Stem Cell Projects 2018-09-21T16:45:34+00:00
Engineering airway epithelial cells from stem cells for disease modeling and therapy

Amy Wong

Dr. Amy Wong
Senior Research Associate

Lung disease is the third leading cause of death in North America. The limited availability of differentiated patient-specific lung epithelium remains a major roadblock for the potential development of therapeutic drugs to treat many airway diseases. Cystic fibrosis is a fatal disease that affects airway ciliated epithelial cells caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene ultimately resulting in impaired bacterial clearance in the airways and lung destruction.

In 2012, our lab was the first to develop a method to generate a renewable source of airway epithelial cells that express functional CFTR from human pluripotent stem cells. We identified key growth factors that mimic lung developmental pathways in vivo followed by air-liquid interface culture to direct the differentiation of pluripotent stem cells into mature proximal airway epithelia. The result was the establishment of tight junction-coupled differentiated airway proximal epithelial cells with apical localization of the CFTR protein for proper chloride transport and function. The ability to generate a renewable source of airway cells offers great hope for personalized disease modeling, drug discovery, and tissue regeneration to treat many lung diseases.

Investigating the totipotent state and the progressive restriction of cell fate during early embryonic development

Alexander Murray

Dr. Alexander Murray
Postdoctoral Fellow

After fusion of the sperm and oocyte, both highly specialised cell types, extensive remodelling of the epigenome as well as activation of the zygotic genome is required to generate a fully totipotent zygote. Two-cell blastomeres also maintain totipotency, however this capacity to generate both embryonic and extra-embryonic lineages is progressively lost during subsequent development. Stem cells of the early embryo, including pluripotent embryonic stem (ES) cells derived from the inner cell mass (ICM) and multipotent trophoblast stem (TS) cells from the trophectoderm (TE) faithfully maintain their developmental trajectories in vitro and have been invaluable in elucidating the key regulatory networks that irrevocably set aside the pluripotent ICM/epiblast cells from the placental trophoblast lineage.

A number of studies have reported a sub-population of two-cell like ES cells, which display totipotent characteristics yet the derivation of a stable totipotent cell line has not been reported and is an intriguing prospect. This would enable in vitro characterization of the first cell fate decision between the embryonic and trophoblast lineages. During this project, I will seek to characterize these rare two-cell like ES cells and explore their developmental plasticity to determine the extent to which they can be considered totipotent. Furthermore, I will investigate pathways and manipulations that promote the expansion of this rare population with the aim of identifying novel regulators of totipotency as well as conditions that can maintain this state in vitro.

100 Cell Project – Characterization of CFTR mutations across Canada

Zoe Ngan - Lab TechZoe Ngan
Research Technician

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in impaired airway epithelial cell function, and consequent increase in bacterial infections and lung damage. The challenge of CF research is the wide range of mutations in the CFTR gene, secondary modifier genes, as well as limited access to renewable human samples. To advance this research, our group is part of the 100-cell team grant aimed to generate a resource for a wide variety of CF mutations, furthering a personalized medicine approach for treatment.

The 100 Cell Project aims to capture the prevalence of each CFTR mutation in 100 CF patients from across Canada. From each patient; induced-pluripotent stem cells (IPSC) will be generated to establish a cell biobank. This resource will be accessible to all CF researchers for use in drug screenings as well as tissue regeneration. My role in the project is the culturing and characterization of the cell lines. Furthermore, cell lines from patient specific CFTR mutations will undergo directed differentiation into mature airway epithelial cells. These IPSC cells are a renewable resource available to CF researchers for high-throughput drug screenings of potential CF drugs. Lastly, I will be responsible for running an annual workshop to translate our resources to the larger CF community.

Recent projects

Understanding gene regulatory networks distinguishing pluripotent and trophoblast stem cells

Nicole LyonsNicole Lyons
Former Laboratory Research Project Coordinator

In mammalian early development, the first differentiated cell lineage is the trophectoderm (TE), which eventually gives rise to the placenta. Trophoblast stem (TS) cells form the TE, whereas embryonic stem (ES) cells form the embryo proper. Injecting TS and ES cell populations into blastocysts has confirmed contribution to placenta and embryo proper formation, respectively. Successful conversion of ES to TS cells was first achieved by forced repression of Oct4, a POU-family transcription factor essential for ES pluripotency (Niwa et al. 2000). Interestingly, driving expression of caudal-related homeobox 2 (Cdx2) can also induce the differentiation of ES cells to TS cells. Subjecting ES-derived TS cells via Cdx2 overexpression to chimeric analysis revealed that these cells function properly and successfully contribute to the formation of the placenta (Niwa et al. 2005).

This project aims to understand the differences between the gene regulatory and epigenetic networks of pluripotency versus trophoblast cell fate. Using an inducible system to reprogram ES cells to TS cells, we are interested in the genetic and epigenetic changes over time that underlie this conversion. Preliminary analysis revealed that mesendodermal genes are transiently upregulated during the ES to TS transition. Sorting and characterizing the subpopulations in the ES to TS conversion will elucidate the essential steps required for this fundamental switch in stem cell state.

Investigating differences in pluripotent states to drive differentiation

Jodi Garner

Jodi Garner
Former Lab Manager

Different states of pluripotency exist in stem cell populations. Human embryonic stem (hES) cells share molecular and functional properties similar to those of the mouse epiblast stem cells (EpiSC), a cell line derived from the postimplantation epiblast. It has been suggested that EpiSC represent a primed state of pluripotency distinct from the naïve state represented by mouse embryonic stem (mES) cells. By manipulating the culture conditions for hES cells, we are able to drive the conversion of primed hES cells into a naïve cell-like state, resembling that of mES cells.

Ectopic expression of Cdx2 in mES cells is sufficient to drive the conversion of mES into a TS cell state (Niwa et al. 2005) while similar overexpression in EpiSC is insufficient to drive such cell fate changes, leading us to believe that the different pluripotent states of these cells greatly influences cell fate potential. Our research goal is to determine how transcription factor overexpression in these two states of pluripotency differs in hES cells. In particular, we are interested in the role of CDX2, a gene associated with TS phenotype, in converting naïve hES cells into human trophoblast stem (TS) cells.

Identifying novel modulators of vasculogenesis

Lamis HammoudDr. Lamis Hammoud
Former Research Associate

Mammalian vascular development begins shortly after the initiation of gastrulation. Blood vessels develop by two processes: vasculogenesis and angiogenesis. Vasculogenesis occurs in both the embryo and yolk sac by aggregation of de-novo-forming angioblasts (endothelial precursors) into a primitive network of endothelial tubes known as the primitive vascular plexus. This plexus then undergoes angiogenesis: remodeling involving growth, proliferation, regression, migration and sprouting of new vessels from existing ones, resulting in the formation of the mature circulatory network. VEGF, acting through the Flk1/VEGFR2 receptor, is crucial for blood vessel formation and development. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumour growth and metastasis. While the latest antiangiogenic drugs, such as Avastin (anti-VEGF), have been shown to prolong life expectancy in cancer patients, they have serious side effects. Furthermore, relapses often occur necessitating the need for novel therapeutic targets.

The goals of our research are two-fold; to gain a better understanding of the downstream effectors of the VEGF/Flk1 pathway; and to develop a robust in vitro vascular differentiation assay using mouse embryonic stem (mES) cells suitable for small molecule screens to identify novel modulators of angiogenesis. Embryonic stem cells have the ability to differentiate into many tissues, such as blood vessels, in a manner that resembles embryonic development. To that end, we have optimized an in vitro mES cell-based differentiation assay using Flk-eGFP ES cells. This assay has been validated using a variety of vascular modulators. Furthermore, we developed an algorithm using the Cellomics ArrayScan platform, based on the neuronal profiling software, to quantify total expression of Flk1 as well as the number of fluorescent sprouts. Currently, we are using this vascular differentiation assay to screen a kinome small molecule inhibitor library provided by the Medicinal Chemistry Platform of the Ontario Institute for Cancer Research (OICR). Promising candidates showing quantitative deviation from control cultures have been observed, will be validated in secondary assays, and their efficacy will be tested in preclinical tumour models in mice.

Identification of gene regulatory networks driving endoderm specification

Angela MacDonaldDr. Angela MacDonald
Former PhD Student

The developing embryo contains two distinct endoderm lineages, extraembryonic endoderm (ExEn) and definitive endoderm (DE). ExEn contributes to extraembryonic tissues that function as supportive tissues for the embryo whereas DE contributes to embryonic tissues that give rise to the embryonic gut. The core gene regulatory network (GRN) driving DE specification has been well characterized in sea urchin and xenopus embryos, which lack ExEn tissues. However, the study of mammalian endoderm development is complicated by the presence of ExEn tissues as the ExEn and DE share the expression of a common set of transcription factors. The overall aim of our project is to elucidate unique GRN motifs that act to specify the ExEn and DE. Of particular interest is the transcription factor Sox17. Our lab has previously shown that the overexpression of SOX17 drives commitment of human embryonic stem (hES) cells into self-renewing mesendoderm progenitor cells whereas Sox17 has also been shown to drive ExEn gene expression in mouse embryonic stem (mES) cells. To test the ability of Sox17 to assemble distinct GRNs driving ExEn or DE fate, we have overexpressed Sox17 in various stem cell populations and performed high throughput sequencing studies. To identify dynamic regulatory networks involved in Sox17-mediated lineage specification we have combined numerous computational techniques to predict novel GRN motifs. These bioinformatic analyses allow the generation of putative regulatory interactions and thus testable hypotheses for which we can explore either in vitro using stem cell lines or in early mouse embryos.