Browsing by Subject "Hepatocytes"

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    Effect of obesity on hepatic drug metabolism
    (2013-09) Chiney, Manoj Shriram
    Obesity has increased markedly over the last few decades and is now a major public health crisis in the U.S. affecting over 1/3 of the US population. Optimization of dosing in obese individuals is a challenge due to the lack of knowledge regarding changes in the pharmacokinetics (PK) of therapeutic agents in obese individuals. Thus the aim of this thesis was to determine the effect of obesity on drug metabolism and evaluate methods that could potentially predict changes in pharmacokinetics in the obese population. The impact of obesity on drug metabolism in children has not been determined and our clinical study (Chapter 2) was the first of its kind to examine the effect of childhood obesity on CYP1A2, CYP2D6, CYP3A4, xanthine oxidase, and NAT2 activity using caffeine and dextromethorphan as probe drugs. Our results conclusively indicate that obesity results in an elevation of xanthine oxidase and NAT2 enzyme activities in obese children as compared to lean children, whereas there was no difference in CYP1A2, CYP2D6 and CYP3A4 activity between obese and lean children. This study provides a potential mechanism of altered 6-mercaptopurine exposure in the obese pediatric cancer population. While clinical studies would provide the most optimum method to predict clearance of therapeutic agents in humans, studies have reported that clearance can also be predicted using animal data. In Chapter 3, we examined mouse, rat and porcine model of obesity in order to determine the utility of these animal models to predict PK in obese humans and to identify a model that would best reflect the human obesity mediated changes in drug metabolism. The study indicated species dependent differences in CLint of various drugs that were due to, either changes in expression of drug metabolizing enzymes or changes in enzyme substrate affinity. The study demonstrated that obesity can alter enzyme activity in a species and model dependent manner. Furthermore this study identified that the rat High Fat Diet animal model of obesity is the best representation of the obesity mediated alterations in humans. In Chapter 4, in collaboration with Drs. Scott Rector and Jim Perfield, University of Missouri, Columbia, we demonstrated obesity mediated alterations of drug metabolism enzyme activity can be prevented by sterculic oil dietary supplementation. These effects are mediated through signal transduction pathways which regulate CAR and PXR transcription factors. These results establish that obesity mediated changes are biochemically dependent and not weight dependent. In Chapter 5, we developed a proof of concept that would help generate biochemically obese hepatocytes. In absence of hepatocytes from obese individuals, these hepatocytes can be used as a tool to predict obesity mediated changes in drug clearance. Our studies indicate that individually, leptin, resistin, IL-6 and TNF-α can modulate expression of various DMEs in a concentration dependent and isoform specific manner. This study demonstrates that the obesity microenvironment is important in obesity mediated changes in drug metabolism. Additional studies would help establish a more robust method to generate and validate these obese hepatocytes. In summary, the work in this thesis has helped identify the drug metabolism enzymes that are altered in the obese children, the utility of using animal models of obesity as tools to study the impact of obesity on pharmacokinetics/pharmacodynamics, proven that it is possible to reverse obesity mediated changes in drug metabolism and developed an in vitro model that may be used to predict changes in drug disposition in the obese population. These findings are important for to better develop dosing strategies in obese humans with concomitant disease.
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    A Systems Approach to Studying the Differentiation of Stem Cells Towards Hepatocytes
    (2017-08) Chau, David
    The recent advancements in stem cell biology have allowed for new and exciting opportunities to use stem cells in clinical and industrial applications. Stem cells have the unique ability to self-renew and differentiate into any specialized cell type found in the body. Using certain mechanical and biochemical cues, stem cells can be directed to become any specific cell type, such as hepatocytes. A robust and efficient process for expansion and differentiation to generate large quantities of functional hepatocytes from stem cells will be essential to establishing a stem cell bioprocess in the future for therapeutic and industrial applications of hepatocytes. In this study, a differentiation protocol with soluble growth factors and cytokines was used to mimic the key signaling cues during embryonic development. However, most directed differentiation processes have run into issues with limited scalability and lack of functionality in the differentiated cells. In an effort to bring stem cell therapy closer to reality, our strategy was to use a systems-based approach to enhance the quality and yield of stem cell-derived hepatocytes. To achieve higher cell yield, we modified an existing differentiation protocol to incorporate a cell expansion stage to facilitate simultaneous differentiation and cell growth. Using transcriptome analysis and mass cytometry, we showed how the population of cells changed over time on both the transcript and protein level. Both analyses revealed that with the new expansion stage, we obtained a higher quantity of hepatocytes within the same time frame compared to the conventional method of differentiation. We then showed the capability to scale up our differentiation for larger scale cultures by adapting the expansion stage onto Cytodex 3 microcarriers. Using the same culture volume as a tissue plate culture, we demonstrated the ability to achieve up to a 5-fold increase in cell number with a final cell density in the range of 4-5x106 cells/ml. These strategies show that the demand for large quantities of hepatocytes can be met by translating the conventional method of differentiation to suspension microcarrier differentiation. Encouraged by our ability to yield higher cell density using microcarrier culture, we explored assessing the functional maturity of our stem cell-derived hepatocytes using transcriptome analysis. We showed that stem cell-derived hepatocytes are still clearly different when compared to primary hepatocytes at the transcriptome level. In addition to evaluating cells using transcriptome analysis, we wanted to be able to compare the current in-vitro processes to embryonic liver development to understand the genetic roadblocks. The transcriptome data from hESCs hepatocyte differentiation was integrated with mouse liver development using principal component analysis and batch corrections. This allowed us to create a unified developmental scale to compare samples from different species and in-vitro to in-vivo platforms. The meta-analysis revealed that stem cell-derived hepatocytes are equivalent to the functional maturity of developing cells at E15 in mouse development. From the transcriptome analysis, we observed many different genes in energy metabolism with dynamic behavior over the course of differentiation. We sought to understand the effect of changes in different metabolic genes and the impacts on metabolic transition during differentiation. We characterized the energy metabolism of hESCs and assessed the metabolic demand of cells at different stages of differentiation. hESCs and early differentiated cells exhibited a high glycolytic flux. transitioning towards an oxidative metabolism as the differentiation progressed. Furthermore, using confocal microscopy, we also characterized the activity and morphology of the mitochondria in the cells at different stages of differentiation. Using the consumption rates of different nutrients as an input to our metabolic flux model along with our transcriptome findings, we were able to gain a deeper understanding of the metabolic behavior of cells during differentiation. Our analysis revealed that cells consume lower amounts of glucose over the course of the differentiation but become more efficient at transporting pyruvate into the mitochondria leading to increased oxidative phosphorylation. However, our metabolic and transcriptome data revealed that our stem cell-derived hepatocytes are not capable of mature metabolic functions such as gluconeogenesis, supporting the immature phenotype that has been described in literature. Together, these studies reveal that stem cells can provide a renewable and scalable source of hepatocytes for therapeutic applications. These cells demonstrate some phenotypic and functional properties of primary hepatocytes but have some contrasting elements compared to their in-vivo counterparts that will need further genetic intervention to enhance their maturation before cellular therapy can become a reality. However, this work is invaluable as it contributes to the current status of the field and facilitates the translation of laboratory practices of stem cell culture into a scalable technology.
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    Systems Engineering of Stem Cell Fate
    (2015-08) Raju, Ravali
    Recent advances in the derivation of functional cells from pluripotent stem cells have raised hope for cell therapy to treat liver ailments. They have enhanced the prospects of developing reliable in vitro models for liver diseases and drug toxicity screening. A differentiation protocol mimicking key signaling cues of embryonic development was developed to direct stem cells (ES) towards the hepatic fate and express key hepatic markers and functions. While these results are encouraging, most directed differentiations from stem cells to the target cell types are hampered by lack of functional maturity, cell heterogeneity and low cell yields limiting their translation to the clinic. These cells are therefore refereed to hepatocyte-like cells (HLCs). An integrative strategy was employed including both experimental techniques as well as a systems-based analysis towards enhancing the product quality and yields of HLCs. Functional maturity was enhanced by initiating three dimensional spheroid formation upon differentiation. Enrichment of hepatic cells using selective medium conditions was performed to obtain higher fraction of cells with the desired properties. Cell expansion was incorporated during differentiation to improve cell yields. Several additional strategies have been used to increase hepatocyte maturity in literature including co-culture and transfection with transcription factors. These methods including ours have shown improvement, however a universal gap to maturation is still present when compared to primary hepatocytes. Comparison of transcriptome data of differentiation to embryonic liver development can elucidate the genetic roadblocks preventing ES cells from reaching the functional maturity of their tissue counterparts. Transcriptome data was compiled from various depositories for mouse fetal liver development (from E8.5 to post-natal). Transcriptome data was obtained during the time course of our human hepatic differentiation protocol and was augmented with human in vitro hepatic differentiation data in the public depository. Interestingly, majority of the HLCs are similar irrespective of the cell source and protocol. The entire cohort of HLCs clustered separately from the primary hepatocytes and adult liver indicating an inherent roadblock to maturation. The transcriptome data of human ES hepatic differentiations was then integrated with mouse liver development using a unique approach. This allowed us to identify the corresponding development stage at which the in vitro stem cell differentiation is blocked. The analysis uncovered a pivotal gene set with contrasting profiles in ES differentiation and mouse liver development that merit combinatorial genetic intervention to enhance maturation of ES derived hepatocytes. Thus, one can envision the availability of stem cell based liver therapies in the not so distant future.