Browsing by Author "Larijani, Leila"

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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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    Mesenchymal progenitor cells from non-inflamed versus inflamed synovium post-ACL injury present with distinct phenotypes and cartilage regeneration capacity
    (2023-06-25) Krawetz, Roman J.; Larijani, Leila; Corpuz, Jessica M.; Ninkovic, Nicoletta; Das, Nabangshu; Olsen, Alexandra; Mohtadi, Nicholas; Rezansoff, Alexander; Dufour, Antoine
    Abstract Background Osteoarthritis (OA) is a chronic debilitating disease impacting a significant percentage of the global population. While there are numerous surgical and non-invasive interventions that can postpone joint replacement, there are no current treatments which can reverse the joint damage occurring during the pathogenesis of the disease. While many groups are investigating the use of stem cell therapies in the treatment of OA, we still don’t have a clear understanding of the role of these cells in the body, including heterogeneity of tissue resident adult mesenchymal progenitor cells (MPCs). Methods In the current study, we examined MPCs from the synovium and individuals with or without a traumatic knee joint injury and explored the chondrogenic differentiation capacity of these MPCs in vitro and in vivo. Results We found that there is heterogeneity of MPCs with the adult synovium and distinct sub-populations of MPCs and the abundancy of these sub-populations change with joint injury. Furthermore, only some of these sub-populations have the ability to effect cartilage repair in vivo. Using an unbiased proteomics approach, we were able to identify cell surface markers that identify this pro-chondrogenic MPC population in normal and injured joints, specifically CD82LowCD59+ synovial MPCs have robust cartilage regenerative properties in vivo. Conclusions The results of this study clearly show that cells within the adult human joint can impact cartilage repair and that these sub-populations exist within joints that have undergone a traumatic joint injury. Therefore, these populations can be exploited for the treatment of cartilage injuries and OA in future clinical trials.
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    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, Sunghoon
    Abstract 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.