The discovery of human pluripotent stem cells (hPSCs) has opened a new field called regenerative medicine that offers new strategies for curing diseases and drug discovery. It also provides the means of regenerating disease-relevant cells in vitro for disease modeling, and the possibility of cell replacement therapy. Among the most promising applications of hPSCs technology is the generation of blood cells that can be used for engraftment or transfusion in the clinic. Generating engraftable hematopoietic stem cells from hPSCs in vitro can fulfill the promise of using hPSCs to cure human diseases. Making functional HSCs in vitro from hPSCs remains an elusive goal. There are key pathways that are misregulated during hPSCs differentiation, which could impair the engraftment potential of hPSCs. WNT signaling is needed in the early phase of differentiation. However, evidence from mouse models and human development show that WNT signaling is downregulated during the maturation of HSCs. Therefore, we hypothesize that mimicking the dynamics of WNT signaling temporally during the differentiation could improve the functional maturation of differentiated HPCs. To this end, we have established an inducible gene activation system based on dCas9-VPR that can activate endogenous loci. We performed targeted activation of negative regulators of WNT. The system has shown promise in specific activation of WNT negative regulators, AXIN2 and APC2, but it needs further optimization to be able to steer cell fate and obtain functional HSCs.

Muscle homeostasis and regeneration are complex cellular processes orchestrated by muscle stem cells and their interaction with their stem cell microenvironment. The fate of a muscle stem cell is influenced by different conditions such as muscle injury, cold stress, or disease. During extensive muscle repair and in the context of muscular dystrophy, we identified the critical function of the Epidermal Growth Factor Receptor (EGFR) in establishing cell polarity and in turn the efficient formation of myogenic progeny able to repair muscle. Using a novel drug screen, we identified the p53 protein to regulate muscle stem cell fate decision to repress the formation of brown adipose tissue as a means to regulate whole-body metabolism. To increase the impact of our research we also optimized protocols evaluating mouse satellite cell transplantation to delineate stem cell hierarchy and developed a new paradigm to model human muscle stem cell fate to better translate our findings into the clinical arena. These findings reveal the tunable nature of stem cell fate decisions and highlight the development of research tools to accelerate the translation of research findings to improve human health.

Indiana University-Purdue University Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) allow for the unprecedented ability to recapitulate early stages of human development, which are otherwise inaccessible to investigation. This is especially true for one of the earliest events in human development, the establishment of a neuroretinal fate from an unspecified pluripotent population. To test the ability of hPSCs to serve in this capacity, hPSCs were generated using mRNA-reprogamming methods and maintained in xenogeneic-free differentiation conditions. These cells were directed to differentiate using a three-dimensional approach to analyze their ability to successfully recapitulate early events in human development in a temporal and developmentally-appropriate fashion. To do so, hPSCs were first directed to an anterior neural phenotype, which was confirmed by analysis of stage-specific neural transcription factors via immunocytochemistry and quantitative RT-PCR. Next, the cells were directed to an optic vesicle-like stage, where the presumptive retinal cells were identified by the expression of specific transcription factors. Finally, three-dimensional optic vesicle-like retinal organoids were identified, isolated, and further analyzed for the expression of markers associated with some of the differentiated cell types of the neural retina. Upon establishment of hPSC-derived retinal organoids, this system was further utilized to study the neurodegeneration in glaucoma and provide insights into the disease mechanisms. Overall, the results of this study help to demonstrate the suitability of hPSC-differentiation approaches as an effective tool to model retinal development and disease.

No description available.

The Wnt/β-catenin pathway is a fundamental regulator of embryonic development and adult tissue homeostasis. The key effector, β-catenin, is a multifunctional protein that occupies dual roles in signalling and intercellular adherens junctions. β-catenin primarily signals though the TCF/LEF transcription factors; however, many transcription factors, in addition to TCF/LEFs, interact with β-catenin, and the function of these interactions is poorly understood. To investigate novel β-catenin regulated signalling mechanisms with certainty, we developed TCF/LEF quadruple knockout (QKO) mESCs. In vitro differentiation of QKO cells reveals a neural differentiation bias, which is attenuated by overexpression of stabilized β-catenin. Our data indicate the presence of a TCF-independent β-catenin regulated neural differentiation blockade in mESCs. In addition to directly challenging the central dogma of canonical Wnt signalling, this finding has the potential to unveil new therapeutic targets for the treatment of many β-catenin-associated diseases, including forms of brain cancer that may arise from the oncogenic stimulation of neural stem cells. Furthermore, we describe an attempt to identify genome-wide TCF-independent β-catenin binding sites in QKO cells by ChIP-seq. Optimization trials provide proof of concept that the fold enrichment method of interpreting ChIP-qPCR results can be highly misleading when compared to the more comprehensive % input method of analysis. This conclusion has important implications for all fields of scientific research in which ChIP-seq methodology is employed. / Thesis / Master of Science (MSc)

No / Stem cells are known to differentiate in response to the chemical and mechanical properties of the substrates on which they are cultured. Thus, supramolecular biomaterials with tunable properties are well suited for the study of stem cell differentiation. In this report, we exploited this phenomenon by combining stem cell differentiation in hydrogels with variable stiffness and metabolomics analysis to identify specific bioactive lipids that are uniquely used up during differentiation. To achieve this, we cultured perivascular stem cells on supramolecular peptide gels of different stiffness, and metabolite depletion followed. On soft (1 kPa), stiff (13 kPa), and rigid (32 kPa) gels, we observed neuronal, chondrogenic, and osteogenic differentiation, respectively, showing that these stem cells undergo stiffness-directed fate selection. By analyzing concentration variances of >600 metabolites during differentiation on the stiff and rigid gels (and focusing on chondrogenesis and osteogenesis as regenerative targets, respectively), we identified that specific lipids (lysophosphatidic acid and cholesterol sulfate, respectively), were significantly depleted. We propose that these metabolites are therefore involved in the differentiation process. In order to unequivocally demonstrate that the lipid metabolites that we identified play key roles in driving differentiation, we subsequently demonstrated that these individual lipids can, when fed to standard stem cell cultures, induce differentiation toward chondrocyte and osteoblast phenotypes. Our concept exploits the design of supramolecular biomaterials as a strategy for discovering cell-directing bioactive metabolites of therapeutic relevance.

Activation of Ras/mitogen-activated protein kinase (ERK MAPK) signalling controls the differentiation of mouse embryonic stem (ES) cells. An established modulator of the ERK MAPK pathway is the IRS-1 (Insulin Receptor Substrate 1) family adaptor protein Gab1 (Grb2-associated binder 1). Gab1 is ubiquitously expressed and is activated by a wide range of cell surface receptors, mediating growth factor, cell-cell and cell-substratum interactions. The N-terminal region of Gab1 contains a pleckstrin homology (PH) domain required for membrane binding and a nuclear localisation sequence (NLS) that facilitates nuclear translocation. Undifferentiated mouse ES cells preferentially express high levels of a novel form of Gab1 (Gab1β) lacking the N-terminal region. Based on its novel structure and abundance, Gab1β may act in a dominant negative manner by binding and mislocalising downstream effectors. Alternatively, it may have a deregulated function unrestrained by the PH or NLS domains. Data presented here shows that Gab1β is tyrosine phosphorylated in response to the self-renewal factor Leukemia Inhibitory Factor (LIF) and/or Foetal Bovine Serum (FBS) stimulation. This then leads to the formation of complexes with Shp2 and the p85 subunit of PI3K. Experiments comparing the responses of wild-type and Gab1β knock-out ES cells indicate that Gab1β enhances ERK and potentially AKT phosphorylation in response to LIF. In contrast, Gab1β has a negative effect on ERK and AKT phosphorylation in response to IGF-1 (Insulin Growth Factor 1). These results suggest that the contribution of Gab1β to signalling activity is receptor specific and may imply that the response of ES cells to ERK activation is context specific. By reintroducing fluorescently tagged Gab1 proteins into Gab1β knockout ES cells, I investigated the localisation of Gab1β in ES cells. Gab1β localised at the cell membrane as well as in a perinuclear body. I next investigated the potential role of Gab1β in the differentiation of ES cells into neural precursors. A monolayer differentiation protocol was used to differentiate Gab1β wild-type and knock-out cells into neural precursors. Furthermore, the effect of insulin on the emergence of neural precursors from Gab1β-targeted cells was also explored.

No description available.

Cell-to-cell variation in expression of pluripotency- and lineage-determining factors has been proposed to be integral to the process of cell fate commitment in pluripotent cells both in vitro and in vivo. Understanding the sources of this heterogeneity in pluripotent stem cells promises greater insight into the mechanisms underlying cell fate choice. I identify mitochondrial membrane potential as an axis of heterogeneity in mouse embryonic stem cell populations, and show that high mitochondrial membrane potential marks cells that are in a stable self-renewing state. Partial overlap with previously described metastable subpopulations is demonstrated through gene expression analysis. I present evidence that similarly to previous findings in HeLa, heterogeneity in mitochondrial membrane potential is associated with variation in global transcription rate in mESCs. The direct impact of global transcription rate on differentiation propensity is demonstrated through manipulation of RNA Pol II transcription elongation rate. Mitochondrial variability is therefore likely a functionally relevant source of extrinsic gene expression variability in mouse embryonic stem cells.

Embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo and are characterized by the ability to self-renew and differentiate into all cell types of an adult organism, as demonstrated by their transplantation into embryos in the mouse. Isolation of cells with similar properties from human embryos has permitted the study of human cell differentiation in vitro as might occur during development. As such, human ES cells may be useful to assess and predict the developmental toxicity of environmental compounds capable of epigenetic alterations of the genome and its expression. The first objective of my research was to validate the functional significance to maintenance of an undifferentiated human ES cell state of expressed genes whose epigenetic modification is conserved across diverse lines and/or likely to be deterministic of an embryo stem cell associated epigenetic state. The second goal was to determine the sensitivity and relationship of the expression of these genes to environmental factors known to perturb the epigenome, specifically subcytotoxic exposure to diverse organic and metallic compounds and the availability of atmospheric oxygen. siRNA-mediated knockdown of genes previously identified on the basis of the conserved methylation status of gene associated Cytosine-Guanine islands (i.e. GLIS2, HMGA1, PFDN5, TET1 and JMJD2C) and two related family members (TET2 & 3) resulted in induction of cell differentiation in two independent human ES cell lines (RH1 and H9). Differentiation was reflected by morphological changes, reduction or loss of pluripotency associated markers, qualitative and quantitative reduction in genomic 5-hmC and upregulation of diverse germinal lineage markers. Subcytotoxic exposure of the same human ES cell lines to diverse compounds known to alter the epigenome (i.e. 5-azacytidine, sodium arsenite, cadmium chloride and valproic acid) generally induced downregulation of the aforementioned genes, loss of genomic hydroxymethylation and differentiation when applied under normoxia (20% O2), the exception being valproic acid. The same treatment applied under hypoxia (0.5% O2), did not induce differentiation, with the exception of cadmium chloride. Hypoxia is a general feature of developing embryos prior to the establishment of a maternal/fetal placental interface and fetal cardiovasculature. The protective effect of hypoxia was associated with elevation of ROS, expression of the dioxygenases TET1 and JMJD2C, and genomic hydroxymethylation. This research has demonstrated that genes identified on the basis of a conserved pattern of epigenetic modification function in the maintenance of an undifferentiated human ES cell phenotype. Furthermore, a human ES cell-based toxicology test system has been developed with which one can assess the subcytotoxic effects of compounds known to disrupt the epigenome and affect development by assessing their impact on maintenance of an undifferentiated human ES cell state. This is reflected by alterations in pluripotency markers, epigenetically-defined biomarkers and changes in global 5-hmC levels and the expression of genes responsible for this epigenetic modification (TET1-3). The epigenetically-defined biomarkers of pluripotent human ES cell identity (GLIS2, HMGA1, PFDN5, JMJD2C and TET1) could serve as biomarkers for screenings of compounds at an epigenetic level as their expression has been shown to be altered upon compound exposure along with monitoring the expression of 5-hmC.