Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 67-72).

Liver injury stimulates hepatocyte proliferation, regenerating the liver through self-replication. In cases where there is severe, repetitive, parenchymal damage, as seen in human chronic liver disease, hepatocyte mediated regeneration becomes impaired. In this setting it is currently unclear whether endogenous biliary epithelial cells can repopulate the hepatocyte compartment. This thesis therefore aimed to address this point by lineage tracing the main two liver epithelia populations on a background of impaired hepatocyte regeneration. To impair regeneration, an Itgb1 transgene was specifically deleted, conditionally, from the hepatocyte epithelium. Long-term loss of β1-Integrin alone or with additional injury caused an epithelial ductular reaction of biliary origin. Alongside β1-Integrin ablation, the hepatocyte epithelium was also labelled with a heritable ROSA26LSLtdTomato reporter. Impaired hepatocyte regeneration mediated by β1- integrin ablation resulted in 25% of hepatocytes becoming tdTomato negative (non-hepatocyte derived). To verify that the non-hepatocyte mediated regeneration was originating from the biliary epithelium, anti-Itgb1 RNAi was administrated to K19CreERT LSLtdTomato mice. Resulting in tdTomato positive hepatocytes that had differentiated from the labelled tdTomato positive biliary epithelial cells. In summary, this thesis demonstrates that hepatocyte β1-Integrin ablation combined with toxic damage causes marked ductular reactions and results in a substantial regeneration of functional hepatocytes from the biliary epithelium.

Pericytes represent a population of potential mesenchymal stem cells (MSC) that reside within a perivascular niche until they are required in normal homeostasis and the response to injury. Their mesenchymal capacities for multipotent differentiation, immune modulation and release of trophic factors hold great promise for regenerative therapies. Pathological expression of these potentials has been described in disease states, while acute or chronic inflammation following injury can lead to the production of signalling molecules that ultimately drive these progenitors to a fibrotic fate. The aim of this work was to explore how fate decisions of pericytes are regulated by their niche (in the setting of osteogenesis), and in the response to acute and chronic injury (in the setting of fibrosis). It was hypothesized that interactions between pericytes and endothelial cells (EC) within their perivascular niche are responsible for regulating mesenchymal differentiation. The osteogenic, adipogenic and chondrogenic potential of pericytes following isolation from multiple human organs was confirmed. The interactions between pericytes and EC in 2D and 3D coculture and the production of basement membrane proteins in these settings were confirmed. The osteogenic differentiation of pericytes was accelerated by EC but no influence of EC on the adipogenic and chondrogenic differentiation of pericytes was detected. Furthermore, data indicated that the influence on pericyte osteogenic potential by EC may occur through wnt signaling. The activation of TGFβ (transforming growth factor beta) through αv integrins has been suggested as central mediator of fibrosis in multiple organs. We hypothesized that selective αv integrin deletions in PDGFRβ (platelet derived growth factor receptor beta) expressing pericytes identifies a targetable pathway regulating fibrosis in skeletal muscle. We report that PDGFRβ-Cre inactivates genes in murine skeletal muscle pericytes with high efficiency. Deletion of the αv integrin subunit in pericytes protected mice from chemical injury induced skeletal muscle fibrosis. Pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated muscle fibrosis, even when administered after fibrosis was established.

The exogenous expression of master transcription factors (TFs) to drive cell identity changes is an exciting and powerful approach to cell and tissue engineering. Yet, the generation of desired cell types is often plagued by inefficiency and inability to produce mature cell types. Through investigations of the molecular mechanisms of induced pluripotent stem cell (iPSC) generation, I discovered that expression of constitutively active Smad2/3 (Smad2CA/3CA), together with the Yamanaka factors, could dramatically improve the efficiency of reprogramming. Mechanistically, SMAD3 interacted with both co-activators and reprogramming factors, bridging their interaction during reprogramming. Because SMAD2/3 interact with a multitude of master TFs in different cell types, I tested the conversions of B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. Remarkably, each conversion system was markedly enhanced when the master TFs were co-expressed with Smad3CA. These results revealed the existence of shared molecular mechanisms underlying diverse TF-mediated cellular conversions, and demonstrated SMAD2/3 as a widely applicable cofactor that potentiates the generation of diverse cell types with profound efficiency and maturity.

Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-08Bitstream added on 2014-06-13T19:45:08Z : No. of bitstreams: 1 limaneto_jf_dr_botfmvz.pdf: 5509939 bytes, checksum: 6d585da226479576f95a5b051bde27a2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A célula-tronco (CT) é definida como uma célula com capacidade de gerar diferentes tipos celulares e reconstituir diversos tecidos. Além disso, a CT apresenta propriedades de auto-renovação, gerando cópias idênticas a si mesma. De acordo com sua origem, as células-tronco podem ser chamadas de adultas e embrionárias. As células-tronco adultas (CTA) mais utilizadas nas clínicas de terapia celular são as células-tronco hematopoiéticas e as células tronco mesenquimais, encontradas principalmente na medula óssea, tecido adiposo e no sangue do cordão umbilical. As células-tronco embrionárias (CTE) são derivadas da massa celular interna de embriões no estágio de blastocisto. Desta maneira este trabalho teve como objetivo desenvolver uma metodologia adequada para o isolamento, cultivo e caracterização de células tronco embrionárias e mesenquimais de eqüinos, além de verificar a capacidade que as células possuem em se diferenciar in vitro em outros tipos célulares. Foi coletado sangue da medula óssea de eqüinos entre 8 e 15 anos de idade. As células tronco mesenquimais foram isoladas após a primeira e segunda passagem. As células foram caracterizadas com marcadores de superfície CD34 (mononucleares) e CD44 (mesenquimais). Após isolamento e caracterização, as células tronco mesenquimais foram diferenciadas para as linhagens osteogênica, adipogênica, condrogênica e neurogênica. A confirmação da diferenciação das células tronco foi realizada por marcadores teciduais específicos. Estas células também, foram capazes de expressarem marcadores neurais. Para o isolamento das células tronco embrionária eqüina, embriões com oito a nove dias foram coletado e a massa celular interna (MCI) isolada mecanicamente. Após o isolamento, a MCI foi transferida para a placa de cultivo previamente preparada com monocamada de fibroblastos para o desenvolvimento... / The stem cell (SC) is defined as cells with the capacity of generate different cellular types and rebuild various tissues. Moreover, the SC has a selfregenerate ability, generating identical copies of itself. According to its origins, the SC can be named as “adult” or “embryonic”. The adult stem cell (ASC) more often used in clinical trials and cellular therapy, are the hematopoietic stem cells and the mesenchymal stem cells, isolated mainly from the marrow bone, adipose tissue and umbilical cord blood. The embryonic stem cells (ESC) are obtained from the inner cell mass of embryos at the blastocyst stage. In this way the present study had as objective to develop an adequate methodology of isolation, culture and characterization of embryonic and mesechymal stem cells from horses, verifying the capacity of those cells to differentiate in vitro into different cells types. Bone marrow blood was collected from horses, aging from 8 to 15 years and filtered with a donation blood kit filter, to avoid clots. The mesenchymal stem cells were isolated after the first and the second passage. The SC were characterized using surface markers CD34 (monuclear) and CD44 (mesenchymal). After the isolation and characterization, the mesenchymal stem cells were differenced into osteogenic, adipogenic, condrogenic and neurogenic lineage. The cells differentiations were confirmed using specific tissue markers. To isolate the embryonic stem cells equine embryos with 8 to 9 days were used. The inner cell mass (ICM) were extract mechanically and transferred to a culture dish previously prepared with fibroblasts monolayer to colony formation and development. The colonies were characterized with pluripotency markers and then submitted to a differentiation process into neurogenic lineage, confirmed by specific neural tissue markers

Hypothermia is potently neuroprotective, but the molecular basis of this effect remains obscure and the practical challenges of cooling have restricted its clinical use. This thesis was borne on the premise that considerable therapeutic potential may lie in a deeper understanding of the neuronal physiology of cooling. Rodent studies indicate that hypothermia can elicit preconditioning wherein a subtoxic stress confers resistance to an otherwise lethal injury. This cooling-induced tolerance requires de novo protein synthesis – a fundamental arm of the cold-shock response, for which data in human neurons is lacking. Since cooling protects the human neonatal brain, experiments herein address the molecular effects of clinicallyrelevant cooling using functional, maturationally-comparable cortical neurons differentiated from human pluripotent stem cells (hCNs). Several core hypothermic phenomena are explored, with particular scrutiny of neuronal tau, since this protein is modified extensively in brains that are resistant to injury. Mild-to-moderate hypothermia produces an archetypal cold-shock response in hCNs and protects them from oxidative and excitotoxic stress. Principal features of human cortical tau development are recapitulated during hCN differentiation, and subsequently reversed by cooling, returning tau transcriptionally and post-translationally to an earlier foetallike state. These findings provide the first evidence of cold-stress-mediated ontogenic reversal in human neurons. Furthermore, neuroprotective hypothermia induces mild endoplasmic reticulum (ER) stress in hCNs, with subsequent activation of the unfolded protein response (UPR). Reciprocal modulation of both tau phosphorylation and the ER-UPR cascade suggests that cold-induced hyperphosphorylation of tau and ER-hormesis (preconditioning) represent significant components of hypothermic neuroprotection. Cooling thus modifies proteostatic pathways in a manner that supports neuronal viability. Historically, hypothermic preconditioning has been limited to the acute injury setting, and tau hyperphosphorylation is an established hallmark of chronic neural demise. More recently however, preconditioning has been proposed as a target for neurodegenerative disease and neuroprotective roles of phospho-tau have emerged. To date, hypothermia has protected hCNs against oxidative, excitotoxic and ER stress, all of which have been implicated in traumatic as well as degenerative processes. This ‘cross-tolerance’ effect places exponential value on the molecular neurobiology of cooling, with the potential to extract multiple therapeutic targets for an unmet need.

Despite advances in treatment, heart failure remains one of the top killers in Canada. This recognition motivates a new research focus to harness the fundamental repair properties of the human heart, with human cardiac stem cells (CSCs) emerging as a promising cell candidate to regenerate damaged myocardium. The rationale of this approach is simple with ex vivo amplification of CSCs from clinical grade biopsies, followed by delivery to areas of injury, where they engraft and regenerate the heart. Currently, outcomes are limited by modest engraftment and poor long-term survival of the injected CSCs due to on-going cell loss during transplantation. As such, we explored the effect of cell encapsulation to increase CSC engraftment and survival after myocardial injection. Transcript and protein profiling of human atrial appendage sourced CSCs revealed strong expression the pro-survival integrin dimers αVβ3 and α5β1- thus rationalizing the integration of fibronectin and fibrinogen into a supportive intra-capsular matrix. Encapsulation maintained CSC viability and expression of pro-survival transcripts when compared to standard suspended CSCs. Media conditioned by encapsulated CSCs demonstrated superior production of pro-angiogenic/ cardioprotective cytokines, angiogenesis and recruitment of circulating angiogenic cells. Intra-myocardial injection of encapsulated CSCs after experimental myocardial infarction favorably affected long-term retention of CSCs, reduced scar burden and improved overall cardiac function. Taken together, cell encapsulation of CSCs prevents detachment induced cell death while boosting the mechanical retention of CSCs to enhance repair of damaged myocardium.

Several studies have proposed the fallopian tube epithelium as a site of origin of ovarian cancer. The discovery of precursor lesions in the fallopian tube in patients at risk for ovarian cancer supports a probable origin for high-grade serous ovarian carcinoma in this tissue. While the fallopian tube epithelium consists of three distinct cell types, the paired box protein 2 (PAX2) positive cells and potentially the CD44 positive stem-like cells are most relevant to ovarian cancer. Loss of PAX2 expression in the fallopian tube cells is considered to be an early event in epithelial transformation, but the specific role of PAX2 in this transition is unknown. The aim of this study was to define the role of PAX2 in oviductal epithelial cells (OVE) cells and in mouse ovarian surface epithelial cells (MOSE), and to understand its contribution to the formation of serous precursor lesions in the fallopian tubes. Herein, we studied the OVE response to transforming growth factor β (TGFβ, a cytokine found in follicular fluid) and provide evidence of its potential involvement in the regulation of stem cell-like behaviors that may contribute to formation of cancer-initiating cells. Treatment of primary cultures of OVE cells with TGFβ at concentrations found in ovulatory follicular fluid induced an epithelial-mesenchymal transition (EMT) with expected changes in proliferation, cell morphology and expression of SNAIL, Vimentin and E-cadherin. EMT was also associated with decreased expression of PAX2 and an increase in the fraction of cells expressing CD44. Pax2 knockdown in OVE cells and overexpression in ovarian epithelial cells confirmed that PAX2 inhibits CD44 expression and regulates the degree of epithelial differentiation of OVE cells. These results suggest that the loss of PAX2 seen in serous tubal intraepithelial carcinomas (STIC) leads to a shift to a more mesenchymal phenotype associated with stem-like features. Pax2 overexpression in MOSE cells also induced the formation of vascular channels both in vitro and in vivo, which indicate a possible contribution of PAX2 to ovarian cancer progression by increasing the vascular channels to supply nutrients to the tumor cells. Furthermore, since loss of PAX2 in STIC was found associated with P53 and BRCA1 mutations, OVE cells with mutations of the tumor suppressor genes Trp53 and Brca1 were studied. We found that loss of Trp53 with or without loss of Brca1 increased cell proliferation and colony formation in vitro. In addition, loss of Trp53 induced OVE cells to undergo EMT and induced the expression of stem cell–associated genes. We therefore suggest a potential contribution of stem cells in initiating the precursor lesions in the fallopian tubes in combination with tumor suppressor gene mutation.

Cultures of peripheral blood mononuclear cells (MNCs) give rise to at least two different variants of endothelial progenitor cells (EPCs), early and late outgrowth EPCs. We investigated whether microRNAs in early and late EPCs could serve as markers of internal processes that can be exploited to distinguish cell identity and functional capacity. We hypothesized that as MNCs give rise to early and late EPCs, there is a gradual change in total microRNA profile, reflecting a total change in processes within the predominant cell population. Using a candidate microRNA array, early and late EPCs showed vastly different microRNA expression profiles. MiR-146a expression increased progressively as early EPCs emerged around 5-7 days (p<0.05). Through targeting TRAF6 and IRAK1, miR-146a conferred inflammatory tolerance in early EPCs, likely contributing to their purported ability to suppress inflammation. MiR-146a knock down (KD) in endotoxin-stimulated early EPCs reduced anti-inflammatory cytokine IL-1RA (p<0.001), and increased expression of pro-inflammatory cytokines IL-1 (p<0.001) and IL-8 (p<0.01). Interestingly, the microRNA expression profile of late EPCs was highly congruent to mature endothelial cells, with 100-fold greater miR-126 expression than monocytes and early EPCs (p<0.01). MiR-126KD in late EPCs abolished matrigel-network formation (p<0.05); while overexpression (OE) in early EPC augmented network formation (p<0.05) and chemotactic migration (p<0.001). We also found that the melanoma cell adhesion molecule or MCAM (CD146) identified late EPC precursors. Only MCAM+MNCs from adult blood (<5% of total MNCs) yielded late EPC-like colonies. Robust miR-126 expression in these cells predicted the generation of late EPCs. Overall, our results suggest that miR-146a in early EPCs likely contributes to repair by suppressing inflammation during cardiovascular injury; while in late EPCs, miR-126 directly promotes angiogenesis and vascular repair. Finally, we highlight a unique method for the efficient generation of late EPCs by using MCAM selection and screening for miR-126.

INTRODUCTION: Damage to the musculoskeletal system through disease, injury, or ageing can have long-lasting, and detrimental effects on one’s overall well-being. By understanding the processes by which the different tissues of the musculoskeletal system function and communicate, we can apply it to a variety of medical interventions that will benefit the patient population. These include reducing the prevalence of injury-inducing ectopic bone formation in muscle and slowing the degeneration of muscle and bone tissue associated with aging. A major focus is the relationship between muscle and bone tissue, specifically the stem cell populations found in each tissue type. Two genes that are thought to mark stem cell populations associated with muscle and bone tissue are Pax7 and Prx1, respectively. OBJECTIVES: Establish the capability and define optimal conditions to culture primary stem cells isolated from the muscle tissue of the reporter animals that fluorescently tag the Pax7 and Prx1 cell populations. Manipulate culture medium conditions to characterize the differentiation potential for multiple lineages, osteogenic, adipogenic, and myogenic. Lastly, assess whether there is more adipogenic cell differentiation in older animal cell cultures. METHODS: The tamoxifen inducible Pax7^tm1(cre/ER2)Gaka/J and Prx1^CreER-GFP were both crossed with B6.Cg-Gt(ROSA)26sor<tm14(CAG-tdTomato)Hze>/J to create the tamoxifen inducible Pax7/Ai14 and Prx1/Ai14 reporter mice. These animals were then crossed to the B6,129S7-Rag1^tm1Mom/J , creating the Pax7/Ai14/Rag and Prx1/Ai14/Rag reporter mice strains. This transgenic mouse model made it possible to fluorescently identify the Pax7 and Prx1 population of cells isolated from the muscle tissue and characterize the differentiation potential to different cell lineages in vitro. Cells were harvested from both male and female mice that received two tamoxifen injections. Cells were then cultured in various culture media conditions. Determination of specific cell densities, culture conditions, and differentiation time points were determined by manipulating these variables, and assessing the levels of proliferation and differentiation. Multiple assays were run in order to quantify and identify the different cell lineages that were grown in culture under different culture conditions. RESULTS: Cells grown on gelatin coated dishes at densities of 2.2x10^4 to 2.2x10^5 showed optimal performance in proliferation and differentiation. Cells grown in Growth Media containing Chick Embryo Extract (GM) and without (GM-) produced myogenic and adipogenic cell types that were positive for Prx1 expression. Prx1 positive cells grown in the Osteo-Inductive Media (GMOI) produced osteogenic cell types validated through tetracycline uptake. Pax7 expression was low in all culture media conditions. Finally, adipogenic cells were present in both younger and older animals. The adipogenic cells for both populations showed positive Prx1 expression. Younger animals showed a larger relative expression of Plin1 in qRT-PCR analysis. CONCLUSIONS: Although Prx1 is thought to be associated with bone tissue, Prx1 positive cells are located within the muscle and able to be cultured. This muscle derived Prx1 cell population is able to differentiate toward the myogenic, adipogenic, and osteogenic cell lineages. By altering the specific components of culture conditions such as extracellular matrices, seeding density, and media constituents, it is possible to force a particular lineage differentiation for Pax7 and Prx1 muscle derived stem cells. Further studies are needed to elucidate the differentiation potential of Prx1 cells isolated from animals of various ages. Additional in vivo studies are needed to understand the mechanisms surrounding the Prx1 and Pax7 population of cells with their roles in healing and regeneration in response to degeneration and trauma.