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No description available.

Embryonic stem cells are defined by two key characteristics; apparently symmetrical self-renewing cell division and the ability to differentiate into cells of all three germ layers. Self-renewal depends on several extrinsic and intrinsic cues including a gene regulatory network centered around Oct4, Sox2 and Nanog that has been hypothesized to be reinforced by positive reciprocal interactions. Studies measuring Nanog expression by fluorescent reporters and immunoflourescence have shown that some undifferentiated Oct4 positive cells do not express Nanog (Chambers et al., 2007). However, the mechanisms responsible for generating this heterogeneity in Nanog expression are unknown. Here I show that Oct4 heterozygote ES cells lack Nanog-negative cells. Consistent with a model in which ES cell differentiation proceeds effectively through Nanog-negative cells, these Oct4 heterozygotes are retarded in their differentiation kinetics. Importantly, restoring Oct4 levels towards wild type reestablished both heterogenous Nanog expression and rapid differentiation. Analysis of ES cells carrying a mutation in the Oct4 binding site in the proximal Nanog promoter showed that Oct4 acts as a positive activator on the endogenous Nanog. Finally, comparison of gene expression in Nanog expressing and Nanog non-expressing ES cells has identified candidate genes that may be responsible for the switch in Nanog expression.

Mesenchymal stem cells are multipotent cells that have the ability to differentiate down multiple lineages as well as secrete trophic and anti-inflammatory factors. These qualities make MSCs a promising cell source for cell-based therapies to treat a variety of injuries and pathologies. Biomaterials are often used to control and direct stem cell behavior by engineering a desired environment around the cells. Recent research has focused on using the naturally derived sulfated glycosaminoglycan (GAG), heparin as a biomaterial due to its negative charge and ability to sequester and bind positively charged growth factors. Engineering a heparin coating that can mimic the native heparan sulfate proteoglycan structure found at cell surfaces can be used as a novel platform to present GAGs to cells to direct cell behavior. The overall goal of this dissertation was to develop GAG-based coatings on MSC spheroids in order to study the role of heparin and its derivatives on MSC culture applications. To investigate the role of heparin in coating form on MSC behavior, the ability of the coating to sequester positively charged growth factors was characterized. Given the role of sulfation in the negative charge density of heparin and growth factor interactions, a desulfated heparin coating was develop and used to examine how presentation of coatings with native and no sulfation levels could potentiate response to growth factors in the surrounding environment. Additionally, heparin and growth factor binding in coating presentation was explored to develop a novel platform to assemble MSC-based microtissues. Together these studies provided valuable insight into a novel approach to direct cell behavior by engineering a coating that harnesses heparin interactions with the surrounding environment.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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Recent evidence suggests that “mesenchymal stem cells” (MSCs) are resident in the perivascular compartment of connective tissues. However, since the definition of a stem cell assumes that these progenitors have clonal self-renewal and multi-lineage differentiation potential, the term “MSC” has been criticised, as it has been impossible to isolate definitive clonally derived “MSCs.” To test for this most basic definition of a stem cell, here it is shown that human umbilical cord perivascular cells (HUCPVCs) are capable of multilineage differentiation in vitro and, more importantly, in vivo, displaying the ability to differentiate into functionally synthetic cells that direct and contribute to rapid connective tissue healing by producing bone, cartilage and fibrous stroma in a mouse injury model. Uniquely, these cells can be enriched to >1:3 clonogenic frequency in early passage culture, making it possible to isolate clones and daughter sub-clones from mixed gender suspensions, determined to be definitively single-cell-derived by Y-chromosome fluorescent in situ hybridization (FISH) analysis. Each clone was assayed for multi-lineage differentiation capacity into the five mesenchymal lineages: myogenic, adipogenic, chondrogenic, osteogenic and fibroblastic (stroma). The observation that daughter sub-clones possess equal or lesser differentiative potential to their respective parent clones demonstrated the two intrinsic properties of stem cells in vitro: clonal self-renewal and multi-lineage differentiation. This evidence provides a new hierarchical structure of robust MSCs self-renewing to produce more restricted progenitors that gradually lose differentiation potential until a state of complete restriction to the fibroblast is reached. The methods described herein combined with recognition of this lineage hierarchy provides a significant advance to the understanding of MSC biology, and will enable interrogation of the properties of robust self-renewal and differentiation of MSCs in serially transplanted living recipients.

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No description available.