Browsing by Author "Rancourt, Derrick E"

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    Investigating the role of splicing in disorders of craniofacial development
    (2017) Lynch, Danielle C; Parboosingh, Jillian S.; Innes, A Micheil; Bernier, Francois P; Childs, Sarah J.; Rancourt, Derrick E; Zimmerly, Steven; Trainor, Paul A
    Following clinical delineation of a rare disease, identification of the causative gene(s) is a crucial first step towards providing enhanced patient care and understanding disease aetiology. Disease gene discovery, especially when considered in the context of related disorders, can also provide new insight regarding normal development and physiology. Herein, I present two novel disease-causing genes: the ribosomal gene RPLP2 in Nager syndrome (NS) and the spliceosomal gene SNRPB in cerebro-costo-mandibular syndrome (CCMS). The mutations identified in SNRPB are the first example of de-regulated alternative splicing-coupled nonsense-mediated decay as a mechanism for human disease. NS and CCMS are both disorders of first and second pharyngeal arch development, with the jaw and ears being affected. Over the past five years, the association between disorders of craniofacial development and mutations in spliceosomal genes has become apparent with the discovery of SF3B4 in NS, EFTUD2 in mandibulofacial dysostosis type Guion-Almeida, and TXNL4A in Burn McKeown syndrome. The specificity of the phenotypes resulting from mutations in such ubiquitous genes has perplexed the scientific community, but it is at least clear that spliceosomal genes play a more prominent yet subtle role in development than previously thought. The association between Treacher Collins syndrome, which overlaps phenotypically with CCMS and NS, and the ribosomal genes TCOF1, POLR1C, and POLR1D has been known for longer. This work establishes NS as having both spliceosomal and ribosomal defects as a cause. In this thesis I discuss potential links between the mechanisms underlying ribosomal and spliceosomal defects in disorders of craniofacial development, particularly increased sensitivity to reactive oxygen species (ROS).
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    Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors
    (2021-01-13) Borys, Breanna S; Dang, Tiffany; So, Tania; Rohani, Leili; Revay, Tamas; Walsh, Tylor; Thompson, Madalynn; Argiropoulos, Bob; Rancourt, Derrick E; Jung, Sunghoon; Hashimura, Yas; Lee, Brian; Kallos, Michael S
    Abstract Background Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production. Methods The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times. Results CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function. Conclusions Taken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.
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    Transdifferentiation of Fibroblast into Chondrocytes
    (2015-01-12) Cota-Elizondo, Perla A; Rancourt, Derrick E
    Great strides have been made in generating autologous induced pluripotent stem cells (iPSCs) via the process of cellular reprogramming. A key step in cellular reprogramming is the opening of chromatin. In newer transdifferentiation schemes, instead of reverting cells all the way to the embryonic state, cells can instead be redirected to specific cell types. One approach recently applied to the generation of cardiac and neural tissue involves partially reprogramming fibroblasts by blocking the JAK-STAT pathway followed by the introduction of specific differentiation factors. We have used a similar approach to transdifferentiate mouse embryo fibroblasts into chondrocytes. Following one day of exposure to the transcription factors Oct4, Sox2, Klf4 and c-Myc, (OSKM factors) and 10 days of exposure to a Jak inhibitor, resulting cells are dissociated and used to form micromass cultures for chondrogenic differentiation in the presence of TGF-b1 , and BMP-2. Cells formed aggregates, which differentiated into chondrocytes within 15 days, eventually formed hyaline and hypertrophic cartilage tissue based upon Alcian Blue, safranin-o and hematoxylin and eosin (H&E) staining. RT-PCR was used to establish a timeline of chondrocyte/cartilage gene expression. We also observed the expression of Col10 and Mmp13, which suggested the development of bone. Jak inhibitor made in all cases transdifferentiation into cartilage more efficient by avoiding bone formation.