Browsing by Author "Rancourt, Derrick"

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    Absence of p21 Expression Sensitizes iPSCs to iCaspase9 Induced Apoptosis While Not Impacting Ability to Participate in Exogenous Cartilage Repair
    (2022-11-01) Larijani, Leila; Rancourt, Derrick; Krawetz, Roman; Childs, Sarah J.; Bob, Argiropoulos
    p21 is a multifunctional protein that is critical to the control cellular proliferation and plays a number of other functions in the cell. In the nucleus, p21 functions as a tumour suppressor, while in the cytoplasm, phosphorylated p21 functions as a proto-oncogene and can also suppress apoptosis. Because p21 deficiency has been linked to increased tissue regeneration, in this thesis, I sought to investigate if p21 mutations can improve the regenerative capacity of induced pluripotent stem cells in a cellular therapy approach for cartilage injuries in mice. However, because p21 is a tumor suppressor and its loss can result in tumorigenesis, I employed the inducible Caspase9 cell suicide system to purge iPSCs through forced apoptosis. In vitro, iCaspase9-mediated cell death resulted in a statistically significant increase in the apoptosis rate in p21-/- iPSCs compared to p21+/+ iPSCs (wild-type). RNA sequencing was undertaken to determine which pathways are involved in p21 mediated apoptosis. Increased expression of 41 apoptotic and 6 healing-related genes were observed in p21-/- iPSCs compared to wildtype. In vitro chondrogenesis of p21-/- iPSCs showed an increase in chondrogenic genes relative to the wild-type cells. When these iPSCs were transplanted into a focal cartilage injury in mice, ectopic cartilage formation was observed. Neither tumors, nor ectopic cartilage formation was observed in mice that were treated with CID drug to induce iCasp9 mediated apoptosis. Finally, I performed a drug screen to discover compounds that promote apoptosis with iCasp9. In addition to identifying several epigenetic drugs that promoted iCasp9 function in vitro, I also demonstrated that p21 inhibitors could phenocopy the enhanced iCasp9 mediate cell killing seen previously in the p21 mutant iPSCs. Overall, my findings indicate that p21 does play a role in protecting iPSCs from apoptosis and its downregulation can be exploited to increase cell-suicide approach effectiveness. I was also able to demonstrate that iPSCs can induce robust cartilage repair in mice regardless of the expression of p21. Therefore, modulation of p21 warrants further research to determine if exploiting this protein can be used in iPSC clinical trials safely in the future.
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    Assessment of the Efficacy of MRI for Detectionof Changes in Bone Morphology in a MouseModel of Bone Injury
    (Wiley, 2013-07-11) Taha, May A; Manske, Sarah; Kristensen, Erika; Taiani, Jaymi; Krawetz, Roman; Wu, Ying; Ponjevic, Dragana; Matyas, John; Boyd, Steven; Rancourt, Derrick; Dunn, Jeffrey F.
    Purpose To determine whether magnetic resonance imaging (MRI) could be used to track changes in skeletal morphology during bone healing using high-resolution micro-computed tomography (μCT) as a standard. We used a mouse model of bone injury to compare μCT with MRI. Materials and Methods Surgery was performed to induce a burr hole fracture in the mouse tibia. A selection of biomaterials was immediately implanted into the fractures. First we optimized the imaging sequences by testing different MRI pulse sequences. Then changes in bone morphology over the course of fracture repair were assessed using in vivo MRI and μCT. Histology was performed to validate the imaging outcomes. Results The rapid acquisition with relaxation enhancement (RARE) sequence provided sufficient contrast between bone and the surrounding tissues to clearly reveal the fracture. It allowed detection of the fracture clearly 1 and 14 days postsurgery and revealed soft tissue changes that were not clear on μCT. In MRI and μCT the fracture was seen at day 1 and partial healing was detected at day 14. Conclusion The RARE sequence was the most suitable for MRI bone imaging. It enabled the detection of hard and even soft tissue changes. These findings suggest that MRI could be an effective imaging modality for assessing changes in bone morphology and pathobiology.
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    A Biophysical and Molecular Characterization of Human Embryonic Stem Cell-Derived Exosomes
    (2022-02) Heale, Kali; Rancourt, Derrick; Lees-Miller, Susan; Ungrin, Mark
    Exosomes are endocytic nanovesicles that facilitate intercellular communication via the transfer of biomolecules. There are currently several exciting applications for exosomes being developed in therapeutics and diagnostics, reflected in their increased appearance in the academic and commercial spheres. Despite the prospective uses of exosomes, their utility in research is complicated by their often inadequate characterization. This investigation aimed to characterize the biophysical and molecular properties of exosomes harvested from human embryonic stem cells (hESCs) following the guidelines of the International Society for Extracellular Vesicles. Aim 1 addressed a basic characterization of hESC-derived exosomal properties. This included an examination of exosome morphology, size, and protein marker presence via Transmission Electron Microscopy (TEM) and western blot. In Aim 2, the miRNA content of hESC-derived exosomes was investigated via RNA-Sequencing (RNA-Seq). This report begins with a look at the current and prospective uses of exosomes from stem cells in the academic and patent literature. This provides a robust list of potential applications which can be improved by successful characterizations. In Aim 1, while the data is unable to provide definitive evidence towards the isolation of hESC-derived exosomes, this report provides insight into improving practices for exosomal immunogold labelling and western blotting. Additionally, high-resolution imaging of samples isolated from a common exosome isolation procedure revealed the presence of vesicle-like structures with the morphology and size of exosomes. Finally, in Aim 2, sequencing data provided novel preliminary information on the miRNA contents of hESC-derived exosomes. Over 400 miRNAs were detected from the samples, with several of these being involved in mediating pluripotency and cellular reprogramming.
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    Cell Surface Receptors Expression Profile Of Human Synovial Mesenchymal Stem Cells In-Situ And In-Vitro From Normal And Osteoarthritic Knee Joints
    (2016) Al-Jezani, Nedaa; Krawetz, Roman; Rancourt, Derrick; Duncan, Neil
    Osteoarthritis is a chronic disease that results in the degeneration of the articular cartilage, eventually affecting the whole joint. Due to the lack of regenerative capacity in the articular cartilage, synovial mesenchymal stem cells, having chondrogenic capacity and derived from the synovium (SMSCs) are an attractive source for articular cartilage regeneration. However, it is important to identify the optimal SMSCs that have tri-lineage potential, which will help the regeneration of the whole joint. Unfortunately, MSC populations are phenotypically heterogeneous, in terms of their cell surface expression. In this project, SMSCs derived from OA joint with the optimal differentiation capacity display a marker profile of CD90+, CD44+, and CD73+. However, the same profile was expressed on clones that didn’t present with multi-potential differentiation capacity. Therefore, additional markers are necessary to accurately purify SMSCs that would may have increased regenerative capacity for use in cell therapies for patients with OA.
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    Computational Fluid Dynamics Modeling of Scalable Stirred Suspension Bioreactors for Pluripotent Stem Cell Expansion
    (2016-02-03) Le, An; Kallos, Michael; Gates, Ian; Rancourt, Derrick; De Visscher, Alex
    Pluripotent stem cells (PSCs) including embryonic and induced pluripotent stem cells are known for their potential use in cell-based therapy, disease model study, and drug screening. One of the key challenges in pluripotent stem cell research is to establish scalable bioprocesses that reliably produce cells with high quality at any desired quantity. Stirred suspension bioreactors (SSBs) are known to provide a controlled and well-mixed environment for aggregate-forming cells, such as murine and human PSCs. Hydrodynamic environment of SSBs, particularly shear stress and small eddies, have been shown to have a significant impact on the expansion and pluripotency of pluripotent stem cells. However, the exact mechanism has not been fully understood. In this project, computational fluid dynamic (CFD) simulation was employed to model the hydrodynamic environment within SSBs with various configurations and physical conditions. Understanding the hydrodynamics is one of the first key steps in bioprocess development of PSCs using SSBs.
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    Developing a novel biomimetic bioreactor for bone graft engineering with murine embryonic stem cells
    (2012) Lee, Poh Soo; Rancourt, Derrick; Krawetz, Roman
    Following blood, bone graft transplantation is the second most common tissue transplant. Although tissue engineering holds great potential to fulfill demands for better treatment outcomes, it remains technologically challenging to produce bone grafts with normal physiological properties. During skeletal development, endochondral ossification initiates long bone formation and fracture healing. In this study, I aimed to build an in-vitro biomimetic bioreactor to recapitulate physiological niches and processes essential for endochondral ossification to grow bone tissues with anatomical and mechanical properties similar to the native tissues. Here, I have built a prototype capable of generating a dynamic cultivation environment and producing an ivory-toned construct with a stiff texture. The engineered construct greatly resembles a hyaline cartilage model undergoing initial stages of endochondral ossification during skeletal development. In the future, a possible engineered vasculature system may be integrated into the existing bioreactor design to enhance further maturation of constructs to form compact bone.
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    Enhanced bone repair using embryonic stem cells in a mouse model of impaired fracture healing
    (2012) Taiani, Jaymi Tiffany; Matyas, John R.; Rancourt, Derrick
    Fractures that occur in osteoporotic bone typically demonstrate impaired healing or non-union of the bony ends and current treatment methods for these types of injuries are inadequate. The field of tissue engineering offers promising treatment alternatives for diseases and injuries that affect tissues with a limited capacity for repair. Embryonic stem (ES) cells offer advantages over other stem cells types in that these cells are pluripotent, possessing the ability to generate any cell type in the body, and have a high capacity for self-renewal. This thesis describes the development of a novel product for the augmentation of bone fracture repair. Using protocols previously developed by our group, we initially attempted to differentiate bioreactor-expanded ES cells into osteoblasts and chondrocytes in stirred suspension culture systems. Surprisingly, we found that the bioreactor culture environment promoted ES cell pluripotency, resulting in limited cell differentiation. Through further experimentation, we found that exposure to a collagen I extracellular matrix induced osteoblastic differentiation of ES cells with greater efficacy than medium supplementation. Furthern1ore, the cell-loaded collagen constructs formed mineralized tissue nodules following subcutaneous implantation into immune-compromised mice. Subsequently, we developed a fracture model, using both nonnal and osteoporotic mice, to test the efficacy of the constructs to contribute to bone repair at an orthotopic site in vivo. Bone formation was enhanced in the mice treated with cell-loaded collagen constructs and the implanted cells were identified at the fracture site for up to 8 weeks. The formation of a small ectopic soft tissue mass in some of the recipient mice suggests that the collagen I matrix reduced but did not eliminate the tumorigenic potential of the cells. To our knowledge, the studies presented herein are the first of their kind to demonstrate the efficacy of a stem cell therapy for fracture repair in osteoporotic bone in vivo. The findings presented in this thesis highlight the importance of using an orthotopic implantation system to test in vivo functionality of ES cell-derived cells and provide an important foundation for future studies looking to develop stem cell therapies for bone injuries in larger animal models and people.
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    Enhancing osteoporotic fracture healing with p21-/- induced pluripotent stem cells
    (2021-08-25) Olsen, Alexandra B; Krawetz, Roman, J; Rancourt, Derrick; Duncan, Neil, A
    Following a fracture, bone regeneration is dependent on the recruitment of mesenchymal progenitor cells (MPCs). After recruitment of MPCs, regeneration will proceed by one of the two primary osteogenic pathways; intramembranous ossification or endochondral ossification, with the latter being the most common fracture healing pathway. Diseases that disrupt bone homeostasis such as osteoporosis result in prolonged endochondral ossification and as a result, osteoporotic fracture healing results in suboptimal bone regeneration(1). Thus, it is of the utmost importance to determine ways to enhance osteoporotic fracture healing. Of potential interest is the tumour suppressor p21-/- as it has been reported that p21-/- induced pluripotent stem cells (iPSCs) have increased bone and cartilage differentiation potential(2). Our lab has reported that osteoporotic p21-/- mice have decreased trabecular bone loss and also shown that p21-/- mice have increased bone regeneration following a tibia burr hole injury, a model that promotes intramembranous ossification(3). However, the burr hole model is not clinically relevant as it does not mimic commonly occurring fractures. Therefore, the purpose of this thesis was two-fold: first, to assess the regenerative potential of p21-/- mice following a fracture that promotes endochondral ossification and second, to determine the potential therapeutic effects of transplanting p21-/- (iPSCs) into an osteoporotic fracture. It was observed that p21-/- mice demonstrated increased bone regeneration post-fracture under normal or osteoporotic conditions. When p21-/- or C57BL/6 iPSCs were transplanted into the fracture site of an osteoporotic mouse, increased bone regeneration was observed. Furthermore, engraftment of these exogenous iPSCs into the fracture callus was observed. Interestingly, only the transplantation of p21-/- iPSCs resulted in bone that was tougher 4 weeks post-fracture. Taken together, these results suggest that treatment with exogenous iPSCs is a promising option to enhance bone regeneration under osteoporotic conditions. Further elucidating the contributions of iPSCs to bone regeneration is required prior to translating this fundamental knowledge into a clinical setting. Of critical importance, we need to understand the mechanisms by which iPSCs contribute to bone regeneration and determine how best to handle these cells to maximize healing potential and minimize tumorgenicity post-transplant.
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    Expression analysis of the implantation serine proteases
    (2003) Ungarian, Jillian L. R.; Rancourt, Derrick
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    Fluid Shear Stress Promotes Mouse Embryonic Stem Cell Pluripotency via E-cadherin Mechanotransduction
    (2017) Day, Bradley; Rancourt, Derrick; Kallos, Michael; MacNaughton, Wallace; Mains, Paul
    Embryonic stem cells (ESCs) are capable of self-renewal and differentiation into any cell type; this is a powerful tool in generating different cell lineages for regenerative medicine. Stirred suspension bioreactors have been developed as a way to culture ESCs quickly and with minimal labour. Cells are stirred at 100 RPM, which has a maximum tip shear stress of 6 dyne/cm2. We have discovered that cells grown under these conditions maintain pluripotency even in the absence of leukemia inhibitory factor (LIF), an obligate pluripotency maintenance factor. Prior studies have shown that cells can sense physical signals such as shear stress in their environment and modulate a biochemical response through mechanotransduction. In this thesis, I show a link between mechanotransduction and the maintenance of pluripotency. In response to shear stress, β-catenin, a member of the wnt pathway is translocated to the nucleus. Inhibition of β-catenin in the bioreactor results in a decrease in pluripotency gene expression. The disruption of vinculin, a protein recruited to the periphery of the cell in response to shear stress, also leads to a reduction in bioreactor induced pluripotency. Direct manipulation of cells bound to Ecadherin peptides with shear stress generates nuclear β-catenin translocation as well as accumulation of vinculin on the periphery of the cell. These results indicate that shear stress induces bioreactor maintained pluripotency, a phenomenon we have named mechanopluripotency.
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    Generating novel targeting vectors in vivo using phage-plasmid recombination
    (2000) Unger, M. W. Todd; Rancourt, Derrick
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    Identification and characterization of murine Se70-2
    (2006) Minnema, Stephanie E.; Rancourt, Derrick
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    Identification and charaterization of murine se70-2
    (2006) Minnema, Stephanie E.; Rancourt, Derrick
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    ING5 affects mitosis and the DNA damage response in human and murine models
    (2024-04-30) Estanislau Dantas, Arthur; Riabowol, Karl; Luchman, Artee; Biernaskie, Jeff; Rancourt, Derrick; Narendran, Aru; Harkness, Troy
    The INhibitor of Growth family (INGs 1-5) is part of lysine acetyltransferase and lysine deacetylase complexes. They are epigenetic readers that recruit these complexes to the H3K4Me3 mark of active transcription.. Several studies have implicated ING5 in stem cell maintenance in adult and cancer stem cells, cell cycle regulation, and DNA damage response pathways. Here, we investigate the roles of ING5 in two different systems: primary mouse embryonic fibroblasts (MEFs) derived from our Ing5-KO murine model and Brain Tumor Initiating Cells (BTIC cancer stem cells) derived from glioblastoma patients. We show that Ing5-KO MEFs have defects in the cell cycle, with cells accumulating in the G2/M phase of the cell cycle, and carry a higher DNA damage load than their WT counterparts. In addition, after exposure to oxidative stress Ing5-KO cells frequently have abnormal nuclei, defective mitosis and express increased levels of the p21 cyclin-dependent kinase inhibitor compared to WT. RNA-seq analysis of Ing5-KO MEFs revealed significant downregulation of the HOXA family of transcription factors and other transcription factors involved in development such as FOXD1, SOX5 and two transcription factors of the GATA family, indicating that ING5 is part of a network for the regulation of developmental transcription factors. ING5 was also detected on the promoters of SOX4 and GATA6 in primary human fibroblasts, established human embryonic kidney (HEK293) cells and a glioma stem cell.
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    Integration of Silica Sol-Gels into Bioprocesses and Biologically-Derived Pharmaceuticals
    (2013-03-11) Fahr, Jochen; Kallos, Michael; Rancourt, Derrick
    In this thesis work, the use of sol-gel in biotechnology was investigated. This thesis has three independent projects with a common theme of bioprocess engineering. In the first chapter, the high cell density culture of cells in bioreactors was investigated. Specifically, human Foreskin Fibroblasts (hFFs) were cultivated in suspension culture using microcarriers. The goal was to passage hFFs on microcarriers without the use of trypsin. The result was that the yield of hFFs was significantly higher compared to static culture techniques. When cell cultures reach high densities, often the growth is limited by the accumulation of metabolic waste products. In the second chapter, enzyme-laced silica sol-gels were prepared and used for the in situ conversion of lactic acid to pyruvic acid, converting one of the two major metabolic waste products of cell culture into a cell-protecting growth substrate. Silica sol-gels are inorganic matrices that originate from an organic precursor. Sol-gels have been used to protect surfaces, like glass or sheet metal, from corrosion or scratches. In biotechnological applications, sol-gels can be used to encapsulate enzymes and other biologically active ingredients. Encapsulation of enzymes enables an easy recovery and reusability, reducing the cost per unit. One of the benefits of this technology is the simple integration into existing bioprocesses without contamination of protein samples due to the rigid nature of the silica sol-gel particles. This technology also has major implications in the areas of bioprocess engineering, such as tissue engineering and vaccine production by enabling an increase in the medium life span and therefore increasing cultivation times before medium changes, reducing production costs. The final chapter was another application of the sol-gel technology and was completed in an industrial setting. The goal was to develop a coating for safflower-derived oil bodies with silica sol-gel. This coating provided a customizable protection of oil body-associated proteins against digestion along the human gastro-intestinal tract. Overall these developments have examined key issues in current bioprocessing and come up with novel and practical solutions.
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    ISP1 and ISP2: characterization of implantation serine proteinases
    (2006) O'Sullivan, Colleen; Rancourt, Derrick
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    Modulation of murine embryonic stem cell pluripotency markers by fluid flow
    (2011) Lara, Giovanna Gomes; Rinker, Kristina Diane; Rancourt, Derrick