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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.

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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.

Central nervous system injuries often lead to devastating consequences due to an unfavourable environment created after the injury. Current treatments have yet to address the environment for improved prospects of functional recovery. Transplantation of Schwann cells into the lesion site could in part address the issue, promoting nerve regeneration and enhancing functional recovery. Bone marrow stromal cells (BMSCs) promise to be a viable, autologous source for Schwann cell derivation. Fate-committed Schwann cells derived from BMSCs through co-culture with purified dorsal root ganglia (DRG) neurons suggest that the DRG neurons present juxtacrine cues that direct commitment to the Schwann cell fate. We hypothesize that Neuregulin 1 type III (NRG1(III)) is one such juxtacrine cue to which BMSC-derived Schwann cell-like cells (SCLC) respond in the switch to fate commitment. In this study, NRG1(III) was found to be expressed on freshly isolated DRG neurons and that SCLCs expressed both the ErbB2 and 3 receptors. Western blot analysis for phosphorylated Akt and MAPK provided indicators of downstream signalling of NRG1/ErbB complexes. We then tested if both the soluble and membrane bound forms of NRG1 mediate SCLC differentiation towards fate commitment. In contrast to the membrane-bound form on DRG neurons, soluble NRG1 failed to direct the SCLCs towards the Schwann cell fate. HEK293T cells that stably overexpress NRG1(III) were generated and tested as a neuronal surrogate that presents NRG1(III) on the cell surface. In a 5-day co-culture system with HEK293TNrg1(III) cells, SCLCs were found to develop elongated processes, acquiring either unipolar or bipolar morphology that resembles that of Schwann cells. Screening for marker expression by RT-PCR suggested that at this stage of morphological transition, SCLCs were not yet committed to the Schwann cell fate. The co-culture system will be pursued to find ex vivo conditions that direct differentiation of BMSC-derived SCLCs to fate-committed Schwann cells. / published_or_final_version / Biochemistry / Master / Master of Philosophy

MSCs are the most studied subtype of adult stem cells and have been derived from most postnatal organs and tissues. MSCs are defined as having the capacity to self-renew and to differentiate into both mesodermal and non-mesodermal lineages, and are immunosuppressive. For these properties, MSCs have been considered ideal candidates for regenerative medicine and have been used in several clinical trials. The difficulty, however, to preserve the potency of the cells during culture expansion and to monitor differentiation are obstacles in their use in the clinic and have emphasized the need to investigate molecular pathways underlying stem cell fate-decisions during differentiation in more detail. Hippo pathway was recently identified in Drosophila melanogaster and mammals, and controls organ size by regulating cell proliferation, apoptosis and differentiation. It is composed of a core of serine/threonine kinases and scaffold proteins that when activated, phosphorylate and inhibit yes-associated protein (YAP) transcriptional co-factor. Inactivation of YAP in some stem and progenitor cells by this pathway is required for their differentiation. In contrast, failure to inhibit YAP enhances proliferation and may cause oncogenic transformation. In the present study, the expression of multiple YAP variants was confirmed in human and mouse MSCs. In both human and mouse, YAP was inhibited in response to cell-contact inhibition and remained unchanged during in vitro chondrogenic differentiation. Overexpression of human (hYAP1) variant in C3H/10T1/2 cells did not appear to affect colony formation, cell cycle distribution or cell size, but increased cell proliferation, induced cell transformation and reduced the in vitro differentiation capacity of the cells towards the chondrogenic, adipogenic and osteogenic lineages. The effects of hYAP1 overexpression are hypothesized to be either a result of a nuclear co-factor function or indirectly via protein interactions in the cytoplasmic compartment. Hippo pathway and YAP are possible pharmacological targets for modulation of MSC function.

There is emerging evidence to suggest that cardiac myofibroblasts (CMyfbs) participating in cardiac fibrosis represent a heterogeneous population in origin. We hypothesized that bone marrow derived mesenchymal stem cells (MSCs) readily adopt a myofibroblastic phenotype in culture. We assessed and compared human primary MSCs and human CMyfbs with respect to their phenotypic and functional characteristics by examining their gene expression profile, ability to contract collagen gels, and ability to synthesize collagen. We also examined the role of non-muscle myosin II (NMMII) in modulating the myofibroblast function using siRNA and blebbistatin to inhibit NMMII activity. The data revealed that MSCs adopt a myofibroblastic phenotype in culture and demonstrate the capability to contract collagen gels and synthesize collagen similar to human CMyfbs. Inhibition of NMMII activity with blebbistatin completely inhibits gel contractility without affecting cell viability. Thus, MSCs exhibit similar physiological and functional characteristics as CMyfbs, and may contribute to cardiac fibrosis.

There is emerging evidence to suggest that cardiac myofibroblasts (CMyfbs) participating in cardiac fibrosis represent a heterogeneous population in origin. We hypothesized that bone marrow derived mesenchymal stem cells (MSCs) readily adopt a myofibroblastic phenotype in culture. We assessed and compared human primary MSCs and human CMyfbs with respect to their phenotypic and functional characteristics by examining their gene expression profile, ability to contract collagen gels, and ability to synthesize collagen. We also examined the role of non-muscle myosin II (NMMII) in modulating the myofibroblast function using siRNA and blebbistatin to inhibit NMMII activity. The data revealed that MSCs adopt a myofibroblastic phenotype in culture and demonstrate the capability to contract collagen gels and synthesize collagen similar to human CMyfbs. Inhibition of NMMII activity with blebbistatin completely inhibits gel contractility without affecting cell viability. Thus, MSCs exhibit similar physiological and functional characteristics as CMyfbs, and may contribute to cardiac fibrosis.

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.