Papers by Derrick Rancourt
Mutation of the Calcium Channel Gene Cacna1f Disrupts Calcium Signaling and Synaptic Transmission in Mouse Retina
Investigative Ophthalmology & Visual Science, 2004
Embedding Informational Interviews into Postsecondary Curriculum
Canadian journal of career development, Oct 28, 2020
An informational interview is a traditional career exploration technique. Students have a convers... more An informational interview is a traditional career exploration technique. Students have a conversation with professionals where they seek advice on their career (Fiske, 2016). They can also be used to find information on industry and specific workplaces. A form of rapid prototype testing (Burnett & Evans, 2016), after each interview, the interviewer makes changes to their career vision and plan – refining it through additional interviews. These interviews help students build a network of contacts in a specific professional area. They can also benefit the interviewees by helping them to build a candidate pool for future hires. Although it is taboo for interviewers to ask for employment, IIs often lead to employment via planned happenstance (Mitchell, Al Levin, & Krumboltz, 1999). This makes it a more effective job search strategy than conventional applications because there is a significant hidden job market (Burnett & Evans, 2016).
Managing Emergent Knowledge: Addressing the Competency Expectations of Biomedical Employers
Canadian journal of career development, Oct 28, 2020
Orpheus Recombination
Methods in molecular biology, 2008
In recent years, methods to address the simplification of targeting vector (TV) construction have... more In recent years, methods to address the simplification of targeting vector (TV) construction have been developed and validated. Based on in vivo recombination in Escherichia coli, these protocols have reduced dependence on restriction endonucleases, allowing the fabrication of complex TV constructs with relative ease. Using a methodology based on phage-plasmid recombination, we have developed a comprehensive TV construction protocol dubbed Orpheus recombination (ORE). The ORE system addresses all necessary requirements for TV construction; from the isolation of genespecific regions of homology to the deposition of selection/disruption cassettes. ORE makes use of a small recombination plasmid, which bears positive and negative selection markers and a cloned homologous "probe" region. This probe plasmid may be introduced into and excised from phage-borne murine genomic clones by two rounds of single crossover recombination. In this way, desired clones can be specifically isolated from a heterogeneous library of phage. Furthermore, if the probe region contains a designed mutation, it may be deposited seamlessly into the genomic clone. The complete removal of operational sequences allows unlimited repetition of the procedure to customize and finalize TVs within a few weeks. Successful gene-specific clone isolation, point mutations, large deletions, cassette insertions, and finally coincident clone isolation and mutagenesis have all been demonstrated with this method.
British Journal of Cancer, Dec 21, 2010
BACKGROUND: Although the naturally occurring reovirus causes only mild symptoms in humans, it sho... more BACKGROUND: Although the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures. METHODS: We characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts. RESULTS: Sequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated s1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size. CONCLUSION: Despite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy.
Molecular Therapy, May 1, 2016
in order to properly assess for their compatibility with cationic transfection reagent in facilit... more in order to properly assess for their compatibility with cationic transfection reagent in facilitating transfection, we transfected cells at multiple time points (day 2, 3, 5 and 7 days post cell seeding) and found that transfection efficiencies correlated with the time frame in which cells were growing the fastest (Days 2-3); highest efficiencies were seen in cells cultured on Glass, Cytodex 3, CultiSphere S and Hillex II (~12-16%). In summary, we demonstrated here a first step towards the efficient transfection of primary tissue-derived human fibroblasts directly in a suspension microcarrier culture using cationic reagent; additional optimization is expected to bring the efficiencies comparable to those in static culture.
Molecular Therapy, May 1, 2016
presence of polybrene, these vesicles are able to transfer plasmids in a large panel of animal ce... more presence of polybrene, these vesicles are able to transfer plasmids in a large panel of animal cells. Unfortunately, the production of V-VSV-G and their use for nucleic acid delivery is poorly documented. Here we propose to improve this promising method of transfection. At first we developed a V-VSV-G production process by transient transfection of HEK-293 cells using polyethylenimine (PEI). Three modes of production were compared: cells cultivated in adherence, in suspension and on micro-carriers. Also we demonstrated that the quantity of vesicles produced depends on the VSV-G sequence used. The harvest of V-VSV-G from cell culture media was also optimized. Then, several parameters potentially involved in the formation and the transfer efficiency of V-VSV-G/DNA complex were studied: polybrene concentration, order of addition of mix transfection components, incubation time of the complexes, medium of transfection, etc. Stability studies also demonstrated that V-VSV-G are robust particles: DNA transfer capacity of V-VSV-G is efficient after 10 freezing and thawing cycles and V-VSV-G can be stored for long term at +4 °C, -20 °C and -80 °C. Finally, V-VSV-G/DNA ratio was optimized for three different cell types. Transfection efficiency of 70 % and 55 % were obtained for HEK-293 and HeLa cells respectively, with 1 µg of V-VSV-G and 0.4 µg of DNA. Transfection of refractory cells such as human myoblasts, reached 25 % with 5 µg of V-VSV-G and 0.8 µg of DNA. V-VSV-G can also deliver large plasmids (18 kb). Furthermore, no cytotoxicity was observed in cells transfected with these complexes. Presently, the potential of V-VSV-G to transfer siRNA is investigated. In conclusion, V-VSV-G is a powerful tool for nucleic acid delivery which could be useful for several applications oriented toward cell and gene therapies.
Transplacement Mutagenesis
Humana Press eBooks, 2002
Bone, Jul 1, 2014
In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the effic... more In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cellgenerated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (μCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p b 0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p b 0.001) and trabecular number (p b 0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.
Molecular Therapy, May 1, 2016
experimental design consisting of eight formulations. Nanocapsules morphology and particle were i... more experimental design consisting of eight formulations. Nanocapsules morphology and particle were investigated by Transmission Electron Microscope, and Dynamic Light Scattering, respectively. The entrapment efficiency of nanocapsules, the effect of the polymer and preparation procedures on trypsin activity and the release profile in simulated gastric fluid SGF and simulated intestinal fluid SIF were assessed.Applying QbD concepts saved the resources, and provided huge useful results within short time. Nanocapsules were spherical with no distortion, Figure . The encapsulation efficiency has reached up to 80.7%, and it has been affected significantly (P 0.005) by the copolymer blocks ratio. Moreover, the drug release profile in SIF over 24 hours was also affected by the copolymer ratios, with released drug up to 64.01% in a triphasic pattern concluded in the following three steps; the first burst phase with 8% release within 15 minutes, then a plateau for 8 -10 hours, finally, trypsin started to release in a sustained rate over the rest of 24 hours, whilst the release in SGF reached 8% in average; which can protect the macromolecules from the gastric enzymes degradation. Trypsin biological activity was affected by the materials and process parameters and retained only 18% of the original activity. However, adding trehalose and encapsulation of solid protein helped the protein to retain up to 84.65% of the original activity. In conclusion, polymeric nanocapsules showed promising results to potentially deliver active proteins orally in the presence of trehalose especially when it was prepared by s/o/w, and when the copolymer blocks ratio was optimised. The applied rationale could be applicable to further macromolecules including monoclonal antibodies, genes, and cells, in order to obtain close quality attributes.
Molecular and Cellular Biology, Jun 1, 1987
The gene coding for the most abundant antifreeze protein (AFP) in the winter flounder was placed ... more The gene coding for the most abundant antifreeze protein (AFP) in the winter flounder was placed downstream of the Drosophila melanogaster hsp7O promoter and introduced into the D. melanogaster germ line by P-element-mediated transformation. In each of six transgenic strains tested, heat shock treatment induced the expression of two major AFP gene transcripts and one minor one. All three transcripts were spliced despite the lack of an obvious D. melanogaster internal intron-splicing sequence. The variation in transcript length was caused by selection of different polyadenylation sites. Western blots showed the presence of immunoreactive AFP in hemolymph from heat-shocked transformants. The immunoreactive material had a molecular weight of 6,200, which is consistent with the loss of the signal sequence from the primary translation product and the retention of the pro sequence. Thus, all the signals for flounder pre-mRNA and preprotein processing were recognized in D. melanogaster.
Stem Cell Epigenetics and Human Disease
Elsevier eBooks, 2018
Differentiation, Mar 1, 2015
While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensi... more While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N 5 -(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stagespecific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases.
Biotechnology and Bioengineering, Feb 11, 2020
Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain ... more Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast (MEF) cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/mL). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (10 9 cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output
Journal of Cell Science, 2012
Nitric oxide (NO) has been shown to play a crucial role in bone formation in vivo. We sought to d... more Nitric oxide (NO) has been shown to play a crucial role in bone formation in vivo. We sought to determine the temporal effect of NO on murine embryonic stem cells (ESCs) under culture conditions that promote osteogenesis. Expression profiles of NO pathway members and osteoblast-specific markers were analyzed using appropriate assays. We found that NO was supportive of osteogenesis specifically during an early phase of in vitro development (days 3-5). Furthermore, ESCs stably overexpressing the inducible NO synthase showed accelerated and enhanced osteogenesis in vitro and in bone explant cultures. To determine the role of NO in early lineage commitment, a stage in ESC differentiation equivalent to primitive streak formation in vivo, ESCs were transfected with a T-brachyury-GFP reporter. Expression levels of T-brachyury and one of its upstream regulators, b-catenin, the major effector in the canonical Wnt pathway, were responsive to NO levels in differentiating primitive streak-like cells. Our results indicate that NO may be involved in early differentiation through regulation of b-catenin and T-brachyury, controlling the specification of primitive-streak-like cells, which may continue through differentiation to later become osteoblasts.
Viruses
Oncolytic viruses (OVs) are an emerging cancer therapeutic that are intended to act by selectivel... more Oncolytic viruses (OVs) are an emerging cancer therapeutic that are intended to 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 is undergoing advanced clinical trials and has received FDA approval in selected circumstances. However, the mechanisms governing reoviral selectivity are not well characterised despite many years of effort, including those in our accompanying paper where we characterize pathways that do not consistently modulate reoviral cytolysis. We have earlier shown that reovirus is capable of infecting and lysing both certain types of cancer cells and also cancer stem cells, and here we demonstrate its ability to also infect and kill healthy pluripotent stem cells (PSCs). This led us to hypothesize that pathways responsible for stemness may constitute a novel route for the modulation of reoviral tropism. We find that reovirus is cap...
Author response for "Computational fluid dynamic characterization of vertical‐wheel bioreactors used for effective scale‐up of human induced pluripotent stem cell aggregate culture
Many students find it difficult to transition into the workforce successfully. They often accept ... more Many students find it difficult to transition into the workforce successfully. They often accept unsuitable positions through happenstance rather than actively planning their career trajectories. This is due to their unawareness of career exploration and mapping. We use informational interviewing as an in class experiential learning assignment to solve this problem. The assignment gives students the opportunity to source information from industry experts and expand their professional network. It helps students develop their professional presence, find careers of interest, and ultimately succeed in their transition into the workforce. It is no surprise that the job market for postsecondary graduates is highly competitive. Only 10% of PhD students will enter tenure track positions meaning most of these highly qualified individuals will not find work in academia. With the number of tenure track positions remaining constant and the number of graduate students doubling, especially in physical and life science fields, it is hardly surprising that graduates are embracing alternate career choices . Many graduate students are then confronted with the task of pivoting and moving into public and private sectors with little guidance on how to successfully make this transition. Undergraduates finishing their degrees face a similar challenge when transitioning into the workforce (Greenbank, 2014). We have been using informational interviews (IIs) as an in class experiential learning assignment for both graduate (biomedical engineers) and undergraduate (third-and fourth-year health sciences) courses over the past four years. This assignment is geared toward helping address the professional skills awareness gap while encouraging career exploration. We believe that higher education is best served when students pursue wayfinding in parallel with learning. IIs are essentially a conversation with a professional. The professional can serve as a model upon which students can base their future work selves. The interviews are purely to gather information and ask for career advice; they are not a request for a job. These interviews allow students to gain first-hand knowledge of what a position truly requires. Students essentially learn vicariously through the experiences of others . This process encourages students to critically analyze their future self. This helps the student envision how they would fit into their role of interest. This makes IIs both an excellent learning approach and a highly effective career exploration tool. Too often, students postpone career exploration and networking until it is too late. This is why we make students perform IIs as part of a course requirement. In this paper we argue that an II assignment is a unique experiential learning tool that can help students learn to rapidly prototype potential careers and develop the essential skills required for effective career mapping and exploration.
Communications biology, Feb 16, 2024
Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine p... more Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.
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Papers by Derrick Rancourt