LMP student seminars: 22 October

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

Britney Tian

• Title: Bioengineered 3D hPSC-cholangiocyte ducts with physiological signals for biliary disease modelling
• Supervisor: Dr. Shinichiro Ogawa

Weisheng (Alice) Liang

• Title: High-resolution insight into the human balance organ to drive therapies for vestibular disorders
• Supervisor: Dr. Alain Dabdoub

Group 5: Infectious Diseases, Inflammation and Immunology

Location: MSB 4279

Juliette Blais-Savoie

• Title: Evolution of vaccine seed influenza A viruses in emrbyonated chicken egg and mammalian cell culture propagation
• Supervisor: Dr. Samira Mubareka & Dr. Nicole Mideo

Maya Aisha

• Title: Unbiased and Spatial Proteomics of Kidney Allograft Biopsies: Terminal Complement Proteins Dominate Chronic Active Antibody Mediated Rejection
• Supervisor: Dr. Ana Konvalinka

Abstracts

Britney Tian: Bioengineered 3D hPSC-cholangiocyte ducts with physiological signals for biliary disease modelling

Despite recent advances in cholangiocyte organoid systems, no human cell-based platform yet faithfully recapitulates intrahepatic cholangiocyte physiology in vitro, limiting our ability to model complex biliary disease pathogenesis. Traditional 3D bile duct-on-chip devices are mostly based on primary human patient or murine extrahepatic cholangiocytes and lack sufficient throughput for investigating complex interplay between multiple biliary constituents. As such, there is an urgent need to intergrade these factors into a human intrahepatic cholangiocyte–based microfluidic platform with increased throughput to systematically examine cholangiocyte response and disease progressing under physiologically relevant signals. To address these limitations, we engineered a perfusable, hPSC-derived 3D bile duct in within the AngioPlate384 platform. This device can hold up to 128 tissues within a single plate to enable the parallel testing of multiple complex biliary microenvironmental signals and features an open-top design to allow easy tissue retrieval for downstream flow cytometry and molecular profiling. The hPSC-bile duct displays robust primary ciliation, apical-basal polarity and high cystic fibrosis transmembrane conductance regulator (CFTR) function. We show that fluid flow and stromal cell incorporation jointly enhances hPSC-cholangiocyte function, and that bile acid exposure, alone or in combination with a pro-inflammatory cytokine interferon-gamma, can synergistically disrupt cholangiocyte function. Lastly, we incorporated proliferative murine or hPSC-derived hepatic mesenchymal cells to model biliary fibrosis, a key feature of biliary disease. By integrating models of both healthy and disease-associated conditions, our system serves as a powerful platform to mechanistically evaluate cholangiocyte function, informing the development of future precision medicine approaches in biliary disease.

Weisheng (Alice) Liang: High-resolution insight into the human balance organ to drive therapies for vestibular disorders

The utricle is a sensory organ in the inner ear crucial for balance. Its sensory epithelium consists of mechanosensory hair cells (HCs) that sense head tilting and linear acceleration, along with nonsensory supporting cells (SCs) that play a key role in maintaining the integrity of the epithelium. HC degeneration is a primary cause of progressive and irreversible balance disorders with no approved therapies. SCs can regenerate lost HCs through transdifferentiation, but regeneration is only robust in early development and drastically declines as the utricle matures. Furthermore, there is a limited understanding of the cellular and molecular characteristics of the human utricle. To address this, we employed single-nucleus multiomic sequencing (snRNA- and snATAC-seq) to profile the fetal human utricle at an early stage with regenerative potential (gestational week 15) and a late stage where regenerative capacity is lost (GW18-19). We identified distinct subpopulations of HCs and SCs, each defined by unique gene expression signatures. Building on these findings, we designed a customized 300-gene panel for high-resolution imaging-based spatial transcriptomics. This enabled validation and visualization of gene expression within the intact utricle, allowing us to identify spatially distinct cell populations. We also uncovered epigenomic differences between the two stages and mapped regulatory networks between active transcription factors. Our integrative multiomic and spatial approach provides the first high-resolution view of the human fetal utricle. These insights lay the groundwork for future work on understanding human-specific mechanisms of sensory cell regeneration to inform strategies for treating balance disorders.

Juliette Blais-Savoie: Evolution of vaccine seed influenza A viruses in emrbyonated chicken egg and mammalian cell culture propagation

Introduction: The majority of seasonal influenza vaccines contain viral antigens from viruses propagated in ovo within the embryonated chicken egg (ECE) system. However, in vitro methods of influenza propagation have also been developed for vaccine production. Although egg-adaptive mutations are often thought to be problematic for disease control, recent theory demonstrates that diversity across a population can instead lead to increased population immunity, ergo greater overall vaccine effectiveness. Yet it remains unclear how much diversity is generated over the course of vaccine production, what frequency new mutations can achieve during the process, and whether the answers to these questions depend on if the viruses are grown in eggs (in ovo) or in vitro.

Methods: To investigate this, we propagated WHO-recommended vaccine seed strain A/Michigan/45/2015 H1N1 (2017-18, 2018-19 flu seasons) and pandemic influenza vaccine strain A/dairy cattle/Texas/24-008749-002/2024 H5N1 in ECEs and Madin-Darby canine kidney (MDCK) cells. We then sequenced the output at each passage on the Illumina Miniseq platform and analyzed the number of non-synonymous mutations arising in each sample as well as the abundance of those mutations relative to the original reverence amino acid at that site.

Results: We found that after several passages in ovo and in vitro, A/Michigan/45/2015 H1N1 which was propagated in ECEs showed numerous mutations on the HA gene, while the same virus propagated in MDCK cells showed no amino acid changes. Comparatively, A/dairy cattle/Texas/24-008749-002/2024 H5N1 acquired significantly less diversity via in ovo propagation. Of the mutations observed at high frequency in A/Michigan/45/2015, two (N179S and S207R) are located on antigenic sites. Furthermore, the mean pi nucleotide diversity index of progeny virus HA sequences increased incrementally across four passages in ECEs, and remained consistently low across three passages in MDCK cells (figure 1). These preliminary findings suggest that, while they are linked to egg-adaptive mutations, ECE-based vaccine manufacturing methods may lead to more antigenically diverse seasonal influenza vaccines than in vitro propagation in MDCK cells, while this may not be the case for avian influenza pandemic vaccines such as those for H5N1.

Maya Allen: Unbiased and Spatial Proteomics of Kidney Allograft Biopsies: Terminal Complement Proteins Dominate Chronic Active Antibody Mediated Rejection

Transplantation is the optimal treatment for end-stage kidney disease, but many grafts fail due to antibody mediated rejection (AMR). AMR is caused by donor-specific antibodies (DSA) against antigens on the graft endothelium. Diagnostic criteria for AMR in kidney allografts consider graft injury, evidence for antibody interaction with the endothelium (i.e. C4d staining in the microvasculature), and circulating DSA. AMR is further stratified into active (aAMR) or chronic-active (caAMR) by the presence of chronic glomerulopathy in the latter. Intriguingly, 50% of patients exhibiting microvascular inflammation consistent with AMR do not have DSA (DSA-AMR). To gain insights into the molecular underpinnings of AMR with and without DSA, we investigated proteomic differences in for-cause biopsies of these groups. Laser capture microdissection was used to separate tubulointerstitium and glomeruli of biopsies from 83 DSA+ and DSA- patients with aAMR or caAMR, followed by unbiased proteomic analysis using a Q-Exactive HFX mass spectrometer. Complement cascade proteins were amongst the most significantly differentially expressed proteins in both compartments. In the tubulointerstitium, terminal complement components were increased in DSA+AMR compared to DSA-AMR, and in caAMR compared to aAMR. In the glomeruli, all quantified complement proteins were increased in caAMR compared to aAMR, with no significant difference between DSA+AMR and DSA-AMR biopsies. Preliminary imaging mass cytometry data demonstrated C5a expression in the basement membranes of biopsies with DSA+caAMR, while C5b-9 co-stained with IgG. Increased complement expression in both DSA+ and DSA- biopsies with caAMR, with C5a expression notable in basement membranes, suggests complement involvement in chronic glomerulopathy.

Contact

No need to register.

Contact lmp.grad@utoronto.ca with any questions.