Transgenic manipulation of exogenous and endogenous gene expression in human embryonic stem cells (hESCs) is a powerful approach to decipher the genetic pathways dictating their developmental fate. Presently used genetic tools face limitations including leakiness in inducibility of expression, epigenetic silencing in long-term cell culture, low genomic integration efficiencies, small genetic cargo limit and lack of high-throughput cloning capabilities. To overcome these limitations, I have constructed R4-Integrase and piggyBac transposon genetic vector systems for stable transgene overexpression and knockdown in hESCs. Preliminary functional testing of the piggyBac vector system in HEK 293T and hESCs demonstrated vector inducibility as well as successful overexpression and knockdown of pluripotency factor OCT4. Concurrently, a cost-effective and high efficiency method for chemical transfection of hESCs was developed. Exogenous overexpression and knockdown of transcription factors in hESCs will aid in the elucidation of gene regulatory networks controlling pluripotency and developmental fate.

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.

Clonal culture is crucial for experimental protocols that require growth or selection of pure populations of cells. Currently, there is no method for deriving neural stem cells (NSCs) clonally from single human embryonic stem cells (hESCs). Bulk derivation of neural progenitors from hESCs for cell therapies can lead to a host of problems including incomplete differentiation leading to proliferation of tumorigenic clusters in vivo. Clonal derivation allows for the screening and selection of only the most suitable cells for culture and expansion. We have developed a clonal, serum free method of generating NSCs and their progenitors directly from hESCs with an efficiency of 1.6%. The NSC colony-forming cell was identified as a TRA-1-60-/SSEA4- cell whose fate becomes specified in maintenance conditions by inhibition of bone morphogenic protein (BMP) signalling. This clonal culture method can be scaled up to produce vast quantities of NSCs for differentiation and use in cell therapies.

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.

We used mouse embryonic stem cells (ESCs) as a model to study the mechanisms that regulate stem cell fate. Using gene expression analysis during a time course of differentiation, we identified 281 candidate regulators of ESC fate. To integrate these candidate regulators into the known ESC transcriptional network, we incorporated promoter occupancy data for OCT4, NANOG and SOX2. We used shRNA knockdown studies followed by a high-content fluorescence imaging assay to test the requirement of our predicted regulators in maintaining self-renewal. We further integrated promoter occupancy data for Polycomb group (PcG) proteins, EED and PHC1 to identify 43 transcriptional networks in which we predict that OCT4 and NANOG co-operate with EED and PHC1 to influence the expression of multiple developmental regulators. Next, we turned our focus to the PcG protein PCL2 which we identified as being bound by both OCT4 and NANOG and down-regulated during differentiation. PcG proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs. Using multiple biochemical strategies, we demonstrated that PCL2 associates with Polycomb Repressive Complex 2 (PRC2) in mouse ESCs, a complex that exerts its effect on gene expression through H3K27me3. Although PCL2 was not required for global histone methylation, it was required at specific target regions to maintain proper levels of H3K27me3. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics and defects in differentiation. Integration of global gene expression and promoter occupancy analyses allowed us to identify PCL2 and PRC2 transcriptional targets and draft regulatory networks. We describe the role of PCL2 in both modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation.

Clonal culture is crucial for experimental protocols that require growth or selection of pure populations of cells. Currently, there is no method for deriving neural stem cells (NSCs) clonally from single human embryonic stem cells (hESCs). Bulk derivation of neural progenitors from hESCs for cell therapies can lead to a host of problems including incomplete differentiation leading to proliferation of tumorigenic clusters in vivo. Clonal derivation allows for the screening and selection of only the most suitable cells for culture and expansion. We have developed a clonal, serum free method of generating NSCs and their progenitors directly from hESCs with an efficiency of 1.6%. The NSC colony-forming cell was identified as a TRA-1-60-/SSEA4- cell whose fate becomes specified in maintenance conditions by inhibition of bone morphogenic protein (BMP) signalling. This clonal culture method can be scaled up to produce vast quantities of NSCs for differentiation and use in cell therapies.

Transgenic manipulation of exogenous and endogenous gene expression in human embryonic stem cells (hESCs) is a powerful approach to decipher the genetic pathways dictating their developmental fate. Presently used genetic tools face limitations including leakiness in inducibility of expression, epigenetic silencing in long-term cell culture, low genomic integration efficiencies, small genetic cargo limit and lack of high-throughput cloning capabilities. To overcome these limitations, I have constructed R4-Integrase and piggyBac transposon genetic vector systems for stable transgene overexpression and knockdown in hESCs. Preliminary functional testing of the piggyBac vector system in HEK 293T and hESCs demonstrated vector inducibility as well as successful overexpression and knockdown of pluripotency factor OCT4. Concurrently, a cost-effective and high efficiency method for chemical transfection of hESCs was developed. Exogenous overexpression and knockdown of transcription factors in hESCs will aid in the elucidation of gene regulatory networks controlling pluripotency and developmental fate.

Adipose-derived stem cells (ASCs) have been extensively investigated for their applicability in the field of tissue engineering due to their multi-lineage differentiation potential and the convenience of cell acquisition. To date, conventional inductive media have been used to induce lineage-specific differentiation of ASCs; however, this general approach has limitations in terms of high costs and unstable differentiation responses. In native tissues, mesenchymal stem cells (MSCs) interact with their surrounding cells (i.e. mature cells) through paracrine and autocrine signaling, which can regulate their metabolism and cell function. Therefore, the author developed in vitro co-culture models to study the interactions between ASCs and three different mature cell types: adipocytes, chondrocytes and osteoblasts. In this work, bovine ASCs (bASCs) from the interdigital fat pad were first isolated and characterized, in terms of in vitro proliferation and multi-lineage (bone, cartilage, fat) differentiation with conventional inductive media and culture conditions. Doubling time calculations and gene expression analysis of stem cell markers indicated a threshold existed for stem cell degeneration at passage 5 (P5) for bASCs when expanded extensively in vitro. The multi-lineage differentiation potentials were compared between passage 2 (P2) and P5. Interestingly, while the P5 bASCs presented significantly lower levels of adipogenesis and chondrogenesis, osteogenesis was maintained or even improved with serial passaging. In the designed indirect co-culture systems, adipogenesis and chondrogenesis were investigated in growth medium without key differentiation factors, whereas osteogenesis was induced in conventional osteogenic medium, to maintain the stable phenotype of the mature osteoblasts in culture. The results were used to demonstrate the general feasibility of mature cell-induced or -enhanced bASC differentiation through soluble, cell-secreted paracrine signaling. When compared to growth factor (GF)-stimulated differentiation, the bASCs in co-culture presented an earlier, but potentially stronger, level of differentiation. Among these paracrine factors, Wnt proteins are known to play essential roles in mediating stem cell self-regulation and fate determination. In the current thesis, the Wnt inhibitors WIF-1 and DKK-1 were used to explore the involvement of the Wnt canonical and non-canonical signaling pathways in the designed co-culture environments. The data showed a strong correlation with the literature, indicating the canonical pathway was upregulated during osteogenesis, but inhibited during adipogenesis. The inhibition of chondrogenesis through the canonical pathway was suggested on a genetic level. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-03-09 13:39:14.553

Cellular Cardiomyoplasty (cell therapy for myocardial regeneration) targets the basic pathophysiology of heart failure and represents a novel technique for augmenting the function of the failing heart. Previous studies have demonstrated massive mechanical losses in the first few minutes. Thus, efforts to reduce mechanical losses may prove more beneficial than those directed against biological losses alone. We believe that "Wash-out" into the disrupted blood vessels is responsible for these early losses. / In the first part of this study we hypothesized that by increasing the size of the injectate, the amount of immediate losses can be reduced achieving better retention. Using Alginate-poly-L-lysine-Alginate (APA) miscrocapsules of two different sizes (200mum&400mum) and comparing retention with bare microspheres (10mum) of similar size to MSCs, we demonstrated that immediate retention rate increased by four folds. The retention rate for group 1 (microspheres only) was 4.28+/-3.46% which was significantly lower than that for groups 2 (microspheres in 200mum microcapsules) at 16.45+/-12.66% and group 3 (microspheres in 400mum microcapsules) at 12.93+/-6.28% for Group (p<0.05). There was no difference between group 2 and 3. / In the second part, we investigated the potential of gradually increasing the cell load on functional improvement and engraftment using conventional intramuscular delivery. Five groups of rats received escalating doses of MSCs after surgically induced ischemia (gp1 no cells, gp2 0.5x 10 6, gp3 1.5x106, gp4 3x106,gp5 5x106 MSCs). At 7 weeks, we observed significant improvement in cardiac function in groups 3 to 5 compared to post-infarction baseline. This was not observed in groups 1 & 2. However, in groups 3 to 5, we observed no functional advantage for increasing the cell load beyond a minimal therapeutic dose. This is consistent with our hypothesis that small cells are washed out into the circulation. / We also showed the ability of Alginate-Poly-l-lysine-Alginate (APA) microcapsules to sustain the viability of encapsulated MSCs in-vitro. Finally, the ability of encapsulated MSCs to improve the function of the heart in-vivo was tested.

A qualitative phenomenological study using van Manen’s human science method was conducted to gain insight into the lived experience of patients and their family members transitioning through one hundred days post haematopoietic stem cell transplantation (HSCT). Three families between zero and five years post HSCT were recruited from a bone marrow transplant unit in central Canada. Multiple in-depth open-ended interviews and field notes were employed to arrive at a detailed description of the lived experience of patients and family members. Awakening from the cocoon emerged as the main essence of patient’s and family members’ experiences, supported by three themes: the disruptions, the chrysalis, and new beginnings. The results from this study provide evidence that the families viewed the HSCT in a positive perspective and highlight the importance of supporting families throughout the acute phase of transplantation.