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Pluripotent embryonic stem cells (ESCs) can differentiate into all somatic cell types, making them a useful platform for studying a variety of cellular phenomenon. Furthermore, ESCs can be induced to form aggregates called embryoid bodies (EBs) which recapitulate the dynamics of development and morphogenesis. However, many different factors such as gradients of soluble morphogens, direct cell-to-cell signaling, and cell-matrix interactions have all been implicated in directing ESC differentiation. Though the effects of individual factors have often been investigated independently, the inherent difficulty in assaying combinatorial effects has made it difficult to ascertain the concerted effects of different environmental parameters, particularly due to the spatial and temporal dynamics associated with such cues. Dynamic computational models of ESC differentiation can provide powerful insight into how different cues function in combination both spatially and temporally. By combining particle based diffusion models, cellular agent based approaches, and physical models of morphogenesis, a multi-scale, rules-based modeling framework can provide insight into how each component contributes to differentiation. I propose to investigate the complex regulatory cues which govern complex morphogenic behavior in 3D ESC systems via a computational rules based modeling approach. The objective of this study is to examine how spatial patterns of differentiation by ESCs arise as a function of the microenvironment. The central hypothesis is that spatial control of soluble morphogens and cell-cell signaling will allow enhanced control over the patterns and efficiency of stem cell differentiation in embryoid bodies.

Introduction: Heart failure remains a major public health problem, with recent estimates indicating that end-stage heart failure with two-year mortality rates of 70-80% affects over 60,000 patients in the US each year. Medical management can be used but success declines for patients with end stage heart failure. Although cardiac transplantation is optimal, less than 2500 cardiac transplants are performed annually due to the severely limited supply of donor organs. Mechanical circulatory support (MCS) devices are now routinely used to bridge patients with end-stage heart failure who become critically ill until a donor heart is available. The use of stem cell therapy to treat heart failure has been gaining significant ground in recent years, specifically due to its regenerative properties, and both animal and human models have shown significant improvements in ventricular mass, ejection fraction, vascularization, wall thickness, and infarct size reduction. Using the patients' HeartWare HVAD device diagnostics, we were able to acquire our response variable; pulsatility. Pulsatility is a variable measure of the differential between minimum and maximum flow and is dependent on device motor speed, power, current, and fluid viscosity. This measurement is important as it relates to the contractility of the heart and could potentially be used as an end point in determining when a patient is healthy enough to have their HVAD explanted. We set out to develop a low cost and effective predictive model to determine amniotic mesenchymal stem cell's ability to repair compromised cardiac tissue of patients using the Total Artificial Heart (TAH) and Donovan Mock Circulation Tank (DMC). Methods: Predictive modelling was performed using the TAH and DMC. The system was set to a range from critical heart failure to a normal operating conditions through the variation of preload, afterload, and ventricular drive pressures with the intent of comparing the results to our patient population. Patients (n=7, 3 dilated, 4 ischemic) received intravenous and intra-myocardial injections of a heterogeneous amniotic mesenchymal stem cells mixture and liquid matrix (MSCs+LM) at HVAD implant. Groups were analyzed based on treatment; control (HVAD only, n=7) versus stem cells (HVAD + MSCs+LM). HeartWare log files were acquired from patients' devices and analyzed in SAS and Matlab. Results from the patient study were compared to the predictive model to determine levels of stem cell response. Results: Pulsatility was found to increase with left drive pressure and afterload. Lower drive pressures resulted in a drop off in pulsatility at higher afterloads while higher drive pressures were able to compensate for any afterload. Pulsatility also increased with preload but lower drive pressures were unable to fully eject at the highest preloads, resulting in a reduced pulsatility. We observed the effects of the stem cell injections on pulsatility and found that patients receiving therapy demonstrated statistically significant increases in pulsatility at 15-20 (p=.0487), 25-30 (p=.0131), 35-40 (p=.0333), and 75-80 (p=0.0476) days post implant. At minimum, when comparing the patient results to the in vitro model, the therapy resulted in a progression from end stage HF conditions to medium cardiac function conditions. At maximum, the therapy resulted in a progression from end stage HF to normal healthy operating cardiac function. Conclusions: Stem cells demonstrated a significantly increased rate of change in pulsatility within the first 40 days and at 80 days post implant when compared to control. They also demonstrated progression from end stage HF to normal healthy cardiac function at two time periods (Days 40, 90). These results justify expansion of the study to encompass a larger patient population to verify the results of the in vitro model to predict cardiac regeneration with multiple functional status indicators.

The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

Traumatic injuries to the spinal cord (SCI) and peripheral nerve (PNI) affect several thousand people worldwide every year. At present, there is no effective treatment for SCI and despite continuous improvements in microsurgical reconstructive techniques for PNI, many patients are still left with permanent, devastating neurological dysfunction. This thesis investigates the effects of mesenchymal stem cells (MSC) derived from adipose (ASC) and dental (DSC) tissue and chitosan/microRNA-124 polyplex particles on regeneration after spinal cord and peripheral nerve injury in adult rats. Dental stem cells were obtained from apical papilla, dental pulp, and periodontal ligament. ASC and DSC expressed MSC surface markers (CD73, CD90, CD105 and CD146) and various neurotrophic molecules including BDNF, GDNF, NGF, VEGF-A and angiopoietin-1. Growth factor stimulation of the stem cells resulted in increased secretion of these proteins. Both ASC and DSC supported in vitro neurite outgrowth and in contrast to Schwann cells, ASC did not induce activation of astrocytes. Stimulated ASC also showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. In a peripheral nerve injury model, ASC and DSC were seeded into a fibrin conduit, which was used to bridge a 10 mm rat sciatic nerve gap. After 2 weeks, both ASC and DSC promoted axonal regeneration in the conduit and reduced caspase-3 expression in the dorsal root ganglion (DRG). ASC also enhanced GAP-43 and ATF-3 expression in the spinal cord, reduced c-jun expression in the DRG and increased the vascularity of the implant. After transplantation into injured C3-C4 cervical spinal cord, ASC continued to express neurotrophic factors and laminin and stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure and the density of the astroglial scar. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the reactivity of OX42-positive microglial cells was markedly reduced. However, ASC did not enhance recovery of forelimb function. In order to reduce activation of microglia/macrophages and the secondary tissue damage after SCI, the role of microRNA-124 was investigated. In vitro transfection of chitosan/microRNA-124 polyplex particles into rat microglia resulted in the reduction of reactive oxygen species and TNF-α levels and lowered expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia. Alternatively, particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury. Microinjections of chitosan/microRNA-124 particles significantly reduced the number of ED-1 positive macrophages after SCI. In summary, these results show that human MSC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for neural regeneration after spinal cord and peripheral nerve injury. The data also suggests that chitosan/microRNA-124 particles could be potential treatment technique to reduce neuroinflammation.

During development, multipotent stem cells establish tissue-specific programmes of gene expression that underlie a process of differentiation into specialized cell types. It was shown in the study that changes in the nuclear architecture during terminal keratinocyte differentiation show correlation with the dynamics of the transcriptional and metabolic activity. In particular, terminal differentiation is accompanied by the decrease of nuclear volume, elongation of its shape, reduction of the number and fusion of nucleoli, increase in the number of centromeric clusters and a dramatic decrease of the transcriptional activity. Global changes in the nuclear architecture of epidermal keratinocytes are associated with marked remodelling of the higher-order chromatin structure of the epidermal differentiating complex (EDC). EDC is positioned peripherally in the epidermal nuclei at E11.5 when its genes show low expression levels and relocates towards the nuclear interior at E16.5 when EDC genes are markedly upregulated. P63 transcription factor serving as a master regulator of epidermal development is involved in the control of EDC relocation in epidermal progenitor cells. The epidermis of E16.5 p63KO exhibits significantly more peripheral positioning of the EDC loci, compared to wild-type. The genome organizer Satb1 serving as a direct p63 target controls higher order chromatin folding of the central part of EDC and Satb1 knockout mice show alterations of epidermal development and expression of the EDC encoded genes. Thus, this study shows that the programme of epidermal development and terminal differentiation is regulated by p63 and other factors and include marked remodelling of three-dimensional nuclear organization and positioning of tissue specific gene loci. In addition to the direct involvement of p63 in controlling the expression of tissue-specific genes, p63 via regulation of the chromatin remodelling factors such as Satb1 promotes establishing specific conformation of the EDC locus required for efficient expression of terminal differentiation-associated genes.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy