Haematopoietic stem cells (HSC) are strictly regulated by intrinsic regulators and extrinsic signals from the microenvironment. Nov (CCN3), a matricellular protein of the CCN family, has been reported as a suppressor gene in solid tumours and chronic myeloid leukaemia (CML). Recent study identified Nov as a positive regulator in human cord blood CD34+ stem cells. However, the functions of Nov in haematopoiesis and adult HSC remain largely unknown.

Since being identified as the cause of AIDS in 1983, HIV-1 infection has reached pandemic proportions. Despite public awareness about prevention, the growing incidence of HIV-1 infection and the limitations of current antiretroviral therapy underscore the imperative need for a vaccine. Understanding the basis of an immune response that controls infection or provides sterilizing immunity remains a major goal in the search for effective vaccines or immunotherapies. Research into correlates of immunity to HIV-1 have largely focused on CD8⁺ T cells or neutralising antibodies (NAbs) but to date these responses have not proved effective in containing viral replication in vaccinees who become infected. Natural killer cells (NKs), monocytes (MCs), and neutrophils (PMNs) are cells of the innate immune system with intrinsic cytotoxic function that can be enhanced by antibodies (Abs) in what is termed antibody-dependent cellular cytotoxicity (ADCC). In my studies I investigated the production of PMNs from human stem cells, the elimination of HIV-1 infected cells by these effector cells, the modulation of cellular cytotoxicity by Ab, and characterized how Abs facilitate a potent ADCC response. I developed a novel flow cytometry assay to measure cytotoxic activity against HIV-1 infected CD4⁺ T cells. Using this, effector cells were shown to have different cytotoxic capacities which were enhanced by Ab. Comparing ADCC mediated by patient serum revealed that higher levels correlated with IgG binding to infected cells. I observed no correlation between serum-mediated ADCC and markers of disease progression including patient status, viral RNA load, CD4⁺ T cell count, or NAb titers. The data presented here have implications for acquisition and control of early HIV-1 infection by NKs, MCs, and PMNs prior to activation of an adaptive immune response, at later stages in the presence of HIV-1-specific Abs, and are relevant to vaccine-induced anti- HIV-1 Ab-based effector mechanisms.

Transwell inserts with microporous membranes, available from multiple commercial sources, have been widely used for various mammalian cell culture applications, including the reduction of cell culture mixing. In this study, we examined the feasibility and functionality of using this technology for separating human embryonic stem cells (hESCs) from their respective feeder cells. We found that when hESCs were propagated on transwell inserts positioned directly above feeder cells grown in a separate dish, the hESCs could be maintained in an undifferentiated state for over 10 passages with no change in their basic pluripotent characteristics. In parallel with our transwell insert experiments, we also evaluated the ability of a new defined, xeno-free medium, HEScGRO™, to enhance the animal-free characteristics of the transwell insert-based culture system. Results from our studies demonstrate that HEScGRO™ medium assists in maintaining the pluripotent characteristics of hESCs propagated in the transwell insert- based culture system. These combined results represent a significant development in properly segregating stem cells from their feeders, thus eliminating cell mixing, contamination, and providing the cells with a superior environment for nourishment and controlled self-renewal. Overall, this development in hESC propagation could have wide-reaching applications for self-renewal and differentiation studies within the field of stem cell biology.

Há um grande interesse no estudo de células-tronco em função de sua capacidade de auto-renovação e plasticidade. Estas características capacitam as células-tronco a produzirem células de diferentes linhagens que participam ativamente do processo de homeostase, da resposta à injúria e da regeneração e reparação tecidual. A polpa dental é o tecido mais estudado na Odontologia em relação a células-tronco, mas diversos estudos já mostraram a presença dessas células também na região periodontal. É importante salientar, que dependendo de sua origem, as células-tronco apresentam comportamentos diversos, especialmente no que tange ao transplante in vivo. Além disso, os microambientes onde as células-tronco residem (nichos), têm um papel fundamental no comportamento das mesmas, pois controlam aspectos essenciais como o estado de indiferenciação e a auto-renovação. Esse projeto de pesquisa teve como objetivos duas análises distintas. Na primeira, verificar se existem células-tronco mesenquimais derivadas da curetagem do alvéolo dental humano após extrações dentais. Na segunda, analisar o nicho de células-tronco nas polpas dentais de camundongos. Em comum, as duas análises se basearam no uso de marcadores previamente utilizados na literatura para o estudo de células-tronco. Como não existem marcadores únicos e específicos para a identificação dessas células, diferentes combinações desses marcadores entre si e de técnicas laboratoriais foram empregadas. No primeiro estudo, após o isolamento das células, deu-se especial atenção à sua caracterização, que foi realizada através de ensaios que avaliaram propriedades e comportamentos que sabidamente ocorrem em células-tronco. Já na segunda parte desse estudo, utilizamos a polpa dental de camundongos para a análise in vivo de nichos. Em camundongos, a localização do nicho responsável pelo crescimento continuo dos incisivos é conhecida. De uma maneira geral, nossos resultados demonstram que: 1) É possível isolar células-tronco/progenitoras a partir de tecidos curetados de alvéolo dental após extrações dentárias. Esse fato é inédito e abre novas perspectivas em termos de oportunidade de coleta e futura aplicação clínica. 2) É possível observar a diversidade populacional nas células que formam o nicho de células-tronco em polpa dental de camundongos, e através de uma organização hierárquica, propusemos um modelo para este nicho. Esse modelo servirá de base para estudos subsequentes que visem avaliar o comportamento das células que formam o nicho, quando isoladas das demais, e abrirá possibilidade para análises em outros tecidos dentais e não dentais. / There is great interest in the study of stem cells due to their ability to produce mature cells of different lineages that participate actively in the process of homeostasis, injury response, regeneration and tissue repair. The dental pulp is the most studied tissue in dentistry, but several studies have shown the presence of these cells in the periodontal tissue region. It is important to note that depending on their origin, these cells exhibit different behaviours, especially in regard to in vivo transplantation. Furthermore, the microenvironment in which these cells are found (niches) have an essential role in their behaviour as they control important aspects such as differentiation and self-renewal. This research project aimed for two distinct analyses. The first was to verify if there are mesenchymal stem cells derived from human dental socket curettage after dental extractions. The second, to analyse the stem cell niche in mouse dental pulp. In common, the two analyses were based on the use of markers previously used in the literature for the study of stem cells. As there are no specific and unique markers to identify these cells, an extensive combination of these markers among themselves, and laboratory techniques were performed. In the first study, after the isolation of the cells, special attention was given to their characterization by combining assays to evaluate stem cells properties and behaviours. In the second part of this study, we used the mouse dental pulp model for in vivo analysis of stem cell niches. In mice, the location of the niche responsible for the incisors continuous growth is known. In general our results showed that: 1) It is possible to isolate progenitor/stem cells from tissue curettage of the alveolus after tooth extractions. This fact is unprecedented and opens new perspectives for the obtention of stem cells for future clinical applications. 2) It is possible to observe the cells population diversity of the mouse dental pulp niche and through a hierarchical organization, we proposed a model for this niche. This model will provide basis for further studies aimed at evaluating the behavior of cells that form the niche, when isolated from the others, and opens the possibility for the analysis of the niche structure in other dental and non-dental tissues.

O tecido ósseo tem sido amplamente estudado devido às suas inúmeras funções e capacidade de auto-regeneração. Entretanto, muitas vezes a reparação óssea completa pode ser prejudicada quando as fraturas ósseas são graves. Muitas pesquisas visando a regeneração do tecido ósseo estão sendo realizadas tanto nas abordagens que envolvem a utilização de enxertos, quanto na aplicação de terapia celular. O objetivo deste trabalho foi obter e caracterizar uma linhagem celular proveniente do tecido ósseo de fetos caninos. Para isso, foi realizado método de dissociação enzimática do osso fetal canino para obtenção da cultura celular, estabelecendo assim o cultivo das células OSTBN6. Além disso, foram realizados técnicas do teste de viabilidade e proliferação celular por MTT, de imunofenotipagem das células, expressão gênica, diferenciação celular em linhagens adipogênica, osteogênica e condrogênica, e análise do potencial tumorigênico das células derivadas de tecido ósseo fetal canino. Sendo assim, a população isolada de células derivadas de tecido ósseo fetal canino isolada apresentou duas morfologias distintas: formato fibroblastóide e formato triangular. Essas células são viáveis e possuem ótima taxa de proliferação, como indicou o ensaio de MTT. As células foram positivas para pluripotência e para células de origem mesenquimal, e foram negativas para marcadores de células de origem hematopoiéticas. As OSTBN6 foram capazes de se diferenciar em linhagem adipogênica, osteogênica e condrogênica, característica de células mesenquimais. Por fim, foi realizado o teste do potencial tumorigênico em camundongos nude, não havendo formação de tumores. Desse modo, concluimos que a célula proveniente do tecido ósseo fetal canino constitui uma fonte segura para utilização na medicina regenerativa e terapia celular / Bone tissue has been widely studied due to its numerous functions and capacity for self-regeneration. However, often the complete bone repair may be impaired when bone fractures are severe. Many research aiming to regenerate the bone tissue are being conducted in both approaches involving the use of grafts, as the application of stem cell therapy. The objective of this study was to obtain and characterize a cell line derived from the canine fetuses bone. To do so, it was carried out an enzymatic dissociation method for obtaining canine fetal bone cell culture, thus establishing OSTBN6 cells culture. In addition, there were carried out technical viability and cell proliferation by MTT test, immunophenotyping of cells, gene expression, cellular differentiation in adipogenic, osteogenic and chondrogenic lineages, and analysis of tumorigenic potential of cells derived from canine fetal bone. Thus, the population of cells derived from isolated canine fetal bone tissue showed two distinct morphologies: fibroblastoid shape and triangular shape. These cells are viable and have optimal rate of proliferation, as indicated by the MTT assay. Cells were positive for pluripotency and as mesenchymal cells and were negative for hematopoietic origin cell markers. The OSTBN6 were able to differentiate into adipogenic, osteogenic and chondrogenic lineage, characteristic of mesenchymal cells. Finally, it was performed the tumorigenic potential test in nude mice, with no tumor formation. Thus, it was concluded that the cell derived from canine fetal bone is a reliable source for use in regenerative medicine and cell therapy

Chronic patellar tendinopathy is a degenerative tendon disorder characterized by chronic activity-related, anterior knee pain associated with localized tenderness, swelling and impaired performance, which is a common clinical problem in athletes. The pathogenesis of patellar tendinopathy is still largely unknown, although tendon overuse is the most commonly suggested etiological factor, and treatment is usually symptomatic. / Histopathologically, the predominant feature of patellar tendinopathy is tendinosis, which is characterized by progressive tissue degeneration with a failed healing response and the absence of inflammatory cells. Hypercellularity with non-tenocyte phenotype cells and tissue metaplasia, including hyaline metaplasia, fibrocartilaginous metaplasia and bony metaplasia were observed in clinical patellar tendinopathy samples. The degeneration of patellar tendon in patellar tendinopathy is an active cell-mediated process rather than a passive degenerative process. Using a patellar tendinopathy animal model, we observed the presence of chondrocytic and osteoblastic phenotype / markers in patellar tendinopathy samples with or without ossification, which was consistent with the findings in clinical samples. Interestingly, chondrocyte makers were expressed by healing tendon cells at week 2 which became round prior to their expression in the chondrocyte-like cells at week 4. This leads us to speculate that erroneous differentiation of tendon-derived stem cells (TDSCs) identified recently in tendon tissues by our group, to chondrocyte / osteoblasts, due to alteration of mechanical and biological microenvironment during overuse, may lead to the ectopic chondro-ossification and failed healing in patellar tendinopathy. Osteo-chondrogenic BMPs, such as BMP-2, BMP-4 and BMP-7 might be possible factors regulating the osteo-chondrogenic differentiation of TDSCs in the pathogenesis of patellar tendinopathy. / In conclusion, our results have provided new insights about the pathological mechanisms of patellar tendinopathy involving the resident stem cells, osteo-chondrogenic BMPs and mechanical overloading. Erroneous differentiation of TDSCs to chondrocytes / osteoblasts due to ectopic osteo-chondrogenic BMP-2 expression, which were induced by repetitive tensile loading stimulation, might account for the chondro-ossification and failed healing in patellar tendinopathy. Re-directing of stem cells for tenogenic differentiation by blocking the ectopic expression of osteo-chondrogenic BMPs may help to promote tendon healing in patellar tendinoapthy. / In this study, we hypothesized that (1) TDSCs isolated from pathological patellar tendon of the CI model will exhibit higher osteogenic and chondrogenic differentiation potential but lower proliferative capacity compared to TDSCs isolated from healthy patellar tendon. Rat pathological tendon in our collagenase-induced failed healing animal model will harbor more TDSCs compared to healthy patellar tendon. (2) Osteo-chondrogenic BMPs, such as BMP-2, BMP-4, and BMP-7, will be expressed ectopically in both preclinical and clinical samples of patellar tendinopathy. (3) BMP-2 will promote osteo-chondrogenic differentiation and inhibit tenogenic differentiation of TDSCs in vitro. (4) Repetitive tensile loading will increase the expression of BMP-2 in TDSCs in vitro. / Our results showed that TDSCs isolated from the collagenase-induced tendinopathic patellar tendon of the animal model exhibited higher osteogenic/chondrogenic differentiation potential as well as lower proliferative capacity, supporting that there might be some defects in the TDSCs from the animal model, which might undergo osteo-chondrogenic differentiation and hence reduced the pool of TDSCs for tendon repair in the development of patellar tendinopathy. The higher clonogenicity and increased yield of TDSCs in tendinopathic patellar tendon might be caused by a compensation for the impaired differentiation potential and proliferative capacity of TDSCs. The histopathological features of our clinical patellar tendinopathy were characterized by tissue degeneration. Non-tenocyte phenotype cells and tissue metaplasia, such as chondrocyte-like cells and endochondral ossification were also observed. We observed the ectopic expression of osteo-chondrogenic BMP-2, BMP-4 and BMP-7 in both our animal model and clinical samples of patellar tendinopathy, which might trigger the erroneous differentiation of TDSCs to non-tenocytes. Indeed, we further showed that BMP-2 could promote the osteo-chondrogenic and inhibit tenogenic differentiation of TDSCs in vitro, which might provide a possible explanation for ectopic chondro-ossification and failed healing in patellar tendinopathy. In addition, our results also showed that in vitro repetitive cyclic tensile loading could increase the expression of BMP-2 in TDSCs, which might provide a possible explanation for the ectopic expression of BMP-2 in patellar tendinopathy. / This study aimed to compare the osteogenic / chondrogenic differentiation potential, proliferative capacity and yield of TDSCs isolated from rat healthy patellar tendon and pathological tendon in our collagenase-induced failed tendon healing animal model of patellar tendinopathy in vitro. The histopathological characteristics of our clinical patellar tendinopathy with or without ossification were examined. The ectopic expression of BMP-2, BMP-4, and BMP-7 in both human and rat samples of patellar tendinopathy was also examined. The effects of BMP-2 on the osteogenic, chondrogenic and tenogenic differentiation of TDSCs was further investigated in vitro. The effect of repetitive tensile loading on the expression of BMP-2 in TDSCs was studied in vitro. / Rui, Yunfeng. / Advisers: Kai Ming Chan; Po Yee Lui. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 172-193). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Choi, Pui Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 101-122). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Tse, Chi Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.X / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Regenerative Medicine --- p.1 / Chapter 1.2 --- Embryonic Stem Cells and Reprogramming --- p.3 / Chapter 1.3 --- Transdifferentiation --- p.6 / Chapter 1.4 --- The Cerebellum --- p.7 / Chapter 1.4.1 --- Functions of the cerebellum --- p.7 / Chapter 1.4.2 --- Structure and organization of the cerebellum --- p.8 / Chapter 1.4.3 --- Principle cellular components in the cerebellum --- p.12 / Chapter 1.4.3.1 --- Purkinje cells --- p.12 / Chapter 1.4.3.2 --- Granule cells --- p.12 / Chapter 1.4.3.3 --- Mossy fibres --- p.13 / Chapter 1.4.3.4 --- Climbing fibres --- p.13 / Chapter 1.4.3.5 --- Deep cerebellar nuclei --- p.13 / Chapter 1.4.3.6 --- Other cerebellar neurons --- p.14 / Chapter 1.4.3.7 --- Neuroglia of the cerebellum --- p.16 / Chapter 1.4.4 --- Circuitry of the cerebellum --- p.17 / Chapter 1.5 --- Development of the Cerebellum --- p.21 / Chapter 1.5.1 --- Anatomical changes during cerebellar development --- p.21 / Chapter 1.5.2 --- Molecular control of cerebellar development --- p.25 / Chapter 1.5.2.1 --- Specification of the cerebellar region --- p.25 / Chapter 1.5.2.2 --- Neurogenesis from the ventricular zone --- p.26 / Chapter 1.5.2.3 --- Neurogenesis from rhombic lip --- p.29 / Chapter 1.6 --- Scope of the Thesis --- p.33 / Chapter CHAPTER 2 --- Materials and General Methods / Chapter 2.1 --- Materials for Molecular Biological Work --- p.35 / Chapter 2.1.1 --- Enzymes --- p.35 / Chapter 2.1.2 --- Chemicals and others --- p.35 / Chapter 2.1.3 --- Plasmid vectors and plasmid --- p.36 / Chapter 2.1.4 --- Solutions and media --- p.36 / Chapter 2.2 --- Materials for Tissue/Cell Culture --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Culture media and solutions --- p.38 / Chapter 2.2.3 --- Culture cells --- p.39 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Materials for Immunocytochemistry --- p.40 / Chapter 2.5 --- Oligonucleotide Primers --- p.41 / Chapter 2.6 --- RNA Extraction --- p.44 / Chapter 2.7 --- Generation of cDNA from mRNA --- p.44 / Chapter 2.8 --- Preparation of Recombinant Plasmid DNA --- p.45 / Chapter 2.8.1 --- Small scale preparation of DNA --- p.45 / Chapter 2.8.2 --- QLAGEN plasmid midiprep kit method --- p.46 / Chapter 2.9 --- Preparation of Specific DNA Fragment from Agarose Gel --- p.46 / Chapter 2.10 --- Subcloning of DNA Fragments --- p.47 / Chapter 2.10.1 --- Preparation of cloning vectors --- p.47 / Chapter 2.10.2 --- Subcloning of DNA fragment --- p.48 / Chapter 2.10.3 --- Transformation of DNA into competent cells --- p.48 / Chapter 2.11 --- Preparation of Competent Cells --- p.48 / Chapter CHAPTER 3 --- Generation and Characterization of Induced Neurons / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Experimental Procedures --- p.51 / Chapter 3.2.1 --- Construction of expression vector --- p.51 / Chapter 3.2.1.1 --- Preparation of insert DNA --- p.51 / Chapter 3.2.1.2 --- Construction of entry vector --- p.52 / Chapter 3.2.1.3 --- Construction of destination vector --- p.52 / Chapter 3.2.1.4 --- Construction of expression vector --- p.52 / Chapter 3.2.2 --- Generation of induced neural cells --- p.57 / Chapter 3.2.2.1 --- Culture of mouse embryonic fibroblasts (MEF) --- p.57 / Chapter 3.2.2.2 --- Production of expression vector containing retroviruses --- p.57 / Chapter 3.2.2.3 --- Transfection and induction of neural fate of MEF --- p.57 / Chapter 3.2.3 --- Immunocytochemcial analysis --- p.58 / Chapter 3.2.4 --- Efficiency calculation --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- A screen for cerebellar Purkinje and granule cell fate-inducing factors --- p.62 / Chapter 3.3.2 --- Characterization of the induced neurons --- p.67 / Chapter 3.3.2.1 --- Granule cell induction --- p.67 / Chapter 3.3.2.2 --- Purkinje cell induction --- p.71 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.4.1 --- Roles of inducing factors in Purkinje cells and granule cells development --- p.102 / Chapter 3.4.2 --- Mechanism of neural transdifferentiation --- p.107 / Chapter CHAPTER 4 --- Future Directions / Chapter 4.1 --- Complete Induction of Purkinje Cell Fate --- p.111 / Chapter 4.2 --- Induced Neurons of Different Subtypes --- p.112 / Chapter 4.3 --- Mechanism of Transdifferentiation --- p.114 / Chapter 4.4 --- Transdifferentiation and Regenerative Medicine --- p.114 / Bibliography --- p.116

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-06-10T13:30:39Z No. of bitstreams: 1 Dissertação - Nadja Pinto Garcia.pdf: 2876829 bytes, checksum: 836b1ac1a926c0202497b6aaef613039 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-06-11T19:15:18Z (GMT) No. of bitstreams: 1 Dissertação - Nadja Pinto Garcia.pdf: 2876829 bytes, checksum: 836b1ac1a926c0202497b6aaef613039 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-06-11T19:20:53Z (GMT) No. of bitstreams: 1 Dissertação - Nadja Pinto Garcia.pdf: 2876829 bytes, checksum: 836b1ac1a926c0202497b6aaef613039 (MD5) / Made available in DSpace on 2015-06-11T19:20:53Z (GMT). No. of bitstreams: 1 Dissertação - Nadja Pinto Garcia.pdf: 2876829 bytes, checksum: 836b1ac1a926c0202497b6aaef613039 (MD5) Previous issue date: 2011-11-24 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The mesenchymal stem cells (MSCs) have regenerative potential by its plasticity and ability to modulate the immune response with immunossupressive effects and secretion of a broadspectrum cytokines. The G-CSF is a potent cell growth factor and has the ability to mobilize SCs into peripheral blood. The aim of this study was to evaluated the influence of different doses of G-CSF on the kinetics at HSC and MSCs mobilization into peripheral blood and the G-CSF effect on the cytokine profile produced by these cells in vitro. We used six groups of 12 female Swiss mice, control group and five doses groups 1, 2, 3, 4 and 5 of G-CSF. The HSCs and MSCs mobilized were identified using cell markers performed by flow cytometry. The peripheral blood (PB) and bone marrow (BM) samples were used of each group to obtain the HSCs and MSCs, which were cultivated in vitro. Cytokines were measured in supernatants of PB and BM cultures by Cytometric Bead Array (CBA). The MSCs mobilized peak occurred with 4 doses of G-CSF in BM and 5 doses in PB. The HSC peaked occurred with 2 doses of G-CSF in BM and 4 doses in PB. There was a greater mobilization of MSCs than HSC, but that reason MSC/HSC was even greater in BM. The BM Cultures doses 3, 4 and 5 of G-CSF showed fibroblastoid adherent cells while in the PB cultures it was in cultures doses 2 and 3 doses of G-CSF. There was an increased production of IL-6, TNF-α in early cultivation of the SCs both BM and PB. The IFN-γ was increased in the initial phase, but peaked at the end of cultivation. The IL-2, IL-4, IL-17A and IL-10 cytokines had similar behavior reaching peak concentration in the late stage of cultivation. In the analysis of high frequency of cytokine producers for each dose of G-CSF in vivo, we observed same behavior cytokine in BM and PB cultures. Most of the cytokines produced in both BM and PB cultures of five doses showed significant differences about lower doses of G-CSF. This study suggested a possible influence of G-CSF to mobilize cells, as is known, but also in the production of several inflammatory and anti-inflammatory cytokines and possibly stimulate and modulate the differentiation of MSCs. / As células-tronco mesenquimais (CTM) apresentam potencial regenerativo não somente pela sua plasticidade, mas também pela sua capacidade de modular a resposta imunológica com efeitos imunossupressores e secreção de um largo espectro citocinas. O G-CSF é um potente fator de crescimento celular e tem a capacidade de mobilizar as CTs para o sangue periférico permitindo fácil obtenção destas células. O objetivo deste estudo foi avaliar a influência de diferentes doses de G-CSF na cinética de mobilização das CTHs e CTMs para o sangue periférico e no perfil de citocinas produzidas in vitro por essas células. Foram utilizados 6 grupos com 12 camundongos fêmeas Swiss, grupo controle e 5 grupos de doses 1, 2, 3, 4 e 5 de G-CSF. As CTHs e CTMs mobilizadas foram identificadas por meio de marcadores celulares específicos por citometria de fluxo. As amostras de CTHs e CTMs, de sangue periférico (SP) e medula óssea (MO) foram cultivadas in vitro e as citocinas foram dosadas nos sobrenadantes destas culturas pela técnica de Cytometric Bead Array (CBA). O pico de CTMs mobilizadas ocorreu com 4 doses na MO e com 5 doses de G-CSF no SP. As CTHs atingiram o pico com 2 doses de G-CSF na MO e com 4 doses no SP. Houve uma maior mobilização de CTMs do que CTHs, porém essa razão CTM/CTH ainda foi maior na MO. As culturas de MO das doses 3, 4 e 5 de G-CSF apresentaram células aderentes fibroblastóides, enquanto que foram observadas nas culturas de sangue de 2 e 3 doses de G-CSF. Houve uma maior produção das citocinas IL-6, TNF-α na fase inicial do cultivo das CTs, tanto MO quanto SP. O IFN-γ apresentou-se elevado na fase inicial, porém atingiu um pico no final do cultivo. As citocinas IL-2, IL-4, IL-17A e IL-10 tiveram um comportamento semelhante atingindo pico de concentração na fase tardia do cultivo. Na análise da frequência de altos produtores de citocinas para cada dose administrada de G-CSF in vivo, observou-se um comportamento semellhante das citocinas das culturas de MO e SP. A maioria das citocinas produzidas nas culturas de 5 doses tanto de MO quanto de SP apresentaram diferença significativa com relação a doses inferiores de G-CSF. Esse estudo sugeriu uma possível influência do G-CSF não somente na mobilização, como já é conhecido, mas também na produção de várias citocinas inflamatórias e anti-inflamatótrias, podendo possivelmente atuar no estímulo da diferenciação e modulação das CTMs.

Há um grande interesse no estudo de células-tronco em função de sua capacidade de auto-renovação e plasticidade. Estas características capacitam as células-tronco a produzirem células de diferentes linhagens que participam ativamente do processo de homeostase, da resposta à injúria e da regeneração e reparação tecidual. A polpa dental é o tecido mais estudado na Odontologia em relação a células-tronco, mas diversos estudos já mostraram a presença dessas células também na região periodontal. É importante salientar, que dependendo de sua origem, as células-tronco apresentam comportamentos diversos, especialmente no que tange ao transplante in vivo. Além disso, os microambientes onde as células-tronco residem (nichos), têm um papel fundamental no comportamento das mesmas, pois controlam aspectos essenciais como o estado de indiferenciação e a auto-renovação. Esse projeto de pesquisa teve como objetivos duas análises distintas. Na primeira, verificar se existem células-tronco mesenquimais derivadas da curetagem do alvéolo dental humano após extrações dentais. Na segunda, analisar o nicho de células-tronco nas polpas dentais de camundongos. Em comum, as duas análises se basearam no uso de marcadores previamente utilizados na literatura para o estudo de células-tronco. Como não existem marcadores únicos e específicos para a identificação dessas células, diferentes combinações desses marcadores entre si e de técnicas laboratoriais foram empregadas. No primeiro estudo, após o isolamento das células, deu-se especial atenção à sua caracterização, que foi realizada através de ensaios que avaliaram propriedades e comportamentos que sabidamente ocorrem em células-tronco. Já na segunda parte desse estudo, utilizamos a polpa dental de camundongos para a análise in vivo de nichos. Em camundongos, a localização do nicho responsável pelo crescimento continuo dos incisivos é conhecida. De uma maneira geral, nossos resultados demonstram que: 1) É possível isolar células-tronco/progenitoras a partir de tecidos curetados de alvéolo dental após extrações dentárias. Esse fato é inédito e abre novas perspectivas em termos de oportunidade de coleta e futura aplicação clínica. 2) É possível observar a diversidade populacional nas células que formam o nicho de células-tronco em polpa dental de camundongos, e através de uma organização hierárquica, propusemos um modelo para este nicho. Esse modelo servirá de base para estudos subsequentes que visem avaliar o comportamento das células que formam o nicho, quando isoladas das demais, e abrirá possibilidade para análises em outros tecidos dentais e não dentais. / There is great interest in the study of stem cells due to their ability to produce mature cells of different lineages that participate actively in the process of homeostasis, injury response, regeneration and tissue repair. The dental pulp is the most studied tissue in dentistry, but several studies have shown the presence of these cells in the periodontal tissue region. It is important to note that depending on their origin, these cells exhibit different behaviours, especially in regard to in vivo transplantation. Furthermore, the microenvironment in which these cells are found (niches) have an essential role in their behaviour as they control important aspects such as differentiation and self-renewal. This research project aimed for two distinct analyses. The first was to verify if there are mesenchymal stem cells derived from human dental socket curettage after dental extractions. The second, to analyse the stem cell niche in mouse dental pulp. In common, the two analyses were based on the use of markers previously used in the literature for the study of stem cells. As there are no specific and unique markers to identify these cells, an extensive combination of these markers among themselves, and laboratory techniques were performed. In the first study, after the isolation of the cells, special attention was given to their characterization by combining assays to evaluate stem cells properties and behaviours. In the second part of this study, we used the mouse dental pulp model for in vivo analysis of stem cell niches. In mice, the location of the niche responsible for the incisors continuous growth is known. In general our results showed that: 1) It is possible to isolate progenitor/stem cells from tissue curettage of the alveolus after tooth extractions. This fact is unprecedented and opens new perspectives for the obtention of stem cells for future clinical applications. 2) It is possible to observe the cells population diversity of the mouse dental pulp niche and through a hierarchical organization, we proposed a model for this niche. This model will provide basis for further studies aimed at evaluating the behavior of cells that form the niche, when isolated from the others, and opens the possibility for the analysis of the niche structure in other dental and non-dental tissues.