem cell line NCL5, as it demonstrated a readiness to adapt to new matrices, better chromosomal stability and higher expression of cell surface markers compared with the other hESC lines. Using in vitro differentiation assays to all three germ layers, NCL5 cultured to late passage (p+20) on human feeder iMRC5, mouse feeder iMEF and feeder free matrix Matrigel, demonstrated the ability to differentiate to ectoderm, endoderm and mesoderm progenitors after induction using three 7 day flat based directed differentiation protocols. Altered differentiation patterns were detected by Real Time PCR and TissueFaxs™ imaging and quantitative analysis, as a consequence of the prolonged culture on the specific matrices used. Such key findings allude to the strong influences of microenvironment and will help to improve the standardisation of in vitro differentiation assays. From these studies, chromosomal changes had no impact on NCL5 stem cell lines‘ ability to form progenitors, however small genetic instabilities may still play a role in terminal differentiation of germ lineage specific cell types. The findings of the programme of work described has led to the successful culture methods and characterisation testing validated in this project being incorporated into routine culture and banking of research grade hESCs at the UK Stem Cell Bank. These protocols will now be made more widely available and should assist stem cell researchers in adopting the most suitable and optimum conditions for culturing stem cells in the undifferentiated and stable state. With the huge surge in stem cell research over the past decade, the development of robust characterisation and culture methods will undoubtedly have significant impact on the exploitation of these cells for regenerative medicine and to assist with this a future aim of the stem cell bank will be to standardise methodologies for clinical grade banking