Browsing by Author "Rancourt, Derrick E."
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Item Open Access Augmenting Genomic Applications Through Simulation and Machine Learning-based Parameter Optimization(2020-04-03) Li, Minghao; Long, Quan; Yang, Guang; De Koning, A. P. Jason; Rancourt, Derrick E.Certain methods for genomic analyses do not take advantage of the full extent of available biological context to address limited sample sizes. Another noted issue is the gulf in bioinformatics software performance between tool authors and end-users. This project explores two cases where genomic applications can be augmented: power estimations for low-prevalence condition studies and haplotype reconstruction. Power is a key statistic for predicting the success of genomic sequencing projects. Low-prevalence conditions are not amenable for usage with existing power estimation frameworks designed for common conditions and consequently will appear underpowered. SimPEL is a tool for simulation-based power estimation for sequencing studies of low-prevalence conditions. It meets an unmet need in the field and augments power estimation through the inclusion of unused genomic aspects of low-prevalence conditions. Elements of low-prevalence condition studies are input into SimPEL and a simulated cohort is applied to calculate the likelihood of identifying the true causal gene(s). SimPEL demonstrates competitive performance on single causal gene conditions and viable performance in instances of heterogeneity. PoolHapX is a haplotype reconstruction tool capable of reconstructing haplotypes and their corresponding frequencies from mixed populations. Its extensive parameter set is laborious to optimize by hand when applied toward unknown use-cases. Pattern recognition algorithms allow for the delegation of this parameter tuning process to machine learning. SLiM, an evolutionary simulation framework, provides the biological basis for the haplotype reconstruction task. Stochastic simulation of PoolHapX parameter values within a defined space generates the large-scale datasets required for supervised learning. Mapping genomic sequencing features to an optimal PoolHapX parameter set is a multi-task learning problem. A novel two-model scaffold has been designed to address this. A gradient boosted decision tree model, mapping PoolHapX parameter sets to a quantitative performance metric, is nested as the cost function of a multi-head feedforward neural network, which in turn takes an input set of summary statistics from aligned genomic data and outputs PoolHapX parameters. Hyperparameter tuning is enabled by Bayesian optimization techniques. This workflow and framework is parallely extendable toward any PoolHapX extension in the future.Item Open Access Cardiac extracellular matrix gel for ESC-derived cardiomyocyte maturation(2020-08-31) Thompson, Madalynn Jade; Rancourt, Derrick E.; Greenway, Steven C.; Krawetz, Roman J.; Ungrin, Mark D.The need for precision medicine has been established and the use of organoids to test individual drug efficacy and develop individual disease models is creating an opportunity for innovation. Human embryonic stem cells (hESCs) are commonly used for these applications but differentiation often results in immature phenotypes that may not be physiologically relevant. While hESCs can be differentiated into any cell type using small molecules, emerging concepts suggest that additional exogenous factors influence differentiation. In situ, stem cells exist in an organ-specific niche which contains extracellular matrix (ECM) proteins that have important paracrine effects to promote the maturity. This project aimed to investigate whether either of two alternative preparations of organ-specific ECM matrices support would offer improved support for tissue-specific differentiation and maturation. ECM matrices from porcine hearts (i.e. cardiogel) were prepared and decellularized either completely or partially. These cardiogel preparations were then studied using mass spectrometry and cytochemical staining before being used to support the maturation of hESCs that had been differentiated into cardiomyocytes (CMs). Moderate decellularization of porcine heart was achieved as shown by histochemical staining and the cardiogels contained variable amounts of important ECM components. However, neither cardiogel preparations appeared to increase the maturity of hESC-derived CMs, as assessed by cellular gene expression after 4 days of culture on cardiogel following differentiation. In conclusion, a protocol to generate cardiogels from porcine heart was achieved but significant batch variability was found. Further optimization may lead to a substrate which promotes cellular maturation and which can be used in conjunction with other technologies to increase cellular yield and homogeneity of resulting cardiac lineages.Item Open Access The Contribution of Endogenous and Exogenous Stem Cells in Fracture Repair(2019-12-17) Ferrie, Leah Elizabeth; Duncan, Neil A.; Krawetz, Roman J.; Matyas, John Robert; Rancourt, Derrick E.Throughout the lifespan, bone remodels in response to damage, such as fracture. However, diseases such as osteoporosis can cause impaired bone healing, increasing the risk of progression to non-repairing defects called fracture non-unions. Promoting the healing of fracture non-unions is a promising target for bone tissue engineering due to the limited success of current clinical treatment methods. There has been significant research on the use of stem cells with and without biomaterial scaffolds to treat bone fractures due to their promising regenerative capabilities. However, the relative roles of transplanted stem cells (exogenous stem cells) and stem cells found naturally in the body (endogenous stem cells) and their overall contribution to in vivo fracture repair is not well understood, thereby delaying the translation of new tissue engineering therapies to the clinic. The purpose of this research was to determine the interaction between exogenous and endogenous stem cells and biomaterials during bone fracture healing. This study was conducted using a burr-hole fracture model in a mesenchymal stem cell lineaging-tracing mouse. Burr-hole fractures were treated with collagen-I biomaterial loaded with and without green fluorescent protein tagged induced pluripotent stem cells. Using lineage-tracing, the roles of exogenous and endogenous stem cells during bone fracture repair could be elucidated. It was determined that in both a normal and impaired model of fracture healing treatment with exogenous stem cells did not result in improved bone formation and did not promote the recruitment of endogenous stem cells. However, treatment with exogenous stem cells in an impaired model of healing may offer healing advantages compared to in a normal model of healing. The outcomes of this study provide fundamental knowledge required for developing more effective stem cell and biomaterial therapies to treat bone fractures.Item Open Access Development of an extracellular matrix to improve pseudoislet survival for the treatment of Type I diabetes(2019-09-12) Wong, Sarah; Ungrin, Mark D.; Kallos, Michael S.; Rancourt, Derrick E.Throughout the islet transplantation process, many islets die due to removal from their native environment. Studies suggest that select extracellular matrices (ECMs) have the potential to support islet survival throughout transplantation. However, there is currently no overview evaluating ECM interactions with pseudoislets. In this study, three common methods of quantifying cell survival were applied to aggregates encapsulated in thin hydrogel sheets and their effectiveness was evaluated. Next, a Design of Experiments approach was used to evaluate composite hydrogel ECMs as a method of improving the survival of INS1 and primary human pseudoislets post-isolation. Encapsulating INS1 pseudoislets in [alginate] = 2wt/v%, [collagen I] ~ 4mg/mL, and [Matrigel] ~ 4mg/mL improved INS1 survival and proliferation when cultured for 3 days. Similarly, collagen I and Matrigel were found to have a neutral or positive effect on primary human pseudoislet survival when encapsulated in 2 wt/v% alginate gels and cultured for 5 days.Item Open Access ING1 Impacts Ovarian Cancer by Altering EMT(2019-08-30) Yang, Yang; Riabowol, Karl T.; Bonni, Shirin; Rancourt, Derrick E.; Muruve, Daniel A.The INhibitor of Growth (ING) proteins are type II tumour suppressors that regulate epigenetic state and transcription by recruiting various chromatin-modifying complexes to chromatin. INGs are involved in multiple cellular processes such as DNA repair, apoptosis and cellular senescence. In this study, we investigated the potential role of ING1 in inhibiting the epithelial-mesenchymal transition (EMT) program that suppresses cancer metastasis and identifies the mechanism of how ING1 regulates gene transcription, as well as its clinical implication in epithelial ovarian cancer. Our analyses revealed that ING1 epigenetically regulates the transcription machinery of the EMT program through binding to the promoter region of Twist1, the critical gene encoding transcription factor (TF) that induce EMT. Subsequently, ING1 promoted the expression of epithelial cell markers, including E-cadherin, while suppressing the expression of mesenchymal markers such as N-cadherin. ING1 antagonized TGF-β or EGF induced EMT and inhibited cancer cell invasion and migration in a Twist1-dependent manner. Integration of ChIP-seq and RNA-seq data revealed the overall genomic binding characteristics of ING1 and its candidate target genes. Gene ontology (GO) and pathway analyses indicated that the genes targeted by ING1 were involved in diverse physiological processes and pathways, mostly associated with the EMT program. The bioinformatical analysis revealed the ING1 binding motif sequence, showing that ING1 recognized target genes through the TEAD family of transcription factors and their co-factor, YAP1, to further influence gene expression. Lastly, we found that high ING1 protein expression was associated with p16 and ARID1A levels and predicted better DSS in ovarian clear cell carcinoma (OCCC). Our data also suggested that high ING1/low N-cadherin expression predicts favourable disease-specific survival (DSS) in epithelial ovarian cancer. These data provide evidence that as a tumour suppressor, ING1 can impact ovarian cancer development through modulating the EMT process and play an essential role in regulating ovarian cancer chemoresistance. In summary, by downregulating the EMT-TF expression and hence and EMT program, ING1 suppresses tumour cell invasion and cancer metastasis. Global genomic profiling revealed new insights into ING1 biology and provided justification for further exploring ING1 functions. This study has also provided critical pre-clinical data that could help establish ING1 as a prognostic and therapeutic agent for ovarian cancer.Item Open Access Long-term co-culture of endothelial and cancer cells within biomimetic microfluidic chips for investigating juxtacrine and paracrine regulatory signaling pathways(2019-01) Soleimani, Shirin; Sanati-Nezhad, Amir; Rancourt, Derrick E.; Ungrin, Mark D.Emerging evidence on how angiocrine factors confer inductive signals to orchestrate a wide range of pathophysiological phenomena (e.g. tumorigenesis, self-renewal of hematopoietic stem cells, regenerative lung alveolization and liver regeneration) lends credence to the concept that endothelial cells are not only the building blocks of vascular networks but also serve as a rich resource of regulatory angiocrine factors. Cancer-associated endothelial cells initiate a series of signaling modes with cancer cells. These signallings determine cancer progression and response to drugs. While it is widely-believed that endothelial cells are only able to affect the cancer cells that are juxtaposed next to them, cells positioned five-cell diameters away from the sinusoid vessels have been shown to be ruled by angiocrine factors. Considering the distance dependency of cancer-endothelium crosstalk, there is a great demand for an in vitro system with high geometrical precision that offers a diverse range of cell-cell distance and cell-cell contact area for studying and distinguishing juxtacrine and paracrine signallings between cancer and endothelial cells in a spatiotemporal manner. In the present study, a three-layer microfluidic chip consisting of a top and a bottom channel separated via a porous membrane is devised, to enable studying the signalling pathways between cancer and endothelial cells under highly-controlled conditions. The porous membranes are variable in terms of pore size, thickness and porous surface area. However, transition from macroscopic to microfluidic platforms for reconstructing in vitro models brings with it a variety of challenges such as the effect of device material (Tissue culture Polystyrene vs. PDMS), surface coating, cell number/surface area unit, cell seeding method and cell culture media exchange schedule. The present work has tried to provide a comprehensive protocol to supply precisely controlled conditions along the microchannels for the cells to grow, using human umbilical vein endothelial cells, E4OR1 transduced endothelial cells and breast cancer MCF-7 cells.Item Open Access Microscale Tissue Engineering and Contributors of the Cellular Niche(2019-05-31) Al-Ani, Abdullah; Ungrin, Mark D.; Biernaskie, Jeff A.; Kallos, Michael S.; Rancourt, Derrick E.; Hirota, Simon Andrew; Gratzer, Paul F.The behaviour of cells is modulated by their microenvironment or ‘niche’. While cellular therapies offer promising curative solutions for many diseases, the efficacy of transplanted cells is often hampered by a suboptimal microenvironment. One strategy to overcome this limitation is to reconstruct the niche of the cells of interest prior to transplantation. The central aim of this thesis is to develop novel tissue engineering approaches to further understand and reconstitute the cellular niche. While these approaches were specifically validated in the retinal and islet systems, they were also designed to be easily implemented in other biological systems. One project showcases a novel scaffold-free, scalable and injectable retinal pigment epithelium (RPE) microtissue for minimally-invasive transplantation. While RPE transplantation holds great potential to cure various retinal degenerative diseases, cells transplanted as cellular suspension exhibit suboptimal survival and function. Conversely, transplanting RPE as coherent cellular sheets has yielded better outcomes, but they are complex to transplant and produce at large scale. Our RPE microtissues were designed to capture the benefit of both approaches: namely, simplicity of production and transplantation, as well as enhanced performance. We found that our RPE microtissue exhibited superior cellular behaviour in terms of gene expression and in vitro function when compared to standard adherent culture. Another project presents a unique approach to produce transplants with a reconstituted cellular niche. This approach aims to repopulate the niche by incorporating finite amounts and proportions of niche cells into transplantable constructs. Using it enabled us to produce size-controlled pseudoislet constructs that contained various proportions of mesenchymal stem cells (MSCs), fibroblast and endothelial cells, and to quantitatively evaluate their in vitro performance. Further, applying this approach led us to discover more than one favourable condition that yielded improvements in islet cell performance in vitro. While the islets of Langerhans were used for biological validation, the approach was designed to be broadly applicable to various biological systems. In sum, this thesis offers several novel approaches for scientists to better understand and enhance the cellular niche. The simplicity, accessibility and scalability of these approaches render them suitable for both scientific applications and clinical translation.Item Open Access Microscale Tissue Engineering for the Study and Treatment of Diabetes(2018-12-12) Yu, Yang; Ungrin, Mark D.; Huang, Carol T. L.; Rancourt, Derrick E.Islet transplantation is a promising approach to the treatment of insulin-dependent diabetes. However, a major clinical challenge is inefficient survival and engraftment of the transplanted material. This has been associated with insufficient oxygen and nutrition delivery after loss of the endogenous capillaries, and stress induced during islet isolation and culture. Quantitative modelling of oxygen delivery predicts significant advantages for smaller islets, and consistent with this concept, smaller human islets have also performed better than larger ones in clinical settings. In order to understand and overcome these limitations for both research and clinical applications, we have established a microscale tissue engineering approach that is capable of consistently and efficiently generating size-controlled pseudoislets from human donor islets, yielding improved survival and function both in vitro and in vivo. We then combined this platform with advanced statistical methodologies and laboratory automation systems to enable assessment of large numbers of modifying factors (and their interactions) identified from the literature in parallel. This project has yielded a substantial improvement in the consistency and efficacy of islet cell packaging for transplantation, and laid a foundation for rapid transition to the clinic.Item Open Access Novel Culture Systems for Studying Proliferation and Maturation of Spermatogonia in vitro(2022-04-20) Sakib, Sadman; Dobrinski, Ina; Rancourt, Derrick E.; Ungrin, Mark D.The work presented in this dissertation aims to establish two novel culture systems for the proliferation and differentiation of spermatogonia. First, we established a suspension culture system for porcine spermatogonia using stirred suspension bioreactors. We showed that porcine pre-pubertal spermatogonia have a higher degree of proliferation in suspension culture compared to static culture. We also show that while spermatogonia proliferate more under ambient O2 compared to lower O2 tension of 10%, they are also likely to undergo differentiation. Our findings indicate that the higher proliferation of spermatogonia that is observed in suspension culture is partially mediated by the Wnt/ ?-catenin pathway. Other signal transduction pathways may also be activated in the stirred suspension bioreactor. This lays the foundation for future work aimed towards better understanding and improvement of suspension culture for spermatogonia. Second, we established a primate, human, mouse, rat and porcine testicular organoid system using the microwell culture system. We find that these organoids have testis-specific morphology and mimic testicular functions. We also present evidence that the mouse and rat organoids can undergo maturation and promote differentiation of spermatogonia. Our proof-of-principle experiments with ciliobrevin D, mono-2-ethylhexyl phthalate and cadmium chloride indicate that testicular organoids can serve as a platform for modeling testis morphogenesis and reproductive toxicity in vitro. In summary, the organoid platform established for this dissertation opens up an exciting avenue of research to further develop and adapt the system for studying the human male reproductive system in vitro in a tissue specific context.Item Open Access Reoviral Cytolysis is Modulated by Stemness(2020-10-13) Bourhill, Tarryn Jackie; Johnston, Randal N.; Rancourt, Derrick E.; Cobb, Jennifer A.; Mahoney, Douglas J.Oncolytic viruses (OVs) are an emerging cancer therapeutic that act by selectively targeting and lysing cancerous cells and by stimulating anti-tumour immune responses, while leaving normal cells mainly unaffected. Reovirus is a well-studied OV that received fast track designation and a special protocol assessment agreement from the FDA for the treatment of metastatic breast cancer. The mechanisms governing reoviral selectivity are not well characterised and are a topic of debate. Reovirus is capable of infecting and lysing cancer cells and, cancer stem cells, and here we demonstrate its ability to also infect and kill healthy pluripotent stem cells (PSCs). This has led us to hypothesize that pathways responsible for stemness modulate reoviral tropism. We find that reovirus is capable of killing murine and human embryonic and induced pluripotent stem cells. Differentiation of PSCs alters the cells’ reoviral-permissive state to a resistant one. In a cancer cell line that was resistant to reoviral oncolysis, induction of pluripotency programming renders these cells permissive to cytolysis. In light of the recent view that pluripotency induction shares similar pathways with carcinogenesis, the same subset of genes that are activated in cancer may also be up-regulated in PSCs (e.g. c-MYC), rendering PSCs permissive to infection. Bioinformatic analysis indicated that the Yamanaka factors may be involved in regulating reoviral selectivity, however this requires further experimental validation. Mechanistic insights from these studies will be useful for the advancement of reoviral oncolytic therapy.Item Open Access Robust bioprocess design and evaluation of commercial media for the serial expansion of human induced pluripotent stem cell aggregate cultures in vertical-wheel bioreactors(2024-07-29) Borys, Breanna S.; Dang, Tiffany; Worden, Hannah; Larijani, Leila; Corpuz, Jessica M.; Abraham, Brett D.; Gysel, Emilie J.; Malinovska, Julia; Krawetz, Roman; Revay, Tamas; Argiropoulos, Bob; Rancourt, Derrick E.; Kallos, Michael S.; Jung, SunghoonAbstract Background While pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified. Methods This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture. Results The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology. Conclusions This study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems.Item Embargo The Roles of ING5 in Regeneration and Maintaining Genomic Stability (in vivo model)(2023-08) Al Shueili, Buthaina; Raibowol, Karl T.; Jirik, Frank R.; Rancourt, Derrick E.In this thesis, we provide the first in vivo description of the functions of ING5, a member of the INhibitor of Growth (ING1-5) proteins that are epigenetic regulators. INGs target histone acetyltransferase (HAT) or histone deacetylase (HDAC) complexes to the H3K4Me3 mark of active transcription. ING5 targets MOZ/MORF and HBO1 HAT complexes to acetylate H3 and H4 core histones, respectively, affecting gene expression. Previous in vitro studies by us and others indicated that ING5 maintains stem cell character in normal and cancer stem cells. Moreover, ING5 has also been implicated in maintaining genomic stability by regulating post-translational modification of DNA repair proteins. Here we find that CRISPR/Cas9 ING5 knockout (KO) mice are sub-fertile but viable and show no decrease in lifespan despite signs of depleted stem cell pools in their brains, skin and peripheral nerves. Cardiac Fibroblasts established from knockout animals had accelerated growth rates, with higher heart fibrosis after an infarct injury. Evidence of chondrogenesis was also obtained in knockout hearts that had compromised heart function, indicating the significance of ING5 for heart integrity. Depletion of ING5 also affected DNA repair, as suggested by the accumulation of DNA damage and apoptosis in the testis, which affected animal fertility. Furthermore, middle-aged to old KO animals develop lymphomas at a rate approximately 6-fold higher than control mice. This may be due to loss of ING5 resulting in p53 instability, with several tissues showing very low or undetectable levels of p53 despite normal levels of p53 mRNA.