Human embryonic stem cells (hESCs) replicate in vitro by the process of self-renewal, whilst maintaining their pluripotency. Understanding the pathways involved in the regulation of this process will assist in developing fully-defined conditions for the robust proliferation of hESCs necessary for therapeutic applications.

Calreticulin, an endoplasmic reticulum (ER)-resident protein, is a calcium buffering chaperone. In this study, with an optimized differentiation protocol from mouse R1 ES cells, we demonstrate a novel role of calreticulin in osteogenic commitment and differentiation. To enhance the efficacy of the method, we manipulated cell density and examined the addition of retinoic acid, dexamethasone and peroxisome proliferator-activated receptor γ. The regimen consisting of seeding 250 cells per embryoid body, with the addition of RA (from day 3 to 5) and Dex (from day 10 to 21) gave the most efficacious output. Using this optimized protocol, we investigated the potential involvement of calreticulin in osteogenesis. Calreticulin knock-out cells displayed impaired osteogenesis compared to wild-type cells. In particular, the nuclear translocation of the runt-domain related transcription factor 2 and Osterix, were impaired in the absence of calreticulin. The stimulatory effect of calreticulin on osteogenesis was mediated by its calcium buffering function.

Calreticulin, an endoplasmic reticulum (ER)-resident protein, is a calcium buffering chaperone. In this study, with an optimized differentiation protocol from mouse R1 ES cells, we demonstrate a novel role of calreticulin in osteogenic commitment and differentiation. To enhance the efficacy of the method, we manipulated cell density and examined the addition of retinoic acid, dexamethasone and peroxisome proliferator-activated receptor γ. The regimen consisting of seeding 250 cells per embryoid body, with the addition of RA (from day 3 to 5) and Dex (from day 10 to 21) gave the most efficacious output. Using this optimized protocol, we investigated the potential involvement of calreticulin in osteogenesis. Calreticulin knock-out cells displayed impaired osteogenesis compared to wild-type cells. In particular, the nuclear translocation of the runt-domain related transcription factor 2 and Osterix, were impaired in the absence of calreticulin. The stimulatory effect of calreticulin on osteogenesis was mediated by its calcium buffering function.

Chapter 1: The pluripotent state is maintained by a network of “core” transcription factors (TF). REX1 (Reduced Expression-1) is a pluripotency related TF derived from retrotransposon-mediated duplication of the zinc finger TF Yin Yang 1 (YY1). Furthermore, expression of REX1 and YY1 induces changes in genes regulated by endogenous retroviral elements (ERV), suggesting an evolutionary origin of REX1 for ERV regulation. Studies suggest that murine REX1 may act in epigenetic regulation of gene expression and ERVs, but the precise mechanism remains unelucidated, so we generated FLAG-tagged REX1 pluripotent stem cell (PSC) lines, as well as a series of truncation mutants to explore the REX1 function. Our studies indicate the presence of previously undescribed isoforms of the full-length REX1 protein, suggesting that regulation by REX1 may be more complex than initially appreciated. We hypothesize that REX1 regulates the expression of a sub-set of ERVs and REX1 isoforms regulate REX1 target genes in pluripotent stem cells. Previously, we performed REX1 ChIP-seq and found enrichment for REX1 binding at specific ERVs. Here, we show that differential expression of REX1 isoforms do not change the expression of ERVs. Furthermore, our REX1 KO lines show changes in expression of ERV family members and together with the ChIP data, suggest that REX1 may act as a negative regulator of some retroviral elements. However, further experiments reveal a potential compensation of REX1 KO, possibly by the homologous factors YY1 and YY2. Due to the limited nature and time constrain of our study, we did not find conclusive evidence to further elucidate the potential compensation mechanism and the characteristics of the REX1 isoforms. Our work provided a new avenue for exploring the functional importance of REX1 isoforms and the potential, YY1 and YY2 independent, regulatory role REX1. Chapter 2: Mitotic bookmarking describes a potential mechanism involved in the stable propagation of cellular identity through the cell cycles. Candidate based studies have identified mitotic bookmarking factors (MBFs) that are retained on the mitotic chromatin and preserve the transcriptional memory of the cell. Nevertheless, there is a poor understanding of which proteins can serve as MBFs, as well as the chromatin dynamics of bookmarked sites during mitosis and the start of G1 phase. Previously, we designed a chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) assay to develop a global unbiased approach for identifying and characterizing novel MBFs. Using ChIP-MS, we identifed putative MBFs associated with the mitotic chromatin in pluripotent stem cells (PSCs) and used ATAC-seq to identify subsets of pluripotency-associated accessible gene regions that appear to be bookmarked by a variety of transcription factors, including PARP1, PSIP1, and HDGF. Here, we characterize the interaction of a putative MBF, not found in our ChIP-MS screen, NFYa, with PARP1 and, inconclusively, another putative MBF, DNMT1. Furthermore, we found that PWWP containing putative MBF, HDGF, has a potential role in pluripotency maintenance but it is not mitosis-specific. Due to the limited nature and time constrain of our study, we did not find conclusive evidence to establish the role of PSIP1 in PSC mitotic bookmarking. Our work provided a new avenue for exploring the functional importance of mitotic bookmarks in pluripotent maintenance. / Thesis / Master of Science (MSc)

It has been hypothesized that tumours are caricatures of normal tissue organization, where a minority cell population, the ‘stem cell’ of cancer, holds the exclusive ability for tumour propagation. These cancer stem cells (CSCs), or tumour-initiating cells, possess extensive self-renewal ability, through which they ensure maintenance of the tumourigenic clone. Such cells have been identified in various cancers, including colorectal, and have been proposed to be the source of tumour re-initiation following therapy. An important and currently unanswered question in solid tumours is whether all CSCs are equal or whether there is a gradient of potency within the CSC compartment. Using primary human colon tumour cells and sensitive in vivo functional assays, we have determined that colon CSCs are not uniform; rather, they vary with respect to their proliferative capacity, which is also linked to their response to chemotherapy. These findings hold therapeutic implications for colorectal cancer treatment since all types of CSCs must be eradicated to remove the risk of tumour relapse. While the CSC model may provide attractive answers to some challenging questions, it remains controversial. Ascertaining the importance of CSCs will come from targeted CSC therapies. Here we demonstrate that human colorectal CSC function is dependent on the self-renewal regulator BMI-1. Down-regulation of BMI-1 inhibits the ability of colorectal tumour-initiating cells to self-renew resulting in the abrogation of their tumourigenic potential. Treatment of primary colorectal cancer xenografts with small molecule BMI-1 inhibitors resulted in colorectal tumour-initiating cell loss with long-term and irreversible impairment of tumour growth. Targeting the BMI-1 related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. Collectively, we have advanced the CSC field in two areas of importance. We show for the first time that the CSC pool encompasses a gradient of proliferative potential linked to chemotherapeutic response. Second, we provide critical proof for the clinical relevance of CSCs by inhibiting tumour growth through targeting of the self-renewal machinery. This body of work significantly advances our understanding of colorectal tumour-initiating cells.

It has been hypothesized that tumours are caricatures of normal tissue organization, where a minority cell population, the ‘stem cell’ of cancer, holds the exclusive ability for tumour propagation. These cancer stem cells (CSCs), or tumour-initiating cells, possess extensive self-renewal ability, through which they ensure maintenance of the tumourigenic clone. Such cells have been identified in various cancers, including colorectal, and have been proposed to be the source of tumour re-initiation following therapy. An important and currently unanswered question in solid tumours is whether all CSCs are equal or whether there is a gradient of potency within the CSC compartment. Using primary human colon tumour cells and sensitive in vivo functional assays, we have determined that colon CSCs are not uniform; rather, they vary with respect to their proliferative capacity, which is also linked to their response to chemotherapy. These findings hold therapeutic implications for colorectal cancer treatment since all types of CSCs must be eradicated to remove the risk of tumour relapse. While the CSC model may provide attractive answers to some challenging questions, it remains controversial. Ascertaining the importance of CSCs will come from targeted CSC therapies. Here we demonstrate that human colorectal CSC function is dependent on the self-renewal regulator BMI-1. Down-regulation of BMI-1 inhibits the ability of colorectal tumour-initiating cells to self-renew resulting in the abrogation of their tumourigenic potential. Treatment of primary colorectal cancer xenografts with small molecule BMI-1 inhibitors resulted in colorectal tumour-initiating cell loss with long-term and irreversible impairment of tumour growth. Targeting the BMI-1 related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. Collectively, we have advanced the CSC field in two areas of importance. We show for the first time that the CSC pool encompasses a gradient of proliferative potential linked to chemotherapeutic response. Second, we provide critical proof for the clinical relevance of CSCs by inhibiting tumour growth through targeting of the self-renewal machinery. This body of work significantly advances our understanding of colorectal tumour-initiating cells.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

It has been suggested that some adult bone marrow cells (BMC) can localize to the injured tissues and develop tissue-specific characteristics including those of the pulmonary epithelium. In Chapter 2 we show that the combination of mild airway injury as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype expressing Clara cell secretory protein (CCSP) and pro-surfactant protein-C. Bone marrow cells from transgenic mice expressing green fluorescent protein driven by the epithelial-specific cytokeratin-18 promoter were injected transtracheally into airway-injured wild-type recipients. BMC retention in the lung was observed to be at least 120 days following cell delivery with increasing transgene expression over time. The results indicate that targeted delivery of BMC can promote airway regeneration. Although bone marrow stem/progenitor cells can develop into lung epithelial cells, the specific subpopulation remains unknown. In Chapter 3 we identify a newly discovered population of murine and human BMC that express CCSP. These CCSP+ cells increase in the bone marrow and blood after airway injury and can be expanded in culture. CCSP+ cells are unique in that they express both hematopoietic and mesenchymal stromal cell markers and can give rise to various lung epithelial lineages in vitro. Importantly, bone marrow transplant of CCSP+ cells to CCSP knockout recipients confirms that bone marrow CCSP+ cells contribute to airway epithelium after airway injury. In Chapter 4 we enrich for a stem/progenitor cell population within the CCSP+ using the stem cell antigen (Sca)-1 as a marker. Here we identified a putative epithelial stem/progenitor cell that can be induced to differentiate into various lung epithelial cell lineages expressing markers exclusive to airway or alveolar epithelial cells when cultured under an air liquid interface. These cells also have self-renewal potential in vitro that can proliferate in vivo and repopulate the injured airway epithelium. This newly discovered epithelial-like cells may play a central role in the bone marrow contribution to lung repair and are exciting candidates for cell-based targeted therapy for treatment of lung diseases.

No description available.

Maintenance of gene dosage is important for proper cellular function and development, as evidenced by the natural silencing of one X-chromosome in mammalian females, and by the embryonic lethality of most autosomal aneuploidy. A notable exception is Down syndrome (DS), which occurs in 1/700 newborns. It has been known for 50+ years that DS is caused by trisomy for chromosome 21 (chr21), yet biological understanding remains wanting; even what cell types and pathways are impacted by chr21 dosage has remained unclear. Given the complexity of DS, better experimental approaches have been needed. This thesis advances understanding of DS pathobiology using an innovative approach that translates the X-inactivation mechanism via the XIST gene, to an inducible system to “silence trisomy” in DS patient-derived iPSCs and their differentiated derivatives. I investigated the most immediate and direct effects of silencing trisomy on mRNAs genome-wide. Initial studies revealed trisomy 21 (T21) impairs early developmental pathways for two major cell type processes: neurogenesis and, surprisingly, angiogenesis. Further analysis of endothelial cells showed chr21 overexpression reduces pathways relating to cell migration, projection, and signaling, and functional assays showed delayed response to angiogenic cues causing a deficit in microvessel formation. The previously unknown cell-autonomous effect of T21 on angiogenesis has broad significance for systems impacted, including brain and heart development, and comorbidities throughout life such as early-onset Alzheimer’s disease. This work also has implications for understanding of dosage sensitivity and genome balance, a fundamental but poorly understood aspect of genome biology.