The ability to control stem cell functions, particularly neuronal progenitors, has long since been believed to be the key to successful treatment of neurodegenerative disorders such as Alzheimer's, Parkinson's and accidents involving head trauma. The neurology field calls for many new solutions to address the controlled neural stem cell seeding and placement of cells for neural tissue regeneration. Self-assembled monolayers (SAM) from the alkanethiol group provide a straightforward applicable, reliable treatment for cell adhesion. An ODT/gold treatment was used to adhere the cells to patterned areas, due mainly to a high confluence of cells attracted to it, as well as the viable environment it produced for the cells. Arrays of micropillars, made of SU-8 photoresist, then covered with a thin film of gold and treated with the ODT, created scaffolding allowing manipulation of neural stem cells. Based on multiple trials of observing varying cross-sectional geometric parameters, metal layer thicknesses and the ODT/Gold treatment, this study explores seeding density control, base and circumferential cell population dependence on those parameters.

White adipose tissue (WAT) is a metabolic organ with flexibility to retract and expand based on energy storage and utilization needs, processes that are driven via the coordination of different cells within adipose tissue. WAT is comprised of mature adipocytes (MA) and cells of the stromal vascular cell fraction (SVF), which include adipose progenitor cells (APCs), adipose endothelial cells (AEC) and infiltrating immune cells. APCs have the ability to proliferate and undergo adipogenesis to form MA, the main constituents ofWAT being predominantly composed of white, triglyceride-storing adipocytes with unilocular lipid droplets. While adiposity and adipose tissue health are controlled by diet and aging, the endogenous circadian (24-h) biological clock of the body is highly active in adipose tissue, from adipocyte progenitor cells to mature adipocytes, and may play a unique role in adipose tissue health and function. To some extent, 24-h rhythms in adipose tissue rely on rhythmic energy intake, but individual circadian clock proteins are also thought to be important for healthy fat. Here we discuss how and why the clock might be so important in this metabolic depot, and how temporal and qualitative aspects of energy intake play important roles in maintaining healthy fat throughout aging.

Vitamin-C (Vit-C) has been shown to modulate hematopoietic stem cells and leukemia stem cell frequency in-vivo. Herein, Vit-C analogue, L-ascorbic acid 2-phosphate (AA2P), was investigated as a new potential HSC expansion agonist. Cord blood CD34+ cells were expanded in cultures with or without AA2P. AA2P induced a 2-fold increase in the expansion of stem and progenitor subsets including lymphoid-primed multi-potential progenitors (p<0.05, n=3) and functional colony forming progenitors. The functional properties of AA2P grafts was evaluated with a xenotransplant model. Superior platelet levels in the periphery (p<0.05) and human bone marrow engraftment (median 75% hCD45+ cells for AA2P Vs. 48% for PBS control at week-22, n=3, p<0.05) was detected in AA2P cohorts Vs. control. In summary, my results demonstrate that AA2P is a new stem and progenitor expansion agonist with AA2P-expanded stem and progenitor cells capable of increased engraftment and higher platelet recovery. These findings may aid to overcome cord blood limitations; thereby, improving clinical relevance.

Introduction: Resistance training improves muscle strength and induces myofiber hypertrophy in young males and females with blunted responses occurring in older adults. These adaptations are partially due to the function of muscle stem cells (MuSCs) and fibro-adipogenic progenitors (FAPs). It remains unknown whether middle-aged males and females respond similarly to resistance training with protein supplementation, specifically at the cellular level. Purpose: The purpose of this study is to investigate the potential sex-specific responses of middle-aged males and females to whole-body resistance training. Methods: Middle-aged adults (N=28), 40-64 years, participated in a 10-week progressive, whole-body resistance training intervention coupled with protein supplementation. Muscle biopsies were collected from the vastus lateralis and stained for fibre morphology, MuSCs, and FAPs. Results: Both sexes increased type II fibre cross-sectional area with training. Myonuclear content, myonuclear domain size, and MuSC content were not altered with training in either sex. Both males and females altered FAP content with training. Interestingly, the change in MuSCs and both FAPs were correlated in males but not females (both P<0.05). It was concluded that there were no sex-specific responses to resistance training in middle-aged males and females; however, MuSCs and FAPs appear to be correlated in males but not females.

Acute myeloid leukemia (AML) is a fatal cancer of the human hematopoietic system characterized by the rapid accumulation of non-functional, immature hematopoietic cells in the bone marrow (BM) and peripheral blood (PB) of affected patients. Limited sources of safe hematopoietic stem/progenitor cells (HSPCs) for transplantation and incomplete mechanistic understandings of disease initiation, progression and maintenance have impeded advances in therapy required for improvement of long-term AML patient survival rates. Toward addressing these unmet clinical needs, the ability to generate induced pluripotent stem cells (iPSCs) from human somatic cells may provide platforms from which to develop patient-specific (autologous) cell-based therapies and disease models. However, the ability to generate iPSCs from human AML patient somatic cells had not been investigated prior to this dissertation. Accordingly, I hypothesized that cellular reprogramming of human AML patient somatic cells to iPSCs is possible and will enable derivation of autologous sources of normal and dysfunctional hematopoietic progenitor cells (HPCs). I first postulated that reprogramming AML patient fibroblasts (AML Fibs) to pluripotency would provide a novel source of normal autologous HPCs. Our findings revealed that AML patient-specific iPSCs devoid of leukemia-associated aberrations found in the matched bone marrow (BM) could be generated from AML Fibs, and demonstrated that this cellular platform allowed for the derivation of healthy HPCs capable of normal differentiation to mature myeloid lineages in vitro. During the tenure of these experiments we also redefined conventional reprogramming methods by discovering that OCT4 transcription factor delivery combined with culture in pluripotent-supportive media was minimally sufficient to induce pluripotency in AML and normal Fibs. Toward capturing and modeling the molecular heterogeneity observed across human AML samples in vitro, we next asked whether reprogramming of AML patient leukemic cells would enable generation of iPSCs and derivative HPCs that recapitulated dysfunctional differentiation features of primary disease. Our results demonstrated that conventional reprogramming conditions were insufficient to induce pluripotency in leukemic cells, but that generation of AML iPSCs could be reproducibly achieved in one AML sample when reprogramming conditions were modified. These AML iPSCs and their derivative HPCs harboured and expressed the leukemia-associated aberration found in the BM leukemic cells and similarly possessed dysfunctional differentiation capacities. Together, this body of works provides the proof of principle that cellular reprogramming can be applied on a personalized basis to generate normal and dysfunctional HPCs from AML patient somatic cells. These foundational findings should motivate additional studies aimed at developing iPSC-based cell therapies and disease models toward improving AML patient quality of life and long-term survival rates. / Thesis / Doctor of Philosophy (PhD)

Organotypic hippocampal slice cultures were used to model the effects of neuroinflammatory conditions following an epileptic state on functional P2X7 receptors (Rs) of subgranular zone (SGZ) neural progenitor cells (NPCs). The compound, 4-aminopyridine (4-AP), is known to cause pathological firing of neurons, consequently facilitating the release of various transmitter substances including ATP. Lipopolysaccharide (LPS) and interleukin-1(IL-1) both potentiated the dibenzoyl-ATP (Bz-ATP)-induced current amplitudes in NPCs, although via different mechanisms. Whereas LPS acted via promoting ATP release, IL-1 acted via its own receptor to directly influence P2X7Rs. Thus, the effect of LPS was inhibited by the ecto-ATPase inhibitor, apyrase, but not by the IL-1 antagonist, interleukin-1RA (IL-1RA); by contrast, the effect of IL-1 was inhibited by IL-1RA, but not by apyrase. Eventually, incubation with 4-AP upregulated the number of nestin/glial fibrillary acidic protein/P2X7R immunoreactive cells and their appropriate staining intensity, suggesting increased synthesis of P2X7Rs at NPCs. In conclusion, inflammatory cytokines accumulating after epilepsy-like neuronal firing may facilitate the effect of endogenous ATP at P2X7Rs of NPCs, thereby probably promoting necrosis/apoptosis and subsequent cell death.

Acute myeloid leukemia (AML) is a deadly cancer of the blood and bone marrow defined by the accumulation of immature and non-functional myeloid progenitor cells. While AML is associated with a high success of chemotherapy-induced remission, it is accompanied by high relapse rates with poor response to subsequent therapies. Therefore, relapsed AML patients only have a 10% probability of long-term survival. An effective postinduction therapy is allogeneic hematopoietic stem cell transplantation (HSCT). However, complications associated with HSCT can be more severe than the AML disease itself. To date, no robust methodology is available to prospectively identify and distinguish AML patients that are more likely to benefit from HSCT. Our group has shown that AML patients with high leukemic progenitor cell content (LPC+) have a significantly lower overall survival (OS) when compared to patients with lower LPC content (LPC-). The objective of this study was to determine whether the LPC assay can be used as a functional predictor of post-HSCT survival. We hypothesized that LPC content correlates to post-HSCT survival times. We performed LPC assays on over 100 primary AML patient samples, showing that HSCT significantly improved OS in both LPC+ and LPC- patients, but LPC+ patients benefited more strongly than LPC- patients. This provides an initial basis to suggest that HSCT can offset the negative prognostic impact associated with high LPC content. To understand the biology of LPCs, we employed the Infinium HumanMethylation450 BeadChip assay to determine whether there are any methylation patterns that distinguish LPC+ and LPC- patients. However, we were not able to discover any uniquely methylated regions that separate the two groups, suggesting for further studies with an increased patient cohort, or extending the analyses to the transcript level. Given the rarity of curative approaches to cancers, a prognostic measure that could determine whether any single patient will benefit from HSCT will have an immediate impact. / Thesis / Master of Science (MSc)

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