LMP student seminars: 3 December
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.
Group 3: Cardiovascular, Physiology and Metabolism
Location: MSB 4171
Carolina Fiallos Herrera
- Title: Monitoring Lung Health During Ex Vivo Lung Perfusion Using Methylation-Sensitive Restriction Enzyme Quantitative-PCR
- Supervisor: Dr. Gavin Wilson
Leo Castiblanco Guzman
- Title: Generation of Functional Airway Epithelium for Tracheal Repair
- Supervisor: Dr. Golnaz Karoubi
Group 5: Infectious Diseases, Inflammation and Immunology
Location: MSB 4279
Hayley Peters
- Title: High-resolution Cell and Transcriptomic Spatial Landscape of Infrapatellar Fat Pad during Knee Osteoarthritis
- Supervisor: Dr. Mohit Kapoor
Abstracts
Carolina Fiallos Herrera: Monitoring Lung Health During Ex Vivo Lung Perfusion Using Methylation-Sensitive Restriction Enzyme Quantitative-PCR
Ex vivo lung perfusion (EVLP) allows for the preservation and treatment of donor lungs prior to lung transplantation (LTx). Methylation analysis of cell-free DNA (cfDNA) from EVLP perfusate provides insight into donor lung health. Tissues have a unique profile of hyper- (~100%) and hypo- (~0%) methylated CpGs which allows for cell-type quantification of cfDNA. Current methylation profiling techniques are expensive and take weeks to complete. This study aims to optimize a methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR) protocol that can be used during EVLP (~3-12 hours). The MSRE-qPCR protocol was tested on known methylation mixtures as a proof-of-concept and on cfDNA from EVLP perfusate from rejected and transplanted lungs. The mixtures and samples underwent a digestion with MSREs or mock digestion, then were quantified using qPCR. The primers targeted differentially hypomethylated CpG regions from four immune and parenchyma cell types. The difference between undigested and digested DNA (ΔCt) represents the relative cell death proportions of each cell type. Methylation percentage was calculated using the formula, 2∆??×100%. The proof-of-concept revealed a strong correlation (r=0.99, n=5, p<.001) between experimental and theoretical methylation values. Additionally, qPCR analysis of perfusate cfDNA revealed unique patterns of cell-type methylation. The methylation percentages of granulocytes from rejected samples were lower than those of transplanted samples. The MSRE-qPCR protocol demonstrates high sensitivity and ability to detect methylation differences. It can be completed within clinically actionable timeframes and will aid the LTx team in making decisions about the donor lung before transplantation.
Leo Castiblanco Guzman: Generation of Functional Airway Epithelium for Tracheal Repair
The tracheal epithelium removes contaminants from inhaled air through mucociliary clearance. Despite its importance in the maintenance of human airways, the development of mucociliary clearance is not fully understood, which is a challenge in tracheal regeneration. Recent human induced pluripotent stem cell (hiPSC) differentiation protocols have demonstrated robust generation of mature epithelial cell types in vitro. However, functional characterization of these cells has been limited. The goal of this project is to quantify ciliary function to better understand the ability of hiPSC-derived airway cultures to model mucociliary clearance, and to explore biomechanical cues to improve functional alignment in vitro and ex vivo. Human iPSC-derived airway basal cells (iBCs) expressing canonical basal cell markers (provided by Hawkins Lab, Boston University) were cultured under air-liquid-interface (ALI) and assessed at 14, 21, and 28 days. iBCs differentiated into ciliated and goblet cells with robust tight junction formation by day 14. Quantification revealed high ciliation throughout day 14-28. Mucociliary transport was negligible at day 14, though transport speed increased by day 28. Patches of locally aligned flow were observed by day 28, and active ciliary beat area and beat frequency did not change significantly from day 14-28. Global unidirectional transport mimicking in vivo conditions was not observed. Further signals are likely necessary to improve mucociliary clearance function. Initial ex vivo tracheal culture demonstrates maintenance of ciliated epithelium. This work provides novel insights into the ciliary beat properties of hiPSC-derived airway cultures and their potential for tracheal regeneration.
Hayley Peters: High-resolution Cell and Transcriptomic Spatial Landscape of Infrapatellar Fat Pad during Knee Osteoarthritis
Objective:
Knee osteoarthritis (KOA) is the most common form of arthritis. The infrapatellar fat pad (IFP) is the largest FPfat pad within the knee; however, its role in KOA is not well understood. Previously, using single-nucleus RNA sequencing, we provided a comprehensive map of the cellular and transcriptomic diversity of the IFP, with considerations for KOA, sex, and obesity status1. In this study, we aim to use Visium HD (VHD) spatial sequencing to develop the first spatial atlas of the IFP, showing cellular heterogeneity at a single cell resolution and how cellular communication may influence KOA.
Methods:
IFPs embedded in OCT underwent histopathological and RNA quality analyses, wherein samples with poor histological features and an RNA Integrity Number <6 were not used. IFPs were subjected to VHD spatial sequencing using the human VHD Spatial Gene Expression kit. Data was processed using SpaceRanger, Seurat was used for and cell segmentation analysis to identifyied individual cells and Loupe Browser visualized identified cells. To date, n=2 KOA-IFPs have been processed, but this is an ongoing project aiming to sequence n=30 IFPs, including healthy donor control IFP.
Results:
Using VHD spatial sequencing, we have identified the spatial location of all major cell types across the IFP at a single cell resolution, with the potential for intercellular communication. Our ongoing efforts are focussed on further characterizing the spatial organization of cells identified in IFP using a larger sample size. We also are employing bioinformatic and functional assays to understand the role of intercellular interaction on KOA pathogenesis.
Conclusions:
Using VHD spatial sequencing, we identified spatial heterogeneity of major cell types across the IFP. Our ongoing efforts will help characterize the role and function of identified cells in KOA pathogenesis.
Contact
No need to register.
Contact lmp.grad@utoronto.ca with any questions.