LMP student seminars: 29 April

Each week during term time, MSc and PhD candidates in the Department of Laboratory Medicine and Pathobiology present their research.

Anyone is welcome. No need to register.

Location: Medical Sciences Building, rooms 4171 or 4279, see below.

As part of the core research curriculum, students taking LMP1001/2/3: Graduate Seminars in Laboratory Medicine and Pathobiology will present their projects. Please see abstracts below.

3. Cardiovascular, Physiology and Metabolism/ Molecular and Cell Biology and Regenerative Medicine

Location: MSB 4279

Ranmal Bandara

• Title: Integration of the LacZ and CFTR transgene using CRISPR/Cas9 mediated homology directed repair or Find and cut-and-transfer (FiCAT)
• Supervisor: Dr. Jim Hu

Randy Van Ommeren

• Title: TBA
• Supervisor: TBA

3. Cardiovascular, Physiology and Metabolism/ Molecular and Cell Biology and Regenerative Medicine

Location: MSB 4171

Ayla Shahid

• Title: Investigating the role of DDR1 in Alveolar Development, Bronchopulmonary Dysplasia and Pulmonary Hypertension
• Supervisor: Dr. Michelle Bendeck

Hayley Peters

• Title: Cell and Transcriptomic Diversity of Infrapatellar Fat Pad during Knee Osteoarthritis
• Supervisor: Dr. Mohit Kapoor

Abstracts

Ranmal Bandara: Integration of the LacZ and CFTR transgene using CRISPR/Cas9 mediated homology directed repair or Find and cut-and-transfer (FiCAT)

Objectives: Cystic Fibrosis (CF) is a monogenic, recessive disorder caused by the mutation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The major caused of morbidity and mortality in CF patients is lung disease. CF Lung gene therapy aims to deliver a functional copy of the CFTR gene to the cells. To have sustained expression of CFTR without repeated delivery of the gene, the integration of the gene into cells is crucial. CRISPR/Cas9 and other nucleases have been shown to enable the integration of genes but the integration efficiency has been low because of the dependence of some of these strategies on homologous recombination. Find and cut-and-transfer (FiCAT) uses CRISPR/Cas9 to create a DNA double stranded break and the piggybac protein linked to Cas9 will then transpose the donor(containing LacZ or CFTR gene) DNA at the target locus. This technology does not rely on homologous recombination for gene integration. In this study we test whether the CFTR and LacZ genes can be integrated using (Aim 1) CRISPR/Cas9 mediated homology directed repair or using (Aim 2) the FiCAT system. 

Methods: Aim 1. 16HBE14O- cells were transduced with HD-Ad carrying CRISPR/Cas9 targeting the human CFTR locus with the LacZ gene flanked by 3 kilobase homology arms. Following transduction, integration was confirmed by junction PCR. The reporter gene expression is analyzed by X-gal staining or quantification using chemiluminescence. The integration efficiency of the LacZ transgene was determined by passaging cells 10 times and then X-gal staining. Aim 2. HEK293 and IPEC-J2 cells were transfected with a 22 KB plasmid containing the FiCAT system with donor (CFTR or LacZ) targeting the AAVS1, GGTA1, human CFTR and pig CFTR loci. Following delivery of the components, site-specific integration is determined using junction PCR and Sanger sequencing. Integration efficiency was determined by passaging cells 10 times and then X-gal staining.

Results: Aim 1. We show the successful transduction of 16HBE14O- cells with CRISPR/Cas9 carrying LacZ HD-Ad. We demonstrated the successful integration of the LacZ transgene at the human CFTR locus. We determined the integration efficiency of the LacZ transgene to be 6%. Aim 2. We demonstrated the successful integration of the LacZ and CFTR transgenes at the AAVS1 locus by junction PCR and Sanger sequencing and confirm integration of the LacZ transgene at the GGTA1 locus by junction PCR. We detected an increase of LacZ expression by chemiluminescence when targeting the AAVS1 locus compared to LacZ donor alone delivered. We are in the process of testing the integration efficiency of the LacZ and CFTR genes at the AAVS1 locus.

Conclusion: Aim 1. The LacZ gene can be successfully integrated at the human CFTR locus at a 6% integration efficiency. The integration of the CFTR transgene is to be determined. Aim 2. These results validated the integration of the CFTR gene using FiCAT which can be used as an alternative strategy for CF therapeutic development.

Randy Van Ommeren: TBA

TBA

Ayla Shahid: Investigating the role of DDR1 in Alveolar Development, Bronchopulmonary Dysplasia and Pulmonary Hypertension

Bronchopulmonary dysplasia (BPD) is a developmental lung disease affecting 80% of premature infants. It is characterized by enlarged alveolar spaces, increased inflammation, and improper development of lung vasculature. A quarter of BPD patients subsequently develop pulmonary hypertension (PH), a condition characterized by elevated pulmonary arterial pressure, right ventricular hypertrophy, and eventual right heart failure. A recent paper from Bonafiglia and colleagues has showed genetic deficiency of Discoidin domain receptor 1 (DDR1) in mice results in BPD-PH like characteristics.

DDR1 is a collagen-binding receptor tyrosine kinase with roles in mechanotransduction, inflammation and cell migration. DDR1 has been previously implicated in conditions like atherosclerosis and fibrosis but little is known about its role in lung development and alveolar epithelial function. We investigated DDR1’s role in the alveolar epithelium using A549 cells and found collagen binding receptors DDR1 and Beta-1-integrin to have redundant roles in supressing expression of transcriptional co-activator TAZ, an important mediator of alveolar epithelial cell differentiation. Furthermore, we found DDR1 regulates expression of pro-fibrotic and angiogenic factors CCN1 and CCN2 that have previously been implicated in BPD-PH. Lastly we found DDR1 to regulate expression of epithelial-to-mesenchymal transition (EMT) markers N-Cadherin, E-Cadherin and Vimentin. This indicates that DDR1 has potential roles in alveolar epithelial differentiation, proliferation and EMT. 

Future work will investigate DDR1’s role in alveolar development using alveolar epithelial cells isolated from neonatal Ddr1-/- mice. Understanding the role of DDR1 in lung development will allow us to better understand BPD-PH pathogenesis and develop downstream therapeutic strategies to improve prognosis

Hayley Peters: Cell and Transcriptomic Diversity of Infrapatellar Fat Pad during Knee Osteoarthritis

Hypothesis: Knee osteoarthritis (OA) is the most common form of arthritis, characterized by cartilage degeneration, synovial inflammation, and fibrosis. The infrapatellar fat pad (IFP) is the largest fat pad in the knee, however its cell composition and involvement in knee OA pathogenesis is not well understood. Additionally, obese individuals (BMI 30-40) are at greater risk of developing knee OA. We hypothesize that differences in the cellular constituents and their functions within the IFP based on knee OA, sex, and BMI status may contribute to distinct molecular mechanisms associated with OA pathogenesis.

Materials and Methods: IFP [n=21; n=6 healthy control donor, n=15 knee OA: n=8 obese BMI 30-40, n=7 normal BMI 18.5-25] underwent single-nucleus RNA sequencing (snRNA-seq), spatial sequencing, advanced bioinformatics, and targeted metabolomics to identify key cell types, subsets, and transcriptomic differences.

Results: Clustering analysis of snRNA-seq data identified fibroblasts, macrophages, adipocytes, and endothelial cells as major cell types, each with multiple subsets and unique transcriptomic signatures. We were also able to spatially locate the major cell types and fibroblast subtypes throughout the IFP. When investigating fibroblast subsets based on knee OA status, sex, or BMI, distinct differences in gene expression were observed and were linked to putative differences in fibroblast function. Targeted metabolomic analysis further determine differences in the function of fibroblasts based on BMI, under normal conditions and when stimulated with profibrobtic or proinflammatory stimuli.

Conclusions: Using snRNA-seq, spatial sequencing and advanced bioinformatics, we have identified distinct cell subsets of fibroblasts, adipocytes, macrophages, and other cell types in IFP, each with unique transcriptomic profiles. We have also uncovered differences in transcriptomic profiles and function within each population based on knee OA status, sex, and BMI.

Contact

No need to register.

Contact lmp.grad@utoronto.ca with any questions