Differentiation and cell cycle regulation in stem cell have a key function for embryonic development, organ homeostasis and tissue repair. Recent results have shown that these two mechanisms are intrinsically connected. Indeed, cell cycle machinery directly controls maintenance of pluripotency and initiation of differentiation. More precisely, the cell cycle regulator Cyclin D appears to control the transcriptional activity of Activin/Nodal signalling during progression of the cell cycle in human Embryonic Stem Cells (hESCs). As a consequence, hESCs can only differentiate into endoderm in the Early G1 phase when Cyclin Ds are expressed at low levels. These results show the mechanisms by which the cell cycle defines differentiation propensity of stem cells. However, these observations also imply the existence of interplays coordinating extra cellular signalling pathways with the epigenetic state, chromatin structure and transcriptional networks during cell cycle progression and these mechanisms remain to be fully uncovered. Here, I have utilised the FUCCI reporter system combined with ATAC-Seq to analyse chromatin dynamics during cell cycle progression in hESCs. Furthermore, I performed ChIP-Seq analyses to define the genomic location of transcriptional regulators during cell cycle progression as well as RNA-Seq to confirm variation in gene expression pattern. Integration of these data shows that the chromatin status in hESCs is highly dynamic and the core pluripotency transcription factors and epigenetic modifiers change genomic location during cell cycle progression. I also showed that hESCs in the Late G1 phase accumulate transcripts that are important for differentiation and development; therefore, indicating this phase represents a unique portion of the cell cycle for cell fate decisions. Taken together, these results uncover that transcriptional networks are unexpectedly dynamic during the progression of cell cycle in stem cells. I hypothesise that these modifications are necessary to prime hESCs for different cell fate choices allowing a diversity of differentiation that is otherwise impossible. Overall these mechanisms underline the need to study transcriptional and epigenetic mechanisms in the dynamic context of the cell cycle and have major implications for adult tissue homeostasis and disease.

Made available in DSpace on 2015-09-17T15:24:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-12-17. Added 1 bitstream(s) on 2015-09-17T15:47:10Z : No. of bitstreams: 1 000844099.pdf: 1423355 bytes, checksum: 8649e82cdb205c9cea62cc32e42d5564 (MD5) / Os processos de reparo da polpa dentária frente a procedimentos conservadores passam, em geral, pelo confronto entre possíveis microrganismos presentes na área da exposição e as células responsáveis pela diferenciação e produção de tecido mineralizado. Entretanto, a literatura ainda necessita de informações sobre o efeito direto de produtos bacterianos sobre este processo. O objetivo deste estudo foi avaliar os efeitos do lipopolissacarídeo (LPS) bacteriano (E. coli) sobre células progenitoras da polpa dental, utilizando-se condições basais e com mineralização préinduzida por meio osteogênico. Para isso, células progenitoras caracterizadas foram submetidas a diferentes concentrações de LPS bacteriano (com ou sem indutor da mineralização [lM]), para observação da atividade de fosfatase alcalina (ALP). A concentração mínima de 200 ng/ml, para se verificar alteração de atividade enzimática de ALP, foi usada para se observar os efeitos funcionais de mineralização (pelo alizarin vermelho - ARS), expressão das citocinas IL-1β e TNF-α, além da expressão dos genes DSPP e DMP-1. Os dados quantitativos foram analisados por ANOVA e teste de Tukey. As células em meio osteogênico com as diferentes concentrações de LPS apresentaram baixa atividade da ALP no curto prazo, comparadas às tratadas com meio α-MEM. Estas apresentaram alta atividade, comparadas ao controle. 200 ng/ml do LPS afetaram o processo de mineralização ao longo do tempo, reduzindo a ação de mineralização dos grupos que o associaram ao indutor de mineralização. Houve expressão de IL-1β e TNF-α para todos os grupos em todos os tempos, além disso, para a IL-1β, o grupo que associou os 200ng/ml de LPS com o meio indutor após 7 dias apresentou maior expressão. Houve expressão do gene DMP-1 e não expressão de DSPP e ocorreu uma maior expressão gênica nos grupos tratados com meio indutor da mineralização no gene DMP-1 . Esses achados / Dental pulp repair processes due to conservative procedures are, in general, affected by the conflicts between possible microorganisms at the area of exposure and cells responsible for differentiation and production of mineralized tissue. However, the literature still lacks information on how bacterial products affect these processes directly. The purpose of this study was to evaluate the bacterial lipopolysaccharide (E. coli LPS) effects on dental pulp progenitor cells, regarding the expression of differentiation genes and function of mineralization, using basal conditions and pre-induced mineralization by osteogenic media. Characterized progenitor cells was initially submitted to different LPS concentrations (with or without osteogenic medium [lM]), to observe alkaline phosphatase activity (ALP). The minimal concentration (200 ng/ml) to observe phosphatase enzymatic activity was used to assess the functional mineralization effects (by alizarin red staining - ARS), cytokine production (IL-1β e TNF-α), besides the gene expression for odontoblast differentiation markers (DSPP and DMP-1). Quantitative data will be statistically analyzed by ANOVA and Tukey's test. Cells in osteogenic medium with different concentrations of LPS showed low ALP activity shortterm compared to those treated with α-MEM. These showed high activity, compared to control. 200 ng/ml of LPS did affect the mineralization process over time, reducing the action of mineralization of the groups that have associated LPS with the IM. There was expression of IL-1β and TNF-α for all groups at all times, additionally, IL-1β for the group that has associated 200 ng/ml of LPS with osteogenic media after 7 days showed higher expression. There was expression of DMP-1 and DSPP genes and the expression in groups treated with IM was greater in DMP-1. These findings suggest that the inflammatory potential of LPS on the pulp progenitor cells contribute to an early mineralization ...

Orientador: Bruno Das Neves Cavalcanti / Banca: Cláudio Antonio Talge Carvalho / Banca: Aletéia Massula de Melo Fernandes / Resumo: Os processos de reparo da polpa dentária frente a procedimentos conservadores passam, em geral, pelo confronto entre possíveis microrganismos presentes na área da exposição e as células responsáveis pela diferenciação e produção de tecido mineralizado. Entretanto, a literatura ainda necessita de informações sobre o efeito direto de produtos bacterianos sobre este processo. O objetivo deste estudo foi avaliar os efeitos do lipopolissacarídeo (LPS) bacteriano (E. coli) sobre células progenitoras da polpa dental, utilizando-se condições basais e com mineralização préinduzida por meio osteogênico. Para isso, células progenitoras caracterizadas foram submetidas a diferentes concentrações de LPS bacteriano (com ou sem indutor da mineralização [lM]), para observação da atividade de fosfatase alcalina (ALP). A concentração mínima de 200 ng/ml, para se verificar alteração de atividade enzimática de ALP, foi usada para se observar os efeitos funcionais de mineralização (pelo alizarin vermelho - ARS), expressão das citocinas IL-1β e TNF-α, além da expressão dos genes DSPP e DMP-1. Os dados quantitativos foram analisados por ANOVA e teste de Tukey. As células em meio osteogênico com as diferentes concentrações de LPS apresentaram baixa atividade da ALP no curto prazo, comparadas às tratadas com meio α-MEM. Estas apresentaram alta atividade, comparadas ao controle. 200 ng/ml do LPS afetaram o processo de mineralização ao longo do tempo, reduzindo a ação de mineralização dos grupos que o associaram ao indutor de mineralização. Houve expressão de IL-1β e TNF-α para todos os grupos em todos os tempos, além disso, para a IL-1β, o grupo que associou os 200ng/ml de LPS com o meio indutor após 7 dias apresentou maior expressão. Houve expressão do gene DMP-1 e não expressão de DSPP e ocorreu uma maior expressão gênica nos grupos tratados com meio indutor da mineralização no gene DMP-1 . Esses achados / Abstract: Dental pulp repair processes due to conservative procedures are, in general, affected by the conflicts between possible microorganisms at the area of exposure and cells responsible for differentiation and production of mineralized tissue. However, the literature still lacks information on how bacterial products affect these processes directly. The purpose of this study was to evaluate the bacterial lipopolysaccharide (E. coli LPS) effects on dental pulp progenitor cells, regarding the expression of differentiation genes and function of mineralization, using basal conditions and pre-induced mineralization by osteogenic media. Characterized progenitor cells was initially submitted to different LPS concentrations (with or without osteogenic medium [lM]), to observe alkaline phosphatase activity (ALP). The minimal concentration (200 ng/ml) to observe phosphatase enzymatic activity was used to assess the functional mineralization effects (by alizarin red staining - ARS), cytokine production (IL-1β e TNF-α), besides the gene expression for odontoblast differentiation markers (DSPP and DMP-1). Quantitative data will be statistically analyzed by ANOVA and Tukey's test. Cells in osteogenic medium with different concentrations of LPS showed low ALP activity shortterm compared to those treated with α-MEM. These showed high activity, compared to control. 200 ng/ml of LPS did affect the mineralization process over time, reducing the action of mineralization of the groups that have associated LPS with the IM. There was expression of IL-1β and TNF-α for all groups at all times, additionally, IL-1β for the group that has associated 200 ng/ml of LPS with osteogenic media after 7 days showed higher expression. There was expression of DMP-1 and DSPP genes and the expression in groups treated with IM was greater in DMP-1. These findings suggest that the inflammatory potential of LPS on the pulp progenitor cells contribute to an early mineralization ... / Mestre

It has long been established that the physical properties of the cell’s surrounding microenvironment has the ability to impose its influence on a range of cell processes. Morphology, differentiation, and proliferation have all been shown to be sensitive to the mechanical cues inherent within the extracellular matrix. Although significant advancements in microfabrication and cell mechanics have been made, questions regarding how physical interactions guide biological systems in three dimensions remain unanswered. By utilizing cocultured systems and microfabricated channeled topographies, we reveal that the three dimensional nature of the environment is capable of driving cell patterning. Contact guidance is the phenomenon by which cells will orient themselves along the geometric patterns of a substrate. Much of its research has focused on the nano/micro scale of two dimensional topographies, affecting alignment along grooves. We have revealed that contact guidance has the ability to impose far more complex cellular behaviour in three dimensional systems. Furthermore, by modulating the elements of confinement surrounding cells, we directed the balance of binding forces between cells and substrate leading to significantly different cell type dependent morphologies. By then altering the geometry of the topography, we revealed the ability to induce cell type separation in cocultured systems. These concepts led to the subsequent discovery that confinement induces three dimensional spheroidal growth of embryonic stem cells. These results reveal that the element of confinement not only influences patterning in three dimensions but guides the fundamental early stages processes essential to all life.

The mammary gland contains a subset of cells with regenerative capacity that is able to generate both luminal and myoepithelial mammary epithelial lineages. Those cells are described as mammary epithelial stem cells. The fate of stem cells is tightly controlled by their microenvironment and adhesion receptors on the stem cells play a vital role in the microenvironment–stem cell communication. They facilitate the interaction of stem cells with the extracellular matrix as well as adjacent cells, and they regulate stem cell homing to their niches, as well as stem cell proliferation, self-renewal, and differentiation. Stem cells express high levels of ECM binding adhesion receptors such as β1 and α6-integrins. Those integrins were used to isolate stem cells from the rest of the differentiated epithelial cells within the mammary gland. However, little is known about the role of those integrins in stem cell self-renewal and differentiation. This project aimed to understand how β1-integrin receptors contribute to stem cell behavior. To achieve this, FACS sorting method of stem cells, the organoid assay, and lentivirus knockdown of β1-integrin using shRNA were optimised. The organoid assay was used as an in-vitro test to assess for the frequency of bi-lineage and luminal progenitor cells in a given mammary epithelial population. It is known that bi-lineage cells produce solid organoids in culture while luminal progenitors produce hollow organoids. The frequency of solid and hollow organoids might therefore be an indication of the stem cells and luminal progenitor frequency respectively. My results showed that cells with the highest solid organoid forming ability were within the basal population, which is high for β1- and α6-integrin. The β1-integrin signaling pathway was shown to be important for maintaining the organoid-forming population in basal and luminal populations. Knocking out β1-integrin in MECs resulted in abolishing their solid and hollow organoid-forming activity. Downstream of β1-integrin, I found that Rac1 but not ILK is important in β1-integrin maintenance of solid organoid-forming cells. Active Rac1 was able to rescue solid organoid formation but was not able to rescue hollow organoids in the β1-integrin knockdown cells. β1-integrin and Rac1 deletion resulted in the down regulation of Wnt/β-catenin signaling, which is important for stem cells. This down regulation was rescued using active Rac1. Activating Wnt/ β1-catenin signaling in primary cells (using Wnt3a ligand or GSK3β inhibitor) resulted in an increase in solid organoid and a decrease in hollow organoid formation. When activating Wnt signaling using GSK3I in β1-integrin knockdown cells, the solid organoid activity was rescued. However, Wnt3a did not rescue solid organoid formation in the β1-integrin knockdown cells. When active Rac1 was overexpressed in β1-integrin null cells, Wnt3a was able to activate solid organoid formation. When inhibiting Rac1 in primary MECs, solid but not hollow organoid activity was significantly decreased. Wnt3a or GSK3I addition did not rescue this reduction. Taken these results together, it can be concluded that β1integrin-Rac1 signaling play a role in controlling stem cells and this is might be achieved through controlling Wnt/β-catenin signaling. These studies are important in understanding the role of integrins in mammary stem cells. They will also provide new insight on how integrins might be controlling breast cancer and thereby, help in providing new targets for cancer therapy.

- anglicky This thesis deals with legal aspects of stem cell research, which in the case of the Czech Republic remains quite disregarded by the general public. The text examines and analyzes the legislation regulating the aforementioned field of research at the international, European and Czech national level in a comprehensive manner. Particular emphasis is placed on legal aspects of embryonic stem cell research, which is also closely related to the legal status of embryo and its protection. The thesis also notes what types of regulatory approaches are adopted in various countries with respect to stem cell research and it subsequently demonstrates these conclusions by examples of Slovak Republic and the United Kingdom. Explanation is set in a broader framework through the introductory chapters that deal with biological and ethical aspects of stem cell research.

Ischaemic heart disease accounts for approximately 7 million deaths worldwide on a yearly basis and this figure is only set to rise as life expectancy in developing countries increases. Although no longer considered a post mitotic organ, the adult heart demonstrates only a very limited capacity for regeneration. Consequently ischaemic injury results in massive loss of contractile cardiomyocytes with damaged myocardium replaced by a non-contractile and poorly conductive collagen scar. This in turn often leads to the development of heart failure. Enhancing or supplementing the myocardial regenerative capacity of the heart is thus a key goal in the development of effective therapies for the treatment of cardiac infarction. Several stem cell populations of non-cardiac origin have been investigated for their capacity to contribute to myocardial repair when therapeutically transplanted into injured hearts. Recent efforts have focused on the “next generation” of donor cells, endogenous cardiac progenitor cells, as these are thought to be better adapted to survival in the cardiac environment and to possess enhanced cardiomyocyte differentiation potential. Pericytes, proposed as the source of the elusive mesenchymal stem cells (MSC) within multiple tissues, are a potential new cell type for use in regenerative medicine. This study tests the hypothesis that pericytes and another perivascular progenitor population, the adventitial cell, from foetal cardiac tissue will positively contribute to the repair of the myocardium post injury. Staining of human foetal ventricular myocardium confirmed the presence of large numbers of both cell types with pericytes tightly associated with capillaries and adventitial cells primarily located in the outer, adventitial layer of muscular arteries. CD146+ CD34- pericytes and CD146- CD34+ adventitial cells were isolated by FACS and expanded in culture. On examination of gene and protein expression both populations stably expressed a similar panel of pericyte markers, MSC markers and cardiac transcription factors as well as c-kit, a cardiac progenitor cell candidate marker. Co-culture with neo-natal rat cardiomyocytes induced expression of an additional cardiac progenitor marker Isl-1 and a mature cardiomyocyte marker ANP in adventitial cells but not pericytes. Labelled, co-cultured, perivascular progenitors readily adhered to rat cells but did not appear to contract independently. De-methylation of perivascular progenitors prior to co-culture resulted in expression of sarcomeric proteins and spontaneous cytoplasmic calcium fluctuations in both populations but more commonly in pericytes. This suggests that cardiac perivascular cells contain a minor sub-population capable of cardiomyocyte differentiation. When these populations were injected into the infarcted hearts of NOD/SCID mice, the animals treated with adventitial cells had significantly reduced cardiac function at 21 days post-surgery on ultrasound examination. An increased scar area and a non-significant trend towards increased scar length and a decreased wall thickness were also observed. Transplanted cells of both groups were detected in low numbers 21 days after injection. Adventitial cells were retained much more readily and in both populations retained cells exhibited three key morphologies: fibroblast type; macrophage type; and cardiomyocyte type. The majority of cells adopted a fibroblast type morphology, lesser numbers a macrophage like morphology and only rare cells a cardiomyocyte like morphology. Both fibroblast and cardiomyocyte type cells had single, human nuclear antigen positive nuclei suggesting true differentiation rather than cell fusion and pericytes exhibited an enhanced ability to differentiate into cardiomyocytes. This supports the in-vitro findings of a minor pro-cardiomyogenic subset within the perivascular cell population. As a result of these findings the starting hypothesis was modified to propose that perivascular cells play a significant role in cardiac fibrosis, largely mediated through expression of surface integrin receptors. This was tested using mice expressing fluorescent proteins under the control of the PDGFR-β promoter and mice in which the αv integrin subunit, common to 5 integrin receptors, had been deleted on the surface of PDGFR-β+ cells. Immunostaining and flow cytometry revealed the PDGFR-β expression to be tightly restricted to perivascular cells and co-expressed with the fibroblast markers, vimentin, PDGFR-α, CD90.2 and CD34 in a subset of cells. Cardiac fibroblasts isolated from reporter mouse hearts revealed strong expression of PDGFR-α and CD34 but PDGFR-β expression in only approximately 20% of the population on flow cytometry. Following angiotensin II induced cardiac hypertrophy and fibrosis approximately 50% of fibroblasts expanding the interstitium were PDGFR-β+. Genetic deletion of the αv integrin subunit on PDGFR-β+ cells resulted in a reduction in cardiac interstitial collagen content of about 50% compared to wild type controls. These findings suggest that the cardiac perivascular PDGFR-β+ population is heterogeneous with a sub-population likely to be fibroblasts or fibroblast progenitors and that the development of cardiac interstitial fibrosis is in part modulated by integrin receptor expression on these cells. In summary this study provides evidence of the existence of a pro-fibrotic progenitor population, which co-express pericyte and MSC markers, within the cardiac perivascular niche. These cells contribute to cardiac fibrosis both on transplantation and endogenously following cardiac injury with the latter mediated via αv integrin expression. Within the perivascular progenitor population however there also appears to be a minor subset of pro-cardiomyogenic cells which are able to adopt a cardiomyocyte phenotype both in-vitro and in-vivo.

M. Tech. / Stem cells are defined as undifferentiated cells that can proliferate indefinitely and have the capacity of both self-renewal and differentiation to one or more types of specialised cells. Traumatic tissue injury and age-related degenerative diseases are a major problem in South Africa and worldwide. Stem cells could be used for tissue engineering and reconstructive surgery. In treating these conditions, the main principle of stem cell therapy is the replacement of damaged and dead cells in injured tissues and organs with new healthy ones expanded in vitro from stem cells (Orlic et al., 2002). These cells can be isolated from adipose tissue in significant numbers and exhibit stable growth and proliferation kinetics in culture and could be differentiated into bone, fat, cartilage and muscle when treated with established lineage-specific factors (Zuk et al., 2002). Low Level Laser Therapy (LLLT) is currently applied in the treatment of numerous diseases and pathological conditions (Gasparyan et al., 2004). LLLT produces positive effects on irradiated cells and tissues such as proliferation of cells, capillary growth and adenosine triphosphate (ATP) activation (Schindl et al., 1998). Low level laser radiation at different intensities has been shown to stimulate as well as to inhibit cellular processes (Moore et al., 2005). Epidermal growth factor (EGF) is a growth factor that plays important roles in the regulation of cell growth, proliferation and differentiation. This study investigated the effect of low level laser radiation alone as well as in combination with EGF on adult adipose derived stem cells (ADSCs) isolated from human adipose tissue. ADSCs were isolated from human adipose tissue through collagenase digestion and cultured in DMEM-F12 containing 10% FBS and antibiotics and incubated at 37°C in a humidified atmosphere of 5% CO2 (Zuk et al., 2001). iii Semi-confluent monolayers of ADSCs were exposed to low level laser at 5 J/cm2 using 636 nm diode laser with a power density of 12.1 mW/cm2 at room temperature in the dark. Cell morphology was monitored at 0, 24 and 48 h using an inverted light inverted microscope. Cell viability was evaluated at 0, 24 and 48 h using the Trypan Blue exclusion test and an adenosine triphosphate (ATP) luminescence assay. bFGF (basic fibroblast growth factor) indirect ELISA and optical density assays were used to monitor cell proliferation at 0, 24 and 48 h post irradiation. In addition the expressions of stem cell markers, β1-integrin and Thy-1, were monitored by immunocytochemical live cell surface labelling and Western blot analysis. Cells were incubated with EGF to enhance proliferation and differentiation and the cell morphology, viability and proliferation were monitored as well as the expressions of stem cell markers, β1-integrin and Thy-1. Morphology of the cells was not altered by irradiating them with 5 J/cm2 using diode laser at 0, 24 and 48 h. Cell viability and proliferation showed an increase at 24 and 48 h post irradiation. At 0 h, there was no significant difference between irradiated and non-irradiated cells in cell viability and proliferation. There was an increase in the expression of β1-integrin and Thy-1 after irradiation as shown by Western blot analysis and immunocytochemical live cell surface labelling. Cell viability and proliferation showed a significant increase at all time points post irradiation with the addition of EGF. There was no noticeable change in cellular morphology at any time point. Low level laser irradiation of human ADSC’s at 636 nm with 5 J/cm2 and 12.1 mW/cm2 increased the viability and proliferation of these cells in vitro. Furthermore, low level laser irradiation appeared to increase the expression of stem cell markers, β1-integrin and Thy-1. In addition, laser irradiation did not alter the morphology of the cultured cells. The addition of EGF to the cells also increased their viability and proliferation as well the expression of the markers, β1-integrin and Thy-1. The study showed that laser irradiation stimulates two important cellular responses namely cell viability and proliferation which indicates that ADSCs may be suitable for tissue engineering and future cell differentiation studies.

M.Tech. / Stem cells possess self-renewal capacity, long-term viability, and multilineage potential. Stem cells play important roles in normal physiological and disease processes, they also have great therapeutic potential. However, there have been controversies surrounding stem cells in political, religious and ethical arenas. Although the use of certain stem cells (i.e. embryonic stem cells) and the means by which they are obtained contravene certain basic ethical laws, researchers have developed methods with which to ethically obtain and create stem cell lines. Stem cells can be classified as either: totipotent, pluripotent, multipotent, oligopotent and unipotent (Moore, 2007). Totipotent cells have the ability to differentiate into all cell types of an embryo, including the extra-embryonic and post embryonic tissues and organs. Pluripotent cells have the potential to differentiate into almost all tissues found in an embryo (including germ cells), but are not capable of giving rise to supporting cells and tissues. Multipotent stem cells have progeny of several differentiated cell types - but all within a particular tissue, organ, or physiological system. A good example of multipotent cells, are the haematopoietic stem cells that produce blood cell-restricted progenitors, as well as all cell types and elements, such as platelets, that are normal components of blood. Oligopotent stem cells produce two or more lineages within a specific tissue, such as neural stem cells that are able to produce subsets of neurons in the brain. Unipotent cells self-renew, as well as give rise to a single mature cell type, a prime example being the spermatogonial stem cells, that give rise to spermatozoa (Moore, 2007). Adult human subcutaneous adipose tissue contains cells with multilineage developmental plasticity like marrow-derived mesenchymal stem cells (Strem et al., 2005, Tong et al., 2000). Adipose derived stem cells can be obtained in abundance and can differentiate into osteogenic, adipogenic, myogenic and chondrogenic lineages when treated with appropriate growth factors.

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