Browsing by Author "Ungrin, Mark"

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    A Comparison of the Developmental Competence of In Vitro- Versus In Vivo-Produced Mouse Embryos
    (2021-10-12) Varghese, Jacob; Thundathil, Jacob; Wong, Benjamin; Ungrin, Mark
    The prevalence of infertility in Canada has substantially increased over the last 30 years with one in six couples seeking medical intervention, many of which rely on assisted reproductive technologies (ARTs) including in vitro fertilization (IVF) and in vitro culture (IVC) of embryos. In clinical IVF programs, embryos undergo extended culture until the blastocyst stage (~6 days) under 5% or 2% O2 concentrations to facilitate success of single embryo transfer. Modest success rates of these culture systems warrant further research to refine culture conditions. The objectives of this study were to characterize stress-related responses of cultured embryos under 5% and 2% O2 in comparison to in vivo-derived blastocysts using a CD1 mouse model. We hypothesized 2% O2 during mouse in vitro embryo culture compromises developmental competence through altered transcriptional profiles of genes involved in the embryonic stress response and apoptosis relative to 5% O2-cultured and in vivo-derived blastocysts. The relative expressions of a cohort of stress-related genes in mouse blastocysts cultured under 5 or 2% O2 were quantified through RT-qPCR and compared to in vivo¬-derived embryos. Apoptotic responses were evaluated using an immunofluorescence assay specific for Caspase-3 as the executioner protein in apoptosis. The mean percentage of blastocysts developed in vivo or cultured under 5% O2 was significantly higher than those cultured under 2% O2. Furthermore, in vivo-derived embryos, or those cultured under 5% O2, had greater total cell numbers relative to those cultured under 2% O2. The expansion status of blastocysts was also greatest in embryos cultured under 5% O2. In general, stress response genes were significantly upregulated in embryos cultured under 2% O2, and the expression of antioxidant-related genes was significantly lower in cultured embryos versus those derived in vivo. Caspase-3 immunofluorescence was significantly higher in cultured embryos versus in vivo-derived embryos.Therefore, we inferred that 5% O2 systems may be optimal for culture of mouse embryos, warranting critical evaluation of culturing human embryos under continuous 2% O2 concentrations during clinical IVF programs.
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    Allogeneic Adipose-Derived Mesenchymal Stem Cells as an Adjunct to Endovascular Treatment of Intracranial Aneurysms
    (2023-06) Belanger, Brooke Lynnae; Mitha, Alim; Sen, Arindom; Liao, Shan; Ungrin, Mark
    Intracranial aneurysms are a condition characterized by the weakening of blood vessels in the brain, affecting approximately 1 in 5 individuals. Rupture of these fragile blood vessels can result significant brain bleeding, also known as subarachnoid hemorrhage, leading to a stroke that can cause severe neurological deficits or death. Current treatment approaches involve either endovascular or open surgery. One of the most common treatments is endovascular surgery, a minimally invasive procedure that entails inserting a small catheter through the femoral or radial artery and navigating it to the location of the aneurysm in the brain. Depending on the anatomical features, the aneurysm sac is filled with small platinum coils, or a mesh metal stent which is placed to cover the aneurysm opening. The objective in both cases is to redirect blood flow and eliminate the risk of stroke. However, recurrent aneurysms occur in up to 30% of these procedures, necessitating multiple surgeries. Recent advancements have introduced novel endovascular devices such as bioresorbable stents to enhance the outcomes of aneurysm treatment. Additionally, allogeneic adult mesenchymal stem cells have shown promise in improving healing processes. This dissertation aims to expand our understanding of the impact of stem cells on aneurysm healing and how they can be effectively utilized to improve the outcomes of brain aneurysm treatments, including endovascular coiling and stenting.
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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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    Bioprocessing of Mesenchymal Stem Cells and Their Derivatives: Toward Cell-Free Therapeutics
    (2018-09-12) Phelps, Jolene; Sanati-Nezhad, Amir; Ungrin, Mark; Duncan, Neil A.; Sen, Arindom
    Mesenchymal stem cells (MSCs) have attracted tremendous research interest due to their ability to repair tissues and reduce inflammation when implanted into a damaged or diseased site. These therapeutic effects have been largely attributed to the collection of biomolecules they secrete (i.e., their secretome). Recent studies have provided evidence that similar effects may be produced by utilizing only the secretome fraction containing extracellular vesicles (EVs). EVs are cell-derived, membrane-bound vesicles that contain various biomolecules. Due to their small size and relative mobility, they provide a stable mechanism to deliver biomolecules (i.e., biological signals) throughout an organism. The use of the MSC secretome, or its components, has advantages over the implantation of the MSCs themselves: (i) signals can be bioengineered and scaled to specific dosages, and (ii) the nonliving nature of the secretome enables it to be efficiently stored and transported. However, since the composition and therapeutic benefit of the secretome can be influenced by cell source, culture conditions, isolation methods, and storage conditions, there is a need for standardization of bioprocessing parameters. This review focuses on key parameters within the MSC culture environment that affect the nature and functionality of the secretome. This information is pertinent to the development of bioprocesses aimed at scaling up the production of secretome-derived products for their use as therapeutics.
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    Canada needs a national COVID-19 inquiry now
    (2024-11-15) Fisman, David; Horton, Jillian; Oliver, Matthew; Ungrin, Mark; Vipond, Joseph; Wright, Julia M.; Zoutman, Dick
    Abstract Background We are now in the fifth year of an ongoing pandemic, and Canada continues to experience significant surges of COVID-19 infections. In addition to the acute impacts of deaths and hospitalizations, there is growing awareness of an accumulation of organ damage and disability which is building a “health debt” that will affect Canadians for decades to come. Calls in 2023 for an inquiry into the handling of the COVID-19 pandemic went unheeded, despite relevant precedent. Canada urgently needs a comprehensive review of its successes and failures to chart a better response in the near- and long-term. Main body While Canada fared better than many comparators in the early years of the COVID-19 pandemic, it is clearly still in a public health crisis. Infections are not only affecting Canadians’ daily lives but also eroding healthcare capacity. Post-COVID condition is having accumulating and profound individual, social, and economic consequences. An inquiry is needed to understand the current evidence underlying policy choices, identify a better course of action on various fronts, and build resilience. More must be done to reduce transmission, including a serious public education campaign to better inform Canadians about COVID and effective mitigations, especially the benefits of respirator masks. We need a national standard for indoor air quality to make indoor public spaces safer, particularly schools. Data collection must be more robust, especially to understand and mitigate the disproportionate impacts on under-served communities and high-risk populations. General confidence in public health must be rebuilt, with a focus on communication and transparency. In particular, the wide variation in provincial policies has sown mistrust: evidence-based policy should be consistent. Finally, Canada’s early success in vaccination has collapsed, and this development needs a careful post-mortem. Conclusions A complete investigation of Canada’s response to the pandemic is not yet possible because that response is still ongoing and, while we have learned much, there remain areas of dispute and uncertainty. However, an inquiry is needed to conduct a rapid assessment of the current evidence and policies and provide recommendations on how to improve in 2025 and beyond as well as guidance for future pandemics.
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    Climbing the mountain: experimental design for the efficient optimization of stem cell bioprocessing
    (2017-12-04) Toms, Derek; Deardon, Rob; Ungrin, Mark
    Abstract “To consult the statistician after an experiment is finished is often merely to ask him to conduct a post mortem examination. He can perhaps say what the experiment died of.” – R.A. Fisher While this idea is relevant across research scales, its importance becomes critical when dealing with the inherently large, complex and expensive process of preparing material for cell-based therapies (CBTs). Effective and economically viable CBTs will depend on the establishment of optimized protocols for the production of the necessary cell types. Our ability to do this will depend in turn on the capacity to efficiently search through a multi-dimensional problem space of possible protocols in a timely and cost-effective manner. In this review we discuss approaches to, and illustrate examples of the application of statistical design of experiments to stem cell bioprocess optimization.
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    Expansion of Skin-Derived Precursor Cells (SKPs) in Stirred Suspension Bioreactors
    (2016) Boon, Kathryn; Kallos, Michael; Biernaskie, Jeffrey; Ungrin, Mark; Hart, David; De la Hoz Siegler, Hector
    Skin-derived precursor cells (SKPs) have potential therapeutic applications for dermal regeneration in patients who have undergone split-thickness skin graft (STSG) surgeries due to severe burns. While these cells are traditionally grown in vitro in static t-flasks, a more standardized, well-controlled culture environment is desirable so that sufficient numbers of cells can be produced for clinical applications. This study outlines a bioprocess which could be used to expand SKPs, and establishes a baseline of how these cells grow in static and bioreactor conditions. Using a 9% total body surface area burn as the baseline, it was determined that a fold expansion of approximately 45 to 270 would be required for a 10 cm² piece of donor skin, assuming all cells isolated proliferate and are of therapeutic benefit. While this is not feasible based on current expansion achieved in bioreactors, this study provides useful information moving forward towards more advanced bioprocess development.
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    Extrinsic Factors and RPE Regeneration
    (2024-01-24) Selje, Sara J.; McFarlane, Sarah; Hocking, Jennifer; Ungrin, Mark
    The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that closely interacts with photoreceptor outer segments of the outer vertebrate retina to maintain visual function. Damage to the RPE, for instance in a disease such as Age-Related Macular Degeneration, results in photoreceptor degeneration and subsequently, vision loss. In contrast to mammals, zebrafish can intrinsically regenerate a functional RPE layer after injury. Specific molecular pathways are known to regulate RPE proliferation in culture, but the pathways that function in vivo to promote RPE regeneration remain largely unknown. My aim is to determine potential pathways that influence RPE regeneration in zebrafish. First, I examine the importance of the secreted ligand Semaphorin 3F (SEMA3F), expressed in the RPE of both mammals and zebrafish, in RPE regeneration. I use a sema3fa homozygous mutant zebrafish on a transgenic RPE injury background (Tg(rpe65a:NTR-EGFP)) where timed application of the drug metronidazole (MTZ) to the bath results in nitroreductase-mediated RPE-specific cell death. My data suggest Sema3fa has no effect on the extent of RPE injury in this model, though RPE apoptosis may be delayed and increased in the absence of Sema3fa. Further, loss of Sema3fa may induce an initial increase in proliferation in the RPE as well as increased proliferation in the photoreceptor outer nuclear layer. Second, I provide an initial assessment of the involvement of additional pathways in zebrafish RPE regeneration. These pathways impact proliferation and/or migration of cells in culture and are expressed within the RPE. I use in situ hybridization to visualize larval RPE expression of 10 candidate genes before and after RPE injury. Genes that may show changes in expression post-injury include bmp7b, caska, foxm1, her4.1, msnb, rpe65a, trpm7, and vrk1. Future work could include using loss-of-function approaches in the RPE injury model to determine potential roles of these genes in RPE regeneration. In the long-term, this work may impact gene therapies for patients suffering from retinal degenerative diseases.
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    Fluid flow and Smad2 affects the response of vascular endothelial cells in vitro and in vivo
    (2016-02-05) Tamez-Vielma, Linda Selene; Rinker, Kristina D.; Moore, Randy; Di Martino, Elena; Proud, David; Ungrin, Mark; Mahinpey, Nader
    Human aortic endothelial cells (HAECs) have been observed to respond to fluid flow and shear stress by activating different signalling molecules both in vitro and in vivo. An important example of these flow-activated molecules is Smad2. Smad2 is a signalling molecule and transcription factor that has shown to be indispensable for the maintenance of vascular integrity. The aim of this study was to understand the effect of shear stress and Smad2 knockdown on endothelial gene expression. HAEC were transfected with Smad2 siRNA, and exposed to steady laminar shear stress (10 dyne/cm2). Our results showed that Smad2 siRNA and shear stress significantly up-regulated genes involved in atherosclerosis, heart dysfunction, and angiogenesis. Furthermore, Smad2 siRNA had a negative impact on athero-protective genes under static conditions. This is the first reported Smad2 siRNA gene expression profile of endothelial cells. Our findings suggest that Smad2 may a have a protective role against cardiovascular diseases.
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    Fluid Force Alterations in Cultured Mammary Epithelial and Breast Cancer Cells: Applications in Breast Cancer Diagnosis
    (2016) Fuh, Kenneth Fuh; Rinker, Kristina; Shemanko, Carrie; Kallos, Michael; Ungrin, Mark; Wang, Edwin; Leask, Richard
    Metastatic progression of breast cancer is characterized by mechanical interactions between tumor cells and various microenvironments, including exposure to fluid flow. Complementing genomic and molecular signaling studies with fluid mechanics holds the promise of providing in-depth knowledge into how these interactions affect the ability of tumor cells to undergo metastasis, and identification of novel biomarkers that can potentially facilitate breast cancer diagnosis and treatment. In this thesis, a bioreactor system was used to expose cultured mammary epithelial and breast cancer cells to fluid shear stress in the physiological range of those experienced in the vascular microenvironment. Genome-wide expression analysis revealed an effect of fluid flow on gene expression patterns and cellular processes involved in metastasis such as EMT, cell migration and adhesion. In addition, TGF-β signaling activity was significantly enriched and several genes belonging to this pathway were overexpressed upon flow exposure. Subsequently, we sought to identify novel flow-responsive biomarkers for breast cancer. For this purpose, bioinformatics and network biology approaches were used to reveal significant enrichment of biological processes involved in metastatic progression. Expression levels of differentially expressed genes were evaluated in clinical expression datasets, and 14 genes were identified as potential biomarkers. Relative expression levels of seven of these biomarkers were quantified in breast cancer patients and healthy volunteers. Five biomarkers passed the threshold for statistical significance and were overexpressed in more than 80% of patients presenting with basal and HER2-enriched breast cancers, which are the most aggressive subtypes of breast cancer. To our knowledge, the studies presented herein are the first of their kind to demonstrate that using an in vitro model to simulate exposure of cells to fluid shear stresses allows for identification of biomarkers for breast cancer. Using this system to study cellular events involved in other types of cancers may lead to new diagnostic and therapeutic approaches for metastatic cancer progression.
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    Generation of Tissue-Engineered Cartilage Constructs in Stirred Suspension Bioreactors
    (2016) Allen, Leah Marie; Sen, Arindom; Hart, David; Matyas, John; Ungrin, Mark; Schmidt, Tannin; Ramirez-Serrano, Alejandro
    Traumatic injuries to articulating joints, such as the knee and hip, can result in the formation of defects within the articular cartilage contained therein. These defects do not heal spontaneously and can initiate a degenerative process, eventually resulting in osteoarthritis (OA). Current cartilage repair options are limited and do not result in the regeneration of durable cartilage. Mesenchymal stem cells (MSCs) isolated from the synovial fluid within joints have an inherent ability to differentiate towards a chondrogenic lineage. Tissue engineered cartilage constructs (TECs) formed from MSCs have been shown to contribute to cartilage repair when implanted into a defect site, thereby providing a potential approach to prevent the onset or the progression of OA. TECs have traditionally been formed in the wells of static culture plates and serum-containing medium. Unfortunately, TECs made in this manner can exhibit variable characteristics which are likely exacerbated by the use of animal-sourced serum in the medium. Therefore, a need still exists to better optimize the generation of uniform TECs to enhance the clinical translatability of this otherwise promising technology. This work investigated the aggregation and culture of human MSCs within suspension bioreactors and serum-free culture conditions for eventual use in filling articular cartilage defects. It also investigated the impact of low-oxygen tension and chondrogenic medium to enhance the MSC differentiation into a chondrogenic phenotype. Expanding on a body of knowledge, this work demonstrated the ability of suspension bioreactors to create a population of aggregates using serum-free culture conditions and non-osteoarthritic human SF-MSCs. Additionally, it demonstrated that the application of low-oxygen tension and chondrogenic growth factors to the suspension bioreactor system is simple. The culture of MSC aggregates in suspension bioreactors under low-oxygen tension resulted in up-regulated gene expression for aggrecan, significantly more collagen/DNA production, and less necrosis on the inside of the aggregates as compared to ambient oxygen tension. The application of chondrogenic medium to the aggregates in the suspension bioreactors resulted in enhanced collagen type-II gene expression and deposition as compared to the static TECs. Although, the overall amount of ECM quantified and staining for glycosaminoglycans was more prominent in the static TECs.
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    Proteomic and transcriptomic analysis of clinical and in vitro flow-exposed breast cancer samples reveals pathways and markers relevant to postpartum breast cancer.
    (2024-01-13) Stibbards-Lyle, Maya; Rinker, Kristina; Dufour, Antoine; Shemanko, Carrie; Ungrin, Mark
    Mechanical forces are an important, yet poorly understood, influence on the metastatic capability of breast cancer cells. They exist in a dynamic interplay with the structural and immune aspects of the cancer microenvironment. Mammary gland involution is a remodeling process that occurs post-lactation and is believed to be responsible for poor outcomes observed in postpartum breast cancer. Fluid shear stress is well-linked to tissue remodeling and is likely increased during this stage of the postpartum period. In this thesis, a bioreactor system was combined with hormonal treatment to expose breast cancer cells to some of the conditions relevant to postpartum involution. We initially validated the model by studying the morphology and viability of cells upon exposure to fluid shear stress and involution and confirming the presence of relevant protein markers. Using machine learning-based analysis of publicly available RNA-seq datasets, we identified numerous genes and pathways upregulated in both postpartum and flow-exposed conditions. We further validated these findings using proteomic analysis of flow-exposed MDA-MB-231 breast cancer cells, which identified similar proteins and pathways to the initial transcriptomic analysis. These included pathways linked to inflammation, wound healing, cell migration, and extracellular matrix organization, which are all pathways critically involved in the action of mammary gland involution. Further investigation revealed that matrix metalloproteinases, a key promoter of the remodeling activities and tumorigenic effect of involution, are significantly upregulated upon exposure to fluid flow. Proteomic analysis of MCF-7 cells treated with hormones associated with lactation revealed markers and pathways related to immune-mediated progression of breast cancer. Finally, we established the utility of our model by validating that the protein expression of chemokine C-X-C motif ligand 13 (CXCL13), which was upregulated in our transcriptomics analysis and is a well-established marker of breast cancer metastasis in young patients, is upregulated in response to flow-exposure. This suggests that our model serves as an efficient method to identify relevant breast cancer biomarkers related to the mechanism of mammary gland involution. To our knowledge, these studies are the first to integrate hormonal and fluid shear stress influences in the context of breast cancer, and to suggest a link between fluid shear stress and mammary gland involution. They serve as an initial proof of concept, indicating that fluid shear stress could serve as a powerful tool in furthering our understanding of postpartum breast cancer and identification of relevant biomarkers for this unique cohort.
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    Rapid prototyping of microfluidic devices for culture of suspended cell sheets
    (2017) Razian, Golsa; Ungrin, Mark
    Embryogenesis is not only interesting as a research base perspective but also from applied tissue engineering point of view because this is how tissues are created naturally. In only a few days, a mammalian fertilized egg undergoes cell divisions and subsequent reorganization to progress through morula and then blastocyst. Some cells of the inner cell mass give rise to a surface layer known as primitive endoderm, and the others epithelialize to form the epiblast layer. This epiblast cell layer, pluripotent tissue, then undergoes gastrulation to develop all tissues and organs of the embryo. As these stages of development are not accessible in many organisms due to ethical and practical challenges, the bulk of our understanding is derived from mice. Therefore, an in vitro model is needed to replicate in vivo structure and microenvironment and study pre-implantation mammalian development in a physiologically relevant form. Overall, we could rapidly prototype a microfluidic platform in an easy, fast and low-cost manner that allows us to control concentration gradients of relevant signals over selected positions of suspended cell sheet, where there is a need. As we aimed to make microfluidic fabrication more accessible to biology labs with no expertise in microfluidic field, this work also led to an easy-to-use kit to make microfluidics available to non-experts. We could also successfully teach this technique to graduate students, professionals at the university level, and younger students (grade 6-8) at summer camp. Cell sheet formation was successful to some extent using an artificial extracellular matrix, StemAdhere, however, these suspended cell sheets could not resist the compression caused by clamping the device.
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    A Response Surface Methodology Based Characterization and Optimization of Pseudoislet Cryopreservation
    (2021-08) Sidhu, Sukhjit; Ungrin, Mark; Rancourt, Derrick; Hollenberg, Morley
    Efficient and consistent cryopreservation of human islets would be a useful tool in generating islet biobanks for transplantation and research uses. However, current islet cryopreservation attempts have an array of challenges due to large islet aggregate size and susceptibility to cryopreservation. Centrifugal-force-aggregation pseudoislets (CFA-PI) offer a promising tool to address these challenges as they can be size controlled and additionally modified to survive the stress associated with cryopreservation. In this thesis, the objective was to use a response surface methodology approach to characterize the effects of cryopreservation manipulated variables on CFA-PI survival post-cryopreservation and use this characterization to optimize (maximize) survival. Two separate cryopreservation processes were characterized, controlled 1 ⁰C/minute cooling rate cryopreservation (CCRC) and vitrification (extremely rapid-cooling using liquid nitrogen). Initial peak post-CCRC CFA-PI survival was 26.9% ± 27.1% of initially cryopreserved islet material with increasing islet purity, FBS concentration of 20%, CFA-PI size of 750 cells/aggregate, 5% DMSO, and 0 additional days of CFA-PI culture time increasing survival. In comparison peak post-vitrification CFA-PI survival was 26.9% ± 6.9% with increasing islet purity, incorporation of fibroblasts into CFA-PI, 10% DMSO, and 0 or 100 µM emricasan increasing CFA-PI survival. Due to constraints on islet supply during the Covid-19 pandemic, a third unifying experiment comparing post-cryopreservation survival directly between CCRC and vitrification, with similar experimental variables, was characterized using a model fibroblast cell line. Validation of this model using human CFA-PI resulted in a survival of 41.0 ± 0.723% using CCRC with intermediate CFA-PI size of 500 cells/aggregate, 5% DMSO, 24% FBS, and no emricasan. While islet survival improved using this approach compared to earlier attempts, there was no statistically significant difference in peak islet survival post-cryopreservation across the different methods of cryopreservation in this thesis. Ultimately, while CFA-PI survival post-cryopreservation did not statistically significantly improve, the effect of cryopreservation manipulated variables on survival was characterized as a modeled and manipulatable mathematical relationship. This relationship establishes a framework for optimizing the survival of CFA-PI, when additional islet tissue becomes available after Covid-19.
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    Serum-Free Culture of Human Mesenchymal Stem Cell Aggregates in Suspension Bioreactors for Tissue Engineering Applications
    (2019-11-07) Allen, Leah M.; Matyas, John; Ungrin, Mark; Hart, David A.; Sen, Arindom
    Mesenchymal stem cells (MSCs) have the capacity to differentiate towards bone, fat, and cartilage lineages. The most widely used culture and differentiation protocols for MSCs are currently limited by their use of serum-containing media and small-scale static culture vessels. Suspension bioreactors have multiple advantages over static culture vessels (e.g., scalability, control, and mechanical forces). This study sought to compare the formation and culture of 3D aggregates of human synovial fluid MSCs within suspension bioreactors and static microwell plates. It also sought to elucidate the benefits of these techniques in terms of productivity, cell number, and ability to generate aggregates containing extracellular matrix deposition. MSCs in serum-free medium were either (1) inoculated as single cells into suspension bioreactors, (2) aggregated using static microwell plates prior to being inoculated in the bioreactor environment, or (3) aggregated using microwell plates and kept in the static environment. Preformed aggregates that were size-controlled at inoculation had a greater tendency to form large, irregular super aggregates after a few days of suspension culture. The single MSCs inoculated into suspension bioreactors formed a more uniform population of smaller aggregates after a definite culture period of 8 days. Both techniques showed initial deposition of extracellular matrix within the aggregates. When the relationship between aggregate size and ECM deposition was investigated in static culture, midsized aggregates (100-300 cells/aggregate) were found to most consistently maximize sGAG and collagen productivity. Thus, this study presents a 3D tissue culture method, which avoids the clinical drawbacks of serum-containing medium that can easily be scaled for tissue culture applications.
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    The Impact of Culture Environment on the Composition and Function of Mesenchymal Stem Cell Derived Extracellular Vesicles
    (2022-05-30) Phelps, Jolene; Sen, Arindom; Duncan, Neil A.; Ungrin, Mark; Sanati-Nezhad, Amir
    Mesenchymal stem cells (MSCs) have attracted research interest due to their ability to induce tissue repair and reduce inflammation. More recently, their therapeutic benefits have been attributed to small membrane bound vesicles called extracellular vesicles (EVs) that they release. The implantation of MSC-EVs offers significant advantages over the implantation of viable MSCs. Their nonliving nature eliminates many of the safety concerns related to cell therapies and enables them to be stored and transported more efficiently. There is a significant need to develop clinically applicable bioprocesses that can produce large numbers of therapeutically relevant MSC-EVs. This involves the translation of small-scale studies to large scale processes, which is limited by considerations such as the medium and platform in which the cells are cultured. Due to the novelty of the EV field, current research in this area is very limited, and thus the goal of this thesis was to evaluate the efficacy of producing MSC-EVs in a clinically relevant medium, and to gain insight into the impact of culture parameters needed for the scale-up of MSC-EVs. This thesis demonstrated, for the first time, that functional MSC-EVs could be produced using a clinically relevant serum-free culture medium for the applications of articular cartilage repair, cerebral ischemia, and intracranial aneurysm repair. Further, it was found that functional MSC-EVs could be produced in scalable stirred suspension bioreactors. These studies were among the first to evaluate the use of such a platform for EV production and were the first to present a scalable model for EV production specifically for the applications of articular cartilage repair and cerebral ischemia. In addition, the culture of MSCs at physiological oxygen conditions was evaluated and found to enhance their angiogenic potential, without damaging physiological function, in the application of cerebral ischemia. Finally, important considerations relevant to the upstream production of MSC-EVs were studied, including the confluence of cultured MSCs and the medium in which the EVs were isolated. This work enabled a proposed workflow for producing EVs in serum-free medium and will contribute highly to the development of clinically applicable bioprocesses aimed at producing MSC-EVs for applications in regenerative medicine.
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    The role of UCH-L1 in mouse progenitor spermatogonia
    (2016) Alpaugh, Whitney; Dobrinski, Ina; Thundathil, Jacob; Natale, David; van der Hoorn, Frans; Ungrin, Mark
    To maintain fertility throughout the life of the adult male, the spermatogonial stem cell (SSC) population of the testis needs to achieve a balance between self-renewal and differentiation. The chemical, molecular and physical environment that supports this balance of fate decision is not completely understood. The deubiquitinating enzyme Ubiquitin C-terminal Hydrolase L1 (UCH-L1) is highly expressed in spermatogonia, but not in further differentiating germ cells of the testis. Previous studies have shown that UCH-L1 is important for normal spermatogenesis; however, its specific role has not been determined. The experiments in this thesis establish that UCH-L1 is specifically expressed in undifferentiated and early differentiating mouse spermatogonia. Examination of the Uch-l1-/- testis revealed that, as mice age, there is an increase in the number of degenerating tubules as well as a decrease in numbers of differentiating cells in the absence of UCH-L1. A combination of gene expression data and analysis of donor-derived spermatogenesis after transplantation of Uch-l1-/- SSCs to germ celldepleted wild-type recipient testes demonstrated that over time, in the absence of UCHL1, the spermatogonia pool expands at the expense of differentiation. Additionally, RNA sequencing of Uch-l1+/+ and Uch-l1-/- spermatogonia showed that the Uch-l1-/- spermatogonia have a gene expression profile closer to that of undifferentiated spermatogonia than to that of differentiating spermatogonia. Finally, 31 novel binding partners for UCH-L1 were identified in spermatogonia which provides a starting point for identifying the mechanisms through which UCH-L1 acts to affect spermatogonia fate decision.