published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Traumatic skeletal muscle injuries that include loss of large amounts of muscle mass are becoming more common in today’s warfare. Traditional treatments often do not prevent long term functional impairments. Using a decellularized extracellular matrix (ECM) as scaffolding to replace lost muscle tissue allows for transmission of force through the injury site, and provides a suitable microenvironment receptive to myofiber growth. Seeding the ECM with progenitor cells improves cellular content in the defect area. Exercise exposes the muscle to improved blood flow as well as higher than normal loading. This results in increased blood vessel density as well as higher levels of cellular content, and near complete restoration of function. / text

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Satellite cells are adult stem cells that contribute to hypertrophy and repair in muscles. It is hypothesized that in muscular dystrophy, the satellite cells population is depleted at a very early age, due to repeated muscle damage and repair. Satellite cell transplantation is a potentially useful therapy for muscle diseases, but the lack of an efficient delivery system has hindered its application. The presented work focuses on two specific aims that address the need for more effective cell delivery methods for cell-based therapy. In Specific Aim 1 enhanced tissue culture techniques, such as heat stress, are used to increase cell survival in satellite cell transplantation studies. Also addressed within this specific aim are methods to label and evaluate performance using real-time PCR techniques.Although much work remains to enhancing the viability of in vitro expanded myoblasts derived from satellite cells, a second important hurdle is the systemic delivery of satellite cells to multiple sites (all muscles, in the case of muscular dystrophies). In vitro and in vivo experiments are being undertaken to explore the physiological role of cell signaling systems involved in directed migration and to determine if these chemokine and growth factors can be manipulated to enhance efficacy of cell-based therapies involving skeletal muscle satellite cells. Specific Aim 2 addresses migration of satellite cells to sites of injury and methods to track transplanted cells within the host. Presented here is the use of FAST SPECT II imaging of 111-Indium oxine radiolabeled satellite cells. The long lifetime of 111-indium oxine and the ability to quantify label using FAST SPECT imaging techniques make this technique ideal for in-vivo tracking of transplanted satellite cells for week long studies. Without in-vivo imaging techniques cell fate studies require sequential animal sacrifice with histological sectioning. This not only increases the number of animals used but also adds a significant inter-animal variability to their assessment. The determination of cell fate after transplantation will have a major impact on cell therapy for treatment of muscle disease as well as other stem cell therapies.

Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- α. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that β-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

Embryonic stem cells (ESCs) are pluripotent cells derived from the early embryo and are able to differentiate into cells belonging to the three germ layers. They are a valuable tool in research and for clinical use, but their applications are limited by ethical and technical issues. In 2006 a breakthrough report described the generation of induced pluripotent stem cells (iPSCs). IPSCs are ESC-like cells generated from somatic cells by forcing the ectopic expression of specific transcription factors. This circumvents the ethical issues about the use of embryos in research and provides multiple opportunities to understand the mechanisms behind pluripotency. The aim of this project was to generate sheep iPSCs and characterise them. In order to learn the technique I initially repeated the original iPSC methodology: the putative mouse iPSCs I have generated display a morphology typical of ESCs, characterised by a high nuclear to cytoplasmic ratio, and form colonies with neat edges and smooth domes. These cells are positive to Nanog, a marker of pluripotency, and can give rise to cells belonging to the mesodermal and the ectodermal lineages when differentiated in vitro. Since the main aim of the thesis was the derivation of sheep pluripotent cells, once established the protocol in mouse, I then moved to the generation of ovine iPSC colonies. The cells I have generated have a morphology similar to that of mouse ESCs, express markers of pluripotency such as alkaline phosphatase and Nanog and can differentiate in vitro and in vivo into cells belonging to the three germ layers. Additionally, these ovine iPSCs can contribute to live born chimeric lambs, although at low level.

The acquisition of renogenic competence (the ability to give rise to kidney) during embryonic development is not yet fully understood. Clarifying the temporal and molecular aspects of this process is equally essential for understanding excretory system development and for devising methods for successful differentiation of embryonic stem cells (ESCs) to renal cells for disease modeling, toxicology screening and potential cell replacement therapies. In embryo development, the metanephric (permanent) kidney arises as a result of inductive interactions between two embryonic structures that arise in the intermediate mesoderm - the ureteric bud (UB, a diverticulum of the Wolffian duct) and the metanephric mesenchyme (MM). The UB develops into the collecting duct system and the MM undergoes an epithelial-to-mesenchymal transition to form the secretory units of the kidney - the nephrons. In this thesis, I used a tissue disaggregation-reaggregation method that allows the reconstruction of mouse organotypic kidney rudiments to place different embryonic cells in the environment of a developing kidney and assess their potential to integrate into kidney epithelia and differentiate to renal cells. First, the suitability of this method was evaluated and a quantitative assay for evaluating the numbers of test cells integrating in various renal compartments was developed. Second, the reaggregation method was used to characterise the renogenic potential of undifferentiated mouse ESCs, ESC-derived cells after Notch inhibition, and cells derived from the presumptive nephrogenic regions of embryos at various stages of development. ESCs are isolated from the inner cell mass of an embryo and have the potential to differentiate to any tissue of the body when injected into mouse blastocysts. Strategies have successfully been devised for ESC differentiation to many lineages, but very few studies reported any success with the differentiation of ESCs to a renal lineage. Undifferentiated ESCs showed a very good ability to form chimeric structures with developing kidney tubules (both nephrons and extending UBs). Nevertheless, the resulting structures were morphologically different from renal epithelia in most cases and integrated ESC-derived cells were not positive for several combinations of kidney markers. These results suggested that the influence of the niche was not sufficient for a successful ESC differentiation to renal cells. Treatment of ESC with an inhibitor of the Notch pathway to increase the proportion of mesodermal cells did not improve this outcome. On the basis of these results, it was speculated that the earliest lineage to which embryonic stem cells must be differentiated in order to become competent to make renal cells should first be identified. I addressed this by determining the developmental stage at which cells able to contribute to the formation of metanephric epithelia first appear in mouse embryo development. When mixed in embryonic kidney reaggregates labelled cells isolated from the nephrogenic regions of E9.5 embryos integrated into various renal compartments. These cells were seen in UBs, nephrons, glomeruli and the condensing mesenchyme. Marker expression studies showed that the exogenous E9.5 cells expressed an array of kidney markers - Pax2 in renal epithelia and the condensing mesenchyme, Wt1 in glomeruli and Six2 in the condensing mesenchyme. Furthermore, exogenous E9.5 cells also co-expressed Pax2/Wt1 in the condensing mesenchyme, Megalin/Ecadherin in the proximal tubule and Pax2/E-cadherin in renal epithelia. This provides evidence that challenges the existing model and suggests that some cells from the intermediate mesoderm at a stage where the metanephric blastema is yet formed are competent to contribute to kidney structures. Furthermore, experiments with E8.5 embryos showed that such a renocompetence could be acquired even before the specification of intermediate mesoderm. These findings contribute to our knowledge about kidney cell specification and provide valuable information to guide future attempts to develop an efficient method for deriving renal cells from ESCs. Furthermore, the reported ability of ESC-derived non-kidney cells to form chimeric structures with renal tubules provides a proof-of-principle that it might be possible to use exogenous types of cells for physiological support to injured kidney tubules, thus offering a possible novel approach for cell replacement therapies.

Embryonic stem (ES) cells were used as a model system to understand the signalling events in pancreas development. ES cells were differentiated through known precursor stages towards the tissue of interest in order to recapitulate development in vitro. Thus, protocols directing differentiation of mouse ES cells towards definitive endoderm and pancreatic β-cells were developed. A combination of activin A and bone morphogenic protein 4 resulted in a population of enriched cells expressing genetic markers of definitive endoderm. In vitro differentiation of ES cells into functional pancreatic β-cells has only been partially successful, as it results in cells that are not fully differentiated or functional. This might be due to a lack of cues emanating from surrounding cells present in the developing pancreas. Conditioned media from the mouse MIN6 β-cell line were used on the basis that differentiated β- cells might send out signals affecting the differentiation of the surrounding islet cells. Mouse ES cells were enriched in definitive endoderm and then treated with MIN6 conditioned medium. Gene expression of the β-cell markers Insulin1, Insulin2, and Glucose transporter 2 was significantly increased relative to the untreated control group after 10 days of treatment with conditioned medium. This result was specific for conditioned medium from MIN6 cells as conditioned medium from a kidney-, a neuronal-, and an exocrine pancreatic cell line had no effect. In order to characterise the secreted factor(s) the conditioned medium was subjected to protein precipitation. The pancreatic differentiation factor was present in a protein fraction, suggesting that the factor(s) was proteinaceous. The protein in question was neither proinsulin nor insulin. This knowledge will support the efficient generation of insulin-secreting cells for diabetes therapy.

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes.  Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development.  Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse ES cell.  By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage-dependent requirement for this transcription factor’s activity.  mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas-like cell phenotype, in which 30% of the cells were beta-cell-like.  Cell markers consistent with the different beta-cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development.  Furthermore, the differential beta-like cells secreted C-peptide (insulin) in response to KC1 and IBMX, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells.  Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non-beta cells in the developing endocrine pancreas.  Collectively, we show that this method of <i>in vitro</i> differentiation provides a template for inducing and studying ES cell differentiation into insulin-secreting cells.