Charles Yoon

Thesis title: Identification of Surface Markers Within the Early Cardiac Lineage

Abstract: Myocardial infarction (MI) is now the leading cause of congestive heart failure and death in the world. Coronary occlusion and the resultant myocardial ischemia rapidly result in myocardial necrosis followed by scar formation. As a result, loss of cardiomyocytes in the adult heart is irreversible and leads to reduced cardiac function. While several studies have now shown that cell transplantation results in small improvements in the infarct area, major challenges such as increasing cell survival, engraftment and functional integration with the host tissue remain. Mouse embryonic stem cells (mESCs) are a promising source of cells as they can differentiate into cardiomyocytes well as into recently identified cardiac progenitor cells. Although, multiple cardiac progenitor stages can be generated, which one or which combination that will result in effective treatment of cardiac disease is uncertain.

Additionally, the ability to isolate and enrich for specific cell types is limited, especially at the progenitor cell stage. In order to address this problem my project involves the identification of new surface markers to allow for the isolation of specific progenitor cell types.

Curtis Woodford
MD/Ph.D Candidate

I am working on a scalable method to differentiate three different human pluripotent stem cell lines (hPSC) into pancreatic progenitor cell populations. The cells generated in this protocol will be transplanted into mice to investigate their potential to differentiate to a beta-cell like population capable of controlling glucose levels. While developing a differentiation protocol for pancreatic progenitors, I am also addressing the variability of endogenous signaling in multiple hPSC cell lines. I am also investigating the effects of endothelial cells on the differentiation of hPSC to pancreatic progenitors. The study of endogenous signaling during differentiation for multiple hPSC lines with or without additional cell populations will reveal how to modify the endogenous signaling environment to induce efficient differentiation, addressing a major problem in current hPSC differentiation protocols. I completed a BASc in chemical engineering at the University of Waterloo, and I'm currently in the MD/PhD program at the University of Toronto.

Elizabeth Csaszar

Umbilical cord blood is an attractive source of hematopoietic stem cells (HSCs) for the therapeutic treatment of hematologic disease. However, low cell numbers currently limit of use of these cells and motivate the study of the ex vivo expansion of HSCs. My project focuses on achieving HSC expansion through the regulation of cell-cell interactions that control HSC fate decisions. In heterogeneous hematopoietic culture systems, soluble signaling factors secreted by differentiating cells inhibit the growth of HSCs. I am developing strategies to manipulate culture systems in order to overcome this inhibition and optimize HSC growth, in a scalable and clinically-relevant manner. This approach combines mathematical modeling, bioreactor design, and in vitro and in vivo experimental assessment with the aim of attaining enhanced HSC expansion and gaining a better understanding of the mechanisms controlling HSC self-renewal.

Hematopoietic stem cells (HSCs) are the only cell type capable of reconstituting long-term blood cell production. Outcomes of clinical marrow transplants depend critically on the number of HSCs in the donor sample, and research strategies aimed at deriving or expanding HSC populations are similarly constrained by the need to monitor their numbers. However, there is currently no direct way of enumerating HSCs. My project is focused on the development of a high-throughput, microdroplet-based PCR platform to quantify rare populations of hematopoietic stem cells (HSCs) based on the expression of HSC-specific transcripts. We aim to identify a transcript expression profile that is unique to long-term reconstituting HSCs and perform single-cell RT-PCR by encapsulating single cells in picolitre droplets using a microfluidics system. Ultimately, this automatable platform will be transformed into a system usable in any diagnostic laboratory for the robust enumeration of rare cell types such as HSC.

Title: Engineering interactions between the immune system and cardiac tissue during the early stages of myocardial infarction

Myocardial infarction (MI) is a leading cause of congestive heart failure, which is the number one cause of mortality worldwide. MI induced inflammatory responses play a critical role in the formation of fibrotic scar in the myocardium, however, very little is known about the key cellular and molecular mechanisms in this process. Utilizing microfluidics, an in vitro inter-tissue MI model will be developed to study immune cell – cardiac tissue interactions . In particular, the activation of signaling pathways involved in the maturation process of dendritic cells and the transition between pro-inflammatory and anti-inflammatory stages with respect to inflammatory cytokine production will be analyzed in hypoxic conditions mimicking ischemia. The findings from this study will enhance our understanding of the role of immune system activation in MI, and could lead to the development of early stage pharmaceutical interventions capable of modulating heart disease progression.

Low cell numbers have limited the use of umbilical cord blood as a source of hematopoietic stem cells (HSCs) for transplantation. In order for an ex vivo bioprocess for HSC expansion to be clinically relevant it should be robust, economical and automated. Currently, such processes have large media and cytokine requirements, limiting their use. Our lab has developed an alternative fed-batch media dilution bioreactor. My project focuses on the design and implementation of automated feedback control to reduce media requirements and improve the reliability of this bioprocess.

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (EpiSCs) are two pluripotent mouse cells. They maintain pluripotency in response to distinct signals; mESCs respond to LIF/BMP4 and EpiSCs respond to Activin/bFGF. The transition between the two, both forward and reverse, depend on the activation of the respective cytokines. We would therefore like to use this system of closely related, but distinct, cells to engineer responsiveness to surroundings such that cell fate decisions can be controlled and also reversed.

Our lab has developed a micro-contact printed high throughput platform for screening the response of human pluripotent stem cells to exogenous signals. Micro-contact printing is a very effective means of patterning cells. However, it does have a few drawbacks. There are many steps in implementing the technique, one of which is cleanroom photolithography (something that isn’t easily available to many biomedical laboratories). Also, the patterns are transferred onto the substrates through a stamping process. This prevents a large-scale production of these patterned substrates. I am developing a lithography based high-throughput technology of producing micro-patterned plates to scale-up the screening capabilities of our lab.

Nafees Rahman

To date, technologies capable of guiding embryonic stem cells (ESC) into specific lineages, such as blood cells, are inefficient. Cells residing in the embryo are exposed to a microenvironment comprised of numerous signaling factors and cell-cell interactions. My research will investigate a scalable bioengineering approach to control the spatial and temporal aspects of ESC differentiation into blood progenitor cells.

Nika Shakiba

Since their discovery in 2006, research into the potential for Induced Pluripotent Stem (iPS) cells to provide a source of patient-specific cells for regenerative medicine applications has shown great promise. However, harnessing iPS technology has proven difficult due to our lack of understanding of the reprogramming process. Insights into the mechanism of reprogramming may not only aid in improving protocols for developing iPS cells, but will likely pave the way for developing a standardized criteria for selecting clinically usable iPS cells. My work aims to take a biomedical engineering approach, by combining mathematical modelling and experimental biology, to understand the state transitions involved in driving cells from a differentiated to iPS cell state. In doing so, I aim to shed light on how this process can be better controlled to improve current iPS production strategies, thus moving these cells one step closer to clinical use.

Nimalan
Thavandiran

Nimalan is designing stimulatory/sensory microbioreactor screening systems for investigating the mechanisms by which human Pluripotent Stem Cells (hPSC) differentiate into mature, adult-like cardiomyocytes within three-dimensional microenvironments (via cell-cell and cell-ECM interactions), and then how they proceed into disease-states caused by Myocardial Infarction (MI). The complex process by which hPSC fate toward a mature and functional heart cell is determined, all the while mediated by biochemical, mechanical, and electrical stimuli, each of which uniquely interact temporally and spatially to induce signaling cues and morphogenesis, still remains elusive. Additionally, it has proven difficult to accurately recapitulate MI in vitro through induction of the same apoptosis signaling pathways as observed in vivo. The goal is to use this microtissue-based high-content screening platform to discover and validate the efficacy and safety of novel small molecules which may be beneficial as cardioprotectants, anti-arrhythmogenics, and promoters of regeneration after MI.

Petra's current project focuses on developing a cure for myocardial infarction, which is the leading cause of death worldwide. A promising new approach is the use of targeted protein therapeutics for in situ regeneration of lost myocardial tissue, which would circumvent the need for cell or tissue transplantation. The drugs would contain growth factors or cytokines that prevent further cell death and that stimulate surviving cardiac cells to regenerate the heart. On a cellular level, two essential steps are necessary for regeneration: initiation of cell cycle re-entry of the remaining mature cardiomyocytes to restore original cell numbers, as well as maturation of newly formed cells to ensure full functional recovery. Using human embryonic stem cell-derived cardiomyocytes as an in vitro model for adult cardiac tissue in combination with sophisticated high-throughput screening tools, the goal of this project is to determine growth factor candidates for protein therapeutics.

The goal of my project is to develop novel ways to drive embryonic and induced pluripotent stem cells (ESCs and iPSCs) towards the hematopoietic (blood forming) lineage to produce T lymphocyte precursors. T cells mediate resistance to opportunistic infections and enhanced T cell reconstitution in immunocompromised patients could improve patient survival. ESCs have been shown to differentiate into committed hematopoietic progenitor cells (HPCs) in clinically viable serum-free conditions in our laboratory (Purpura, K.A., J. Morin and P.W. Zandstra. Exp Hematol 2008). The challenge remains to engineer a controlled serum-free cellular microenvironment with immobilized Delta-like 1 (DL1) ligand to direct the differentiation of ESC/iPSC-derived HPCs to the T cell lineage with high yield and efficiency. It is hypothesized that differentiation of T cells from stem cells can be accelerated by deriving iPSCs from T cells (or T-iPSCs) as they may retain epigenetic memory of the cell type from which they were derived. This hypothesis will be tested by differentiating ESCs and T-iPSCs in a novel hydrogel system with the presentation of immobilized DL1 ligand to robustly and reproducibly study the kinetics of T cell differentiation.