151 research outputs found
PLATFORM FOR CALCIUM IMAGING, ISOLATION, AND CHARACTERIZATION IN PRIMARY COLON EPITHELIAL CELLS
No full textThe colonic epithelium, lining the innermost portion of the large intestine, is subjected to continued mechanical and chemical stress therefore requiring constant self-renewal. This renewal is supplied from the colon crypts which maintain and protect the intestinal stem cells in an undifferentiated state at the base. Through a multitude of growth factors, metabolites, gases, and extracellular matrix gradients along the colon crypt contributing to proliferation at the base and differentiation as cells migrate upwards. Recently, Ca2+ signaling has been identified as an integrator of multiple mitogenic signaling contributing to the regulation of stem-cell differentiation. These findings have yet to be fully explored with primary mammalian colon cells and face challenges in imaging 3D architecture such as organoids. Chapter 2 addresses these challenges performing calcium imaging in 2D primary murine colon monolayers. Adopting imaging strategies originally developed for neuron calcium imaging, and image analysis pipeline was built capable of accurately isolating and extracting Ca2+ cell signals from the colon epithelium. Analyzing the isolate Ca2+ showed a correlation between higher frequencies and lower pulse width with proliferative cells, while broader oscillations occurring at a lower frequency were associated with differentiation. In Chapter 3 the impact of different growth factors is shown on the spatial temporal properties of the colon cell Ca2+ signals, demonstrating a similar relationship between higher frequency Ca2+ signals and proliferation. Chapter 4 improves on the improves on the previous work introducing cells zones undergoing proliferation and differentiation, allowing for more targeted investigation in the Ca2+ signaling found in proliferative colon cells.Doctor of Philosoph
FATTY ACID HANDLING AND LINEAGE MATURATION BY THE HUMAN INTESTINAL EPITHELIUM
No full textThe intestinal epithelium performs a variety of functions that are essential to maintaining human health. These processes are carried out by specialized absorptive and secretory cell types that arise from a common pool of intestinal stem cells (ISCs). The work presented in this dissertation centers around two fundamental cellular processes: 1) fatty acid (FA) handling, and 2) ISC lineage maturation. I will specifically focus on a) mechanisms of FA import, metabolism and export, and b) early cell fate decisions that sort tuft and enteroendocrine lineages through temporal SOX transcription factor expression dynamics. As much conventional work on FA handling and ISC lineage maturation has been carried out in animal models, little is known about the cellular mechanisms regulating these functions in human gut epithelium, thus my work aims to investigate these processes in the context of in vivo and in vitro models using primary human gut tissues. First, I performed single-cell RNA-sequencing to characterize in vivo human small intestinal epithelium. Informed by this analysis, I then developed a high-throughput model capable of mimicking in vivo absorptive enterocyte differentiation, maturation, and FA handling. Using this model, I found that the anti-diabetic drug metformin enhances absorptive enterocyte FA oxidation and export. In a second research focus, I investigated mechanisms of tuft and EEC differentiation and found evidence that suggests human tuft cells undergo a SOX factor relay, where SOX4 is transiently upregulated in secretory progenitors then SOX9 is upregulated and persists for terminal tuft cell maturation. Together these studies provide a framework for investigating human intestinal epithelial functions at the cellular level using in vivo and in vitro gut tissues.Doctor of Philosoph
Resolving Biological Trajectories in Single-cell Data using Feature Selection and Multi-modal Integration
No full textSingle-cell technologies can readily measure the expression of thousands of molecular features from individual cells undergoing dynamic biological processes, such as cellular differentiation, immune response, and disease progression. While computational trajectory inference methods and RNA velocity approaches have been developed to study how subtle changes in gene or protein expression impact cell fate decision-making, identifying characteristic features that drive continuous biological processes remains difficult to detect due to the inherent biological or technical challenges associated with single-cell data. Here, we developed two data representation-based approaches for improving inference of cellular dynamics. First, we present DELVE, an unsupervised feature selection method for identifying a representative subset of dynamically-expressed molecular features that resolve cellular trajectories in noisy data. In contrast to previous work, DELVE uses a bottom-up approach to mitigate the effect of unwanted sources of variation confounding inference and models cell states from dynamic feature modules that constitute core regulatory complexes. Using simulations, single-cell RNA sequencing data, and iterative immunofluorescence imaging data in the context of cell cycle and cellular differentiation, we demonstrate that DELVE selects genes or proteins that more accurately characterize cell populations and improve the recovery of cell type transitions. Next, we present the first task-oriented benchmarking study that investigates integration of temporal gene expression modalities for dynamic cell state prediction. We benchmark ten multi-modal integration approaches on ten datasets spanning different biological contexts, sequencing technologies, and species. This study illustrates how temporal gene expression modalities can be optimally combined to improve inference of cellular trajectories and more accurately predict sample-associated perturbation and disease phenotypes. Lastly, we illustrate an application of these approaches and perform an integrative analysis of gene expression and RNA velocity data to study the crosstalk between signaling pathways that govern the mesendoderm fate decision during directed definitive endoderm differentiation. Results of this study suggest that lineage-specific, temporally expressed genes within the primitive streak may serve as a potential target for increasing definitive endoderm efficiency. Collectively, this work uses scalable data-driven approaches to effectively manage the inherent biological or technical challenges associated with single-cell data in order to improve inference of cellular dynamics.Doctor of Philosoph
MICRORAFT ARRAY PLATFORMS FOR SORTING LIVE-CELL COLONIES BASED ON INTRACELLULAR PROTEIN FLUORESCENCE
No full textCharacterization of intracellular protein states in live cells is necessary to monitor changes in cellular physiology. Protein labeling methods, such as gene-engineered fluorescent proteins or fluorescently tagged antibodies are extensively used to measure intracellular protein abundance, co-localization, and post-translational modification within cells. However, the development of clonal fluorescent reporter cell lines is limited by the low efficiency of gene-engineering, laborious sorting methods, and cell sensitivities; and intracellular immunofluorescence assays are typically end-point assays without straightforward options to relate cell fluorescence measurements to downstream biological studies. Microraft array microdevices may address these methodological issues by miniaturizing cell culture sites, enabling gentle manipulation of cells, and being compatible with automation. Still, no robust microtechnologies. including microraft arrays, have combined pipelines for cell cloning and culture with colony sampling, collection, and assay in parallel. Parallelization of these processes has the potential to stream-line colony screening pipelines, leading to cutting-edge research discoveries and more rapid development of cell lines for biomedical applications. In this dissertation, microraft array-based platforms were advanced to miniaturize cell sampling, bioassay, and banking pipelines for stem and cancer cells. Specifically, the platforms were optimized for the culture, characterization, and sorting of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs) and basophilic leukemia cells. First, microraft arrays were applied to the cloning and sorting of hESCs gene-engineered to express endogenous reporters of pluripotency, resulting in clonal reporter lines that will enable the complex networks involved in pluripotency and differentiation to be investigated. Next, quad microraft arrays were optimized for hiPSC colony maintenance and subculture, and the reprogramming of peripheral blood cells into hiPSCs, enabling preservation and generation of pluripotency in cells, essential for personalized medicine applications. Finally, a semi-automated colony sorting pipeline involving a novel magnetic microwell-based immunostaining plate was developed, enabling the collection of colony fragments, intracellular protein level quantification, and retrieval of viable mother colonies matched to the daughter fragment. Together, these microraft array-based approaches have the potential to advance colony screening pipelines by increasing bioassay throughput and enabling cell sorting by a large number of criteria not currently suitable for viable cell sorting.Doctor of Philosoph
ENGINEERING HIGH-RESOLUTION EXPERIMENTAL AND COMPUTATIONAL PIPELINES TO CHARACTERIZE HUMAN GASTROINTESTINAL TISSUES IN HEALTH AND DISEASE
No full textIn recent decades, new high-resolution technologies have transformed how scientists study complex cellular processes and the mechanisms responsible for maintaining homeostasis and the emergence and progression of gastrointestinal (GI) disease. These advances have paved the way for the use of primary human cells in experimental models which together can mimic specific aspects of the GI tract such as compartmentalized stem-cell zones, gradients of growth factors, and shear stress from fluid flow. The work presented in this dissertation has focused on integrating high-resolution bioinformatics with novel experimental models of the GI epithelium systems to describe the complexity of human pathophysiology of the human small intestines, colon, and stomach in homeostasis and disease. Here, I used three novel microphysiological systems and developed four computational pipelines to describe comprehensive gene expression patterns of the GI epithelium in various states of health and disease. First, I used single cell RNAseq (scRNAseq) to establish the transcriptomic landscape of the entire epithelium of the small intestine and colon from three human donors, describing cell-type specific gene expression patterns in high resolution. Second, I used single cell and bulk RNAseq to model intestinal absorption of fatty acids and show that fatty acid oxidation is a critical regulator of the flux of long- and medium-chain fatty acids across the epithelium. Third, I use bulk RNAseq and a machine learning model to describe how inflammatory cytokines can regulate proliferation of intestinal stem cells in an experimental model of inflammatory hypoxia. Finally, I developed a high throughput platform that can associate phenotype to gene expression in clonal organoids, providing unprecedented resolution into the relationship between comprehensive gene expression patterns and their accompanying phenotypic effects. Through these studies, I have demonstrated how the integration of computational and experimental approaches can measurably advance our understanding of human GI physiology.Doctor of Philosoph
Real-Time Evaluation of Primary Human Colonic Epithelial Responses to Inflammatory Stimuli: Clostridioides difficile Toxins and Cytokines
No full textThe healthy human colonic epithelium (hCE) is exposed to luminal contents from digested meals, ingested medications, and microbial byproducts and toxins. One function of the hCE is to form a barrier against luminal contents while allowing for water and ion absorption. It is thought that one of the early initiators of inflammation, such as in inflammatory bowel disease (IBD), is a breach in the barrier that allows luminal contents to start an uncontrolled cascade of inflammatory events that cannot be naturally resolved. This creates a continual inflammatory environment impairing intestinal stem cell (ISC)-driven regeneration, resulting in epithelial ulcers. Models to investigate inflammation initiation lack physiological relevance as they rely on cancer cell lines or animal models which have fundamental differences from human physiology. Newer organoid models are an improvement but have limited access to the luminal compartment for real-time tracking of cellular processes.This dissertation focuses on the development of new hCE culture platforms and key physiologically relevant readouts that enable real-time monitoring of the earliest events in epithelial disruption due to clinically relevant Clostridioides difficile toxins A and B and inflammatory cytokines. Chapter 1 provides an overview of human small intestinal versus colonic epithelium in homeostasis and disease, in vitro intestinal culture models, and engineering tools to address limitations of existing models. In Chapter 2, I engineer and validate a human ISC reporter toolkit to study inflammation. In Chapter 3, I develop an hCE platform that mimics in vivo absorptive colonocyte transcriptomics using ISC-derived differentiated monolayers and a tight junction reporter to model the impact of C. difficile toxins on intestinal barrier integrity. Chapter 4 combines genetically engineered reporter lines with novel microfabricated platforms that compartmentalize ISC and differentiated cell zones to study ISC proliferation in response to IBD-associated interleukin-22. In Chapter 5, I share conclusions based on results described in this work and reflect on future directions and the broader impact of these findings.Doctor of Philosoph
THE EFFECTS OF ENVIRONMENTAL CHALLENGES ON HUMAN AND MURINE PRIMARY INTESTINAL CELL BEHAVIOR
No full textAs new research continues to support associations between colonic health and overall health, the need for a physiological model of the colon has become a high priority. Current in vitro models are often cumbersome, irreproducible, and seeded with cancerous cells as opposed to primary cells. The following work evaluates the effects of environmental challenges on primary cells using a simple and robust in vitro model of the colonic epithelium. The environmental challenges include adjustments in media composition with regards to glucose and butyrate, inflammatory and anti-inflammatory factors, and oxygenated vs de-oxygenated conditions. Cellular behavior under these varying conditions was evaluated via image analysis, epithelial permeability and electrical resistance, cell morphology, differentiation, ALP and Muc2 secretion, polarity, and cytokine production. This dissertation provides a thorough discussion of the results of these evaluations. While Chapter 1 provides a review on primary cell use in colonic in vitro models, Chapters 2-5 address the aforementioned environmental challenges in different combinations. In Chapter 2, hyperglycemia was paired with inflammatory factor TNFα. The data demonstrate that this combination has a dose-dependent effect on cell morphology. Additionally, TNFα was shown to drive production of mucus, but reduce mucus (muc2) secretion. Lastly, TNFα induced secretion of cytokines IL-8 and IL-1β, which was exacerbated by hyperglycemia. In Chapter 3, hyperglycemia was paired with hypoxia +/- a low level of butyrate. When oxygen was removed from the environment, cellular behavior adjusted to conserve energy through reduction of proliferation and alterations in differentiation that rely on energy source availability (glucose vs butyrate). Additionally, butyrate was shown to improve barrier function and integrity, as well as induce changes in proliferation and differentiation. Chapter 4 focuses on the effect of polyphenols of Buddleja Scordioides on human primary colon cells. Extracts and infusions of B. scordioides as well as exposure to pure polyphenols (quercetin and luteolin) were demonstrated to be non-toxic and shown to promote the maintenance of the healthy epithelial barrier. Finally, Chapter 5 pairs glucose +/- butyrate with hypoxia in primary murine cells. Similar to findings in human primary cells, murine cells were shown to be tolerant of anoxia. Murine cells also demonstrated alterations in proliferation and differentiation when deprived of oxygen. As described in the conclusions (Chapter 6), the observations recorded in this dissertation demonstrate the high value of the in vitro model utilized for these studies. Additionally, each unique study provided valuable insight into cellular behavior under various combinations of environmental stress.Doctor of Philosoph
Targeted disruption of the mouse ferrochelatase gene producing an exon 10 deletion
No full textAbstractProtoporphyria is a disease characterized by a deficiency in ferrochelatase, the terminal enzyme in the heme biosynthetic pathway, which catalyzes the chelation of iron and protoporphyrin to form heme. Clinical symptoms arise from an accumulation of protoporphyrin behind the partial enzyme block and include photosensitivity and sometimes hepatobiliary disease. Protoporphyria is described as an dominant disease, yet patients exhibit decreased ferrochelatase activities of 15–30% of normal, not 50% as might be expected. Missense, nonsense, and splicing mutations have been identified in ferrochelatase cDNA from protoporphyric patients. In this study we introduce an exon 10 deletion, an analogous mutation to that described in some protoporphyric patients, into the mouse embryonic stem (ES) cell genome via homologous recombination. Targeted ES cells were confirmed by Southern blot analysis. Expression of wild-type and exon 10-deleted mRNA was demonstrated by reverse transcriptase–polymerase chain reaction (RT–PCR) and cDNA sequencing. Ferrochelatase levels were analyzed by immunoblotting. Ferrochelatase activity was measured by the chelation of zinc and mesoporphyrin, and by the decrease in protoporphyrin accumulation after adding δ-aminolevulinic acid. In the exon 10+/− ES cells there is expression of both wild-type and exon 10-deleted mRNA, a 50% decrease in cross-reactive material with an anti-ferrochelatase antibody, and an approximate 50% decrease in ferrochelatase activity compared to wild-type ES cells. Therefore, an exon 10 deletion alone is insufficient to decrease ferrochelatase activity to the levels in protoporphyric patients. This suggests the requirement of an additional mutation to decrease the expression of the wild-type allele
Sox9 expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro
No full textThe inability to identify, isolate, and culture intestinal epithelial stem cells (IESCs) has been prohibitive to the study and therapeutic utilization of these cells. Using a Sox9EGFP mouse model, we demonstrate that Sox9EGFP fluorescence signatures can be used to differentiate between and enrich for progenitors (Sox9EGFPsubLo) and multipotent IESCs (Sox9EGFPlo). Sox9EGFPlo cells generate “organoids” in a recently defined culture system that mimics the native IESC niche. These organoids possess all four differentiated cell types of the small intestine epithelium, demonstrating the multipotent capacity of Sox9EGFPlo cells. Our results are consistent with the previously reported observation that single IESCs generate cryptlike units without a detectable mesenchymal cell component. A prospective search revealed that CD24 is expressed in the Sox9EGFPlo population and marks IESCs that form organoids in culture. CD24 represents the first cell surface marker that facilitates fluorescence-activated cell sorting enrichment of IESCs with widely available antibodies without requiring a specialized fluorescent reporter gene mouse model
Placental DEPTOR as a stress sensor during pregnancy
No full textThe author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @ 2012 Portland Press. The article has been made available through the Brunel Open Access Publishing Fund.DEPTOR [DEP-domain-containing and mTOR (mammalian target of rapamycin)-interacting protein] is a modulator of mTOR signalling that binds to mTORC (mTOR complex) 1 and mTORC2. However, to date, the precise functions of DEPTOR are not fully elucidated, particularly in reproductive tissues where mTOR acts as a placental nutrient sensor. Pregnancy is associated with major physiological and psychosocial changes and adaptation to these changes is crucial for normal fetal development. In the present study, we tested the hypothesis that maternal stress can affect mTOR signalling at term, and, as a result, influence placental growth. We first investigated the expression of DEPTOR, mTOR, rictor (rapamycin-insensitive companion of mTOR) and raptor (regulatory associated protein of mTOR) from human placentas (n=23) using Q-PCR (quantitative PCR), and correlated these data to days of pregnancy and maternal stress, as well as placental and fetal weight. Maternal and fetal cortisol levels were also measured. JEG-3 and BeWo cells, used as placental in vitro models, were treated with cortisol and DEPTOR expression was assessed using Q-PCR. DEPTOR appears to be the predominant transcript in the human placenta compared with mTOR, rictor and raptor in both term (n=13) and preterm (n=10) placentas as assessed by Q-PCR. There was a significantly lower level only of log-DEPTOR gene expression in the high stress group (-1.34) than in the low stress group (0.07; t₂₀=2.41, P=0.026). Interestingly, mothers with high stress had significantly elevated levels of cortisol (8555 pg/ml) compared with those with low stress (4900 pg/ml). We then tested the hypothesis that cortisol can directly affect DEPTOR expression. When BeWo cells were treated with cortisol 10, 100 and 1000 nM, the expression of DEPTOR was significantly down-regulated by 50, 41 and 39% (all P<0.05) respectively when compared with basal levels. Treatment of JEG-3 cells with cortisol, led to a significant decrease of DEPTOR expression at 100 nM (39%, P<0.05) and at 1000 nM (73%, P<0.01). These novel findings are indicative of a higher order of complexity of DEPTOR signalling in the human placenta that is affected by maternal stress, which could affect pregnancy outcome
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