Bone is the second most transplanted tissue in the body, with approximately 2.2 million bone graft procedures performed annually worldwide. Currently, autogenous bone is the gold standard for bone grafting due to its ability to achieve functional healing; however, it is limited in supply and results in secondary injury at the donor site. Tissue engineering has emerged as a promising means for the development of new bone graft substitutes in order to overcome the limitations of the current grafts. In this research project, the specific approach for bone tissue engineering involves seeding osteoprogenitor cells within a biomaterial scaffold then culturing this construct in a biodynamic bioreactor. The bioreactor imparts osteoinductive mechanical stimuli on the cells to stimulate the synthesis of an extracellular matrix rich in osteogenic and angiogenic factors that are envisioned to guide bone healing in vivo. Fluid flow, which exerts a hydrodynamic shear stress on adherent cells, has been identified as one of the strongest stimuli on bone cell behavior. It has been shown to enhance the deposition of osteoblastic matrix proteins in vitro, and is particularly important for the delivery of oxygen and nutrients to cells within large scaffolds suitable for bone tissue regeneration. In particular, dynamic flow profiles have been shown to be more efficient at initiating mechanotransductive signaling and enhancing gene expression of osteoblastic cells in vitro relative to steady flow. However, the molecular signaling mechanisms by which bone cells convert hydrodynamic shear stress into biochemical signals and express osteoblastic matrix proteins are not fully understood. Therefore, the overall goal of this research project was to determine the effect of dynamic fluid flow on mechanotransductive signaling and expression of bioactive factors and bone matrix proteins. In the first study, an intermittent flow regimen, in which 5 min rest periods were inserted during fluid flow, was examined. Results showed that signaling molecules, mitogen activated protein kinases (MAPKs) and prostaglandin E2, were modulated with the flow regimen, but that expression of bone matrix proteins, collagen 1α1, osteopontin, bone sialoprotein (BSP), and osteocalcin (OC), were similar under continuous and intermittent flow. Thus, this study suggested that variation of the flow regimen modulates mechanotranductive signaling. In the second study, four flow conditions were examined: continuous flow, 0.074 Hz, 0.044 Hz, and 0.015 Hz pulsatile flow. This study demonstrated that pulsatile flow enhances expression of BSP and OC over steady flow. Similarly, bone morphogenetic protein (BMP)-2 and -7 were enhanced with pulsatile flow, while BMP-4 was suppressed with all flow conditions, suggesting that the mechanism by which fluid flow enhances bone matrix proteins may involve the induction of BMP-2 and -7, but not BMP-4. In the third study, the molecular mechanism by which fluid flow simulates expression of BMPs was examined. Results from this study suggest that this mechanism may involve activation of MAPKs, but BMP-2, -4, and -7 are regulated through multiple different signaling pathways. Overall, the results from this research demonstrate that dynamic flow modulates mechanotransductive signaling and expression of osteoblastic matrix proteins by osteoblast cells. In particular, BMPs, important for formation in vivo, were shown to be induced by fluid flow. Therefore, this work may be beneficial in understanding and developing 3D perfusion culture systems for the creation of a clinically effective engineering bone tissue. / Ph. D.

Rupture of the anterior cruciate ligament (ACL) is one of the most common ligamentous injuries of the knee. Post rupture, the ACL does not heal on itself due to poor vasculature and hence surgical intervention is required to treat the ACL. Current surgical management of ACL rupture consists of reconstruction with autografts or allografts. However, the limitations associated with these grafts have prompted interest in tissue engineered solutions that combine cells, scaffolds and stimuli to facilitate ACL regeneration. This thesis describes a ligament tissue engineering strategy that involves incorporating biomolecules within fibers-based electrospun meshes which mimics the extra-cellular matrix microarchitecture of ligament. However, challenges exist with incorporation of biomolecules. Therefore, the goal of this research project was to develop two techniques to incorporate biomolecules within electrospun meshes: (1) co-axially electrospinning fibers that support surface-grafting of biomolecules, and (2) co-axially electrospinning fibers decorated with biomolecule-loaded microspheres. In the first approach, chitosan was co-axially electrospun on the shell side of poly caprolactone (PCL) and arginine-glycine-aspartate (RGD) was attached to the electrospun meshes. Bone marrow stromal cells (BMSCs) attached, spread and proliferated on these meshes. In the second approach, fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) loaded chitosan-alginate (CS-AL) microspheres were fabricated. The effects of cation to alginate ratio, type of alginate and concentration of CaCl2 on microsphere size, FITC-BSA loading and release were systematically evaluated. The CS-AL microspheres were then incorporated into the sheath phase of co-axially electrospun meshes to achieve microsphere-decorated fiber composite meshes. The results from these model study suggest that both approaches are tractable for incorporating biomolecules within fibers-based electrospun meshes. Both these approaches provide platform for future studies that can focus on ligament-relevant biomolecules such as FGF-2 and GDF-5. / Master of Science

Long-term exposure to pharmacological doses of glucocorticoids has been associated with the development of osteopenia and avascular necrosis. Bone loss may be partially attributed to a steroid-induced decrease in the osteoblastic differentiation of multipotent progenitor cells found in the bone marrow. In order to determine if there is a change in the osteogenic potential of the bone marrow stroma following glucocorticoid treatment, Sprague-Dawley rats were administered methylprednisolone for up to six weeks, then sacrificed at 0, 2, 4, or 6 weeks during treatment or 4 weeks after cessation of treatment. Femurs were collected and analyzed for evidence of steroid-induced osteopenia and bone marrow adipogenesis. Although glucocorticoid treatment did inhibit bone growth, differences in ultimate shear stress and mineral content were not detected. The volume of marrow fat increased with increasing duration of treatment, but returned to near control levels after cessation of treatment. Marrow stromal cells were isolated from tibias, cultured in the presence of osteogenic supplements, and analyzed for their capacity to differentiate into osteoblast-like cells in vitro. Glucocorticoid treatment diminished the absolute number of isolated stromal cells, but did not inhibit the relative levels of bone-like mineral deposition or osteocalcin expression and secretion. Although pharmacological glucocorticoid levels induce bone loss in vivo, physiologically equivalent concentrations have been shown to enhance the formation of bone-like tissue in vitro. However, glucocorticoids have also been reported to inhibit proliferation and type I collagen synthesis in marrow stromal cell cultures. In order to assess the effects of intermittent dexamethasone treatment on the progression of osteogenesis in rat marrow stromal cell culture, this synthetic glucocorticoid was removed from the culture medium after a variable period of initial supplementation. Cell layers were analyzed for total cell number, collagen synthesis, phenotypic marker expression, and matrix mineralization. Prolonged supplementation with dexamethasone decreased proliferation, but did not significantly affect collagen synthesis. Furthermore, increased treatment duration was found to increase bone sialoprotein expression and mineral deposition. The duration of glucocorticoid treatment may be a key factor for controlling the extent of differentiation in vitro. / Master of Science

Human B lymphocytes are notoriously difficult to culture. Two to three days after plating, a sharp decline in viability and cell number can be observed. The objective of this study was to evaluate the effect of support cells on B cell viability in an in vitro human immune system construct. B cells were combined with dendritic cells (DCs) and cultured for various periods of time in the presence of one of three types of support cells: EA cells, HS-5 cells, and HS-27 A cells. The B cells were either in physical contact with the support cells, or allowed to interact through soluble factors in the media in order to determine if the effect on viability was contact dependent or independent. Viability was assessed using flow cytometry. Finally, two functional assays were performed to evaluate the ability of the cultured B cells to respond to an immune challenge. Both recall and nai've antigens were used. The B lymphocytes were then assessed for viability, proliferation and activation using flow cytometry. ELISPOT was also employed to determine if any antigen specific antibodies were produced by the B cells. It was found that while the support cells did improve viability, they did not produce consistent or reliable results. Additionally, B lymphocytes cultured in the presence of support cells or support cell conditioned media had no antigen specific tetanus response and reduced proliferation. Therefore, even though the support cells did under some conditions enhance lymphocyte viability, the lack of a positive functional response negates the value of using them in an experimental system.

Ziel dieser Studie war die Etablierung einer Kokultur aus equinen endometrialen Epithel- und Stromazellen. Nach der erfolgreichen Umsetzung des Kokulturmodells sollte im weiteren Versuchsablauf durch die Zugabe von 17β-Östradiol (E2) und/oder Progesteron (P4) zum Nährmedium der Einfluss der Hormone auf die Zellen untersucht werden. Neben einer lichtmikroskopischen Auswertung der zytomorphologischen Charakteristika beider Zellarten sollte die Expression der Steroidhormonrezeptoren Östrogenrezeptor α und Progesteronre-zeptor sowie der uterinen Proteine Uteroglobin und CalbindinD9k immunzytologisch überprüft werden. Für die Etablierung der Kokultur wurden Endometriumproben von lebenden (n = 5) sowie frischtoten (n = 4) Stuten gewonnen. Eine jeweils parallel entnommene Gewebeprobe von jedem Tier wurde in Formalin fixiert und diente als Referenzmaterial (in situ). Auf die Zelliso-lierung (mechanisch und enzymatisch) folgte die Separation von Epithel- und Stromazellen (EZ/SZ) mittels Filtration, Dichtegradientenzentrifugation und Differenzialadhärenz. An-schließend wurden die EZ auf die Außenseite von Millicell®-Membraneinsätzen aufgebracht. Nach zwei Tagen erfolgte das Einsäen der bis zu diesem Zeitpunkt separat kultivierten SZ auf die Innenseite der Membranen. Als Nährmedium diente ein Gemisch aus DMEM und Ham’s F-12, wobei diesem 2,5 % fötales Kälberserum sowie verschiedene Additive zugesetzt wurden. Ab Kulturtag 4 wurden dem Medium definierte Konzentrationen und Kombinationen von E2 und P4 zugesetzt. Die Kultivierung erfolgte bei einem CO2-Partialdruck von 5 % in 37 °C warmer wasserdampfgesättigter Raumluft. Mit der polarisationsmikroskopisch er-fassbaren Ausbildung durchgehender Zellrasen („scheinbare Konfluenz“) wurden die Kokul-turen in Formalin fixiert und für die Lichtmikroskopie aufgearbeitet. Das Ausgangsgewebe zeigte mehrheitlich eine sekretorische Funktionsmorphologie (n = 6). Einzelne Endometrien befanden sich in einem Übergangsstadium von der Sekretions- zur Proliferationsphase (n = 1), bzw. vice versa (n = 1) oder wiesen eine irregulär proliferative Differenzierung (n = 1) auf. Im Rahmen der Kokultivierung bildeten die EZ innerhalb der Schnittebene vier und die SZ drei verschiedene morphologische Zelltypen aus. Dabei traten rundovale bis polygonale EZ (Typ 1) selten bis gelegentlich, spindelförmige EZ (Typ 2) gelegentlich bis häufig und iso-prismatische (Typ 3) sowie mehrschichtig wachsende EZ (Typ M) jeweils selten auf. Die SZ zeigten innerhalb der Schnittebene selten eine rundovale bis polygonale Zellform (Typ 1), sehr häufig eine spindelförmige Morphologie (Typ 2) und selten ein mehrschichtiges Wachstum (Typ M). Ein Zusammenhang zwischen der endometrialen Funktionsmorphologie zum Zeitpunkt der Zellisolierung oder dem Hormonzusatz und der Häufigkeitsverteilung der Zell-typen sowie der Wachstumsgeschwindigkeit der kultivierten Zellen war nicht offensichtlich. Zytokeratin 19 wurde stets von EZ exprimiert, während es auf Seiten der SZ nur sporadisch in maximal 5 % der Zellen im Bereich mehrschichtig wachsender Zellrasen auftrat. Die Stero-idhormonrezeptoren konnten lediglich in einzelnen Kokulturen aus sekretorisch differenzier-tem Ausgangsgewebe detektiert werden. Uteroglobin wurde in vitro mit einer variablen Häufigkeit in den EZ-Typen exprimiert. Während ein übergreifender Zusammenhang zur hormonellen Supplementierung nicht abgeleitet werden konnte, wurde jedoch ersichtlich, dass im Bereich einschichtig wachsender EZ in Ansätzen aus sekretorisch differenzierten Endometrien unter niedrigen Hormondosen (Zusatz von entweder nur E2 oder nur P4) im Median häufiger Uteroglobin exprimiert wurde. Mit zunehmender Hormonkonzentration im Medium nahm der Anteil immunopositiver Zellen (Typen 1, 2 und 3) deutlich ab. Innerhalb der Stromazellpopulation wurde Uteroglobin selten und ausschließlich in Zellen aus sekretorisch differenziertem Ausgangsmaterial nachgewiesen. CalbindinD9k wurde in vitro vornehmlich intrazytoplasmatisch und sehr vereinzelt intranukleär exprimiert. Insgesamt konnte das Protein in vitro stets in wenigen Typ-1-EZ, sehr selten in Typ-2-EZ und in einer geringen bis mäßigen Anzahl von Typ-3- und Typ-M-EZ beobachtet werden. Innerhalb der Stromazellpo-pulation trat CalbindinD9k ausschließlich in einer geringen (Endometrien aus dem Östrus) bis mäßigen (Endometrien aus dem Interöstrus) Anzahl der Typ-2- und wenigen Typ-M-SZ auf. Insgesamt wurden keine deutlichen Einflüsse der endometrialen Funktionsmorphologie zum Zeitpunkt der Zellisolierung und/oder der hormonellen Supplementierung in vitro auf die im-munzytologischen Charakteristika der kokultivierten Zellen ersichtlich. Abschließend betrachtet, konnte ein Kokultursystem equiner endometrialer Epithel- und Stromazellen erfolgreich etabliert und charakterisiert werden. Es bietet dabei, trotz der z. T. fehlenden Kongruenz zu den Gegebenheiten in situ, Ansätze für potenzielle Folgearbeiten, insbesondere hinsichtlich der Erfassung interzellulärer Wechselwirkungen sowie bezüglich der Vermittlung und Wirkung hormoneller Einflüsse auf zellulärer Ebene. / The aim of the present study was the establishment of a coculture system of equine endome-trial epithelial and stromal cells. Subsequent to the successful development of the coculture model the culture medium should be supplemented with 17β-estradiol (E2) and/or progester-one (P4) in order to study the influence of the hormones on the cellular level. In addition to the examination of cytomorphological characteristics of both cell types via light microscopy, the expression of the steroid hormone receptors (estrogen receptor α and progesterone receptor) as well as of the uterine proteins Uteroglobin and CalbindinD9k was investigated. For the establishment of the coculture system endometrial samples were obtained from living (n = 5) as well as freshly deceased mares (n = 4). A simultaneously taken tissue specimen of each animal was fixed in formalin and served as in situ reference material. After an initial mechanical and enzymatical isolation the epithelial and stromal cells (EC/SC) were separat-ed via filtration, density gradient centrifugation and differential adhesion. Subsequently, the EC were applied to the outer surface of Millicell® inserts. The SC were cultivated separately for 2 days before they were seeded onto the inner surface of the same insert. The culture medium used was comprised of a DMEM and Ham‘s F-12 basis as well as 2.5 % foetal calf serum and different additives. Starting on day 4 of cultivation the standardised medium was supplemented with different concentrations and combinations of E2 and P4. Throughout the study the cultures were kept in a humidified atmosphere of 37°C and a 5 % partial pressure of carbon dioxide. Once the cocultures formed continuous cell layers, as determined via a polarisation microscope (“apparent confluency”), the membranes were fixed in formalin and routinely processed for light microscopical evaluation. The initial tissue samples predominantly showed a secretory functional morphology (n = 6), while single specimens were obtained during the transition from the secretory to the prolifera-tive phase (n = 1) or vice versa (n = 1). One endometrial sample exhibited an irregular proli-ferative differentiation. In the course of cocultivation the EC formed 4 and the SC 3 different cellular morphologies within the section plane. EC with a round-oval to polygonal cell form (type 1) were rarely to occasionally encountered, while spindle-shaped EC (type 2) were occasionally to frequently seen and EC with a cuboidal morphology (type 3) as well as such cells growing in stratified layers (type M) were only infrequently detected. The SC only rarely showed a round-oval to polygonal cell form (type 1) or areas of a stratified cell growth (type M), whereas spindle-shaped SC (type 2) were observed very often. A correlation of the endometrial functional morphology at the time of cell isolation or the hormonal supplementation and the frequency distribution of the cell types as well as the growth rate of the cultivated cells was not evident. The EC always expressed Cytokeratin 19, while on the side of the SC only up to 5 % of the cells in areas of stratified cell growth exhibited this filament. Solely in individual cocultures from secretory differentiated endometrial tissue the steroid hormone receptors could be de-tected. Uteroglobin was expressed in vitro in EC with a variable frequency. An overall corre-lation of the hormonal supplementation and the Uteroglobin expression could not be derived. However, under low hormone doses (only E2 or only P4 supplement) Uteroglobin was detect-ed in EC in areas of single-layered cell growth more often (median value). With an increase in hormone concentration the amount of immunopositive cells (types 1, 2 and 3) diminished noticeably. In SC the protein could only rarely be seen and exclusively in cells from endome-tria with a secretory functional morphology. In vitro CalbindinD9k was predominantly detected intracytoplasmatically, while single cells showed an additional intranuclear expression. Alto-gether, CalbindinD9k could always be observed in a few type-1-EC, rarely in type-2-EC and with a variable frequency in small to moderate numbers of type-3- and type-M-EC. In SC the protein was exclusively expressed in a small (endometrial samples form the oestrous phase) to moderate (endometrial tissue from the interoestrous phase) number of type-2-SC and a few type-M-SC. Generally, no distinct influence of the endometrial functional morphology at the time of tissue sampling and/or of the hormonal supplementation in vitro on the immuno-cytochemical characteristics of the cocultured cells could be observed. In summary, a coculture system of primary equine endometrial epithelial and stromal cells was successfully established and characterised. Despite of the partly absent congruence to the in situ conditions/prerequisites, the present study offers a basic approach and scaffold for further investigations, particularly regarding the ascertainment of intercellular dependencies or the mediation and effectiveness of hormonal influences on the cellular level.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Doctor of Philosophy / Department of Anatomy and Physiology / Mark L. Weiss / Graft Versus Host Disease (GVHD) is the major complication following hematopoietic stem cell transplantation. GVHD is activated by immunocompetent T cells presented in the donor grafted tissue. Due to the increased use of bone marrow transplantation to treat diverse malignancies, the incidence of GVHD has shown a notable increase. Depending of the degree of immunological mismatch between donor and host, 50-70% of patients develop GVHD after allogeneic Bone Marrow Transplantation (BMT). Once GVHD develops, mortality reaches up to 50% in humans. Several studies using Mesenchymal Stromal Cells (MSCs) to prevent and treat GVHD have produced controversial results. It is thought that distinct MSCs sources used in those studies might be an important factor that produces different outcomes. For cellular therapy, the most attractive characteristics of MSCs are their reduced immunogenic potential, and their abilities to modulate immune responses. This dissertation addressed the hypothesis that Wharton’s jelly cells (WJCs) would prevent the pathology and death associated with GVHD after BMT. To accomplish this, I created a clinically relevant model of GVHD by transplanting allogeneic bone marrow across minor histocompatibility antigen (HA) barriers in the rat. To enhance alloreactive T-cell stimulation, bone marrow (BM) was co-administered with a fraction of CD8[superscript]+ cells magnetically selected from spleen to induce GVHD. Bone marrow tissue was isolated from a donor rat Fischer 344 (F344, RT1lv) and transplanted into lethally irradiated (10 Gray) Lewis rat (LEW, RT1l). Once GVHD was induced, MSCs derived from umbilical cord WJCs were either co-transplanted at day 0 with bone marrow, or given on day 2 post-BMT intravenously. The prophylactic potential of WJCs in an in vivo GVHD model was assessed as survival time, clinical symptomatology occurrence, and histopathology injuries in target tissues. Results indicate that while co-administration of WJCs with hematopoietic cells on day 0 failed to alleviate GVHD associated symptomatology and mortality. WJCs administered on day 2 post-induction ameliorated GVHD-associated symptomatology, improved engraftment and survival.

Master of Science / Department of Clinical Sciences / Mary Lynn Higginbotham / Objectives – To isolate and maintain canine Wharton’s jelly mesenchymal stromal cells (WJMSCs) in culture, to determine the effects of micellar nanoparticles containing doxorubicin (DOX) on WJMSCs and canine osteosarcoma (OSA) D17 cell viability, and to determine the effects of conditioned media from WJMSCs loaded with micellar nanoparticles containing DOX on OSA D17 cell viability. Sample Population – Canine WJMSCs containing various concentrations of DOX micelles and canine OSA D17 cells. Procedures – WJMSCs were isolated from canine umbilical cords. Micellar nanoparticles containing DOX were prepared and added to culture plates containing canine OSA D17 cells to determine micelle effects on cell growth and viability. Conditioned media from culture plates containing canine WJMSCs incubated with various DOX micelle concentrations was added to OSA D17 cells for conditioned media experiments. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess OSA D17 cell viability. A trypan blue stain was also utilized to perform cell counts to determine the effect of the DOX micelles on stromal cell growth. Results – WJMSCs were successfully isolated and maintained in culture. Micellar nanoparticles containing DOX decreased OSA D17 cell viability. OSA D17 cell viability was also decreased following incubation with conditioned media from canine WJMSCs loaded with micellar nanoparticles containing DOX. Significant decreases with the conditioned media of canine WJMSCs loaded with 10μM micelles occurred at 48 hours (p < 0.005) and at 72 and 96 hours (p < 0.0001). Significant decreases were also observed with the 1 μM DOX micelles at 72 hours (p < 0.005) and 96 hours (p < 0.0001). WJMSC numbers decreased in a dose dependent manner following incubation with DOX micelles. Changes in WJMSC number was not caused by increased cell death as all variables produced similar percentages of dead cells. Conclusions – Canine WJMSCs were successfully isolated and maintained in culture. Stromal cells containing DOX micellar nanoparticles induced OSA D17 cell cytotoxicity while inducing an anti-proliferative, rather than cytotoxic effect, on the WJMSC. These data support future in vivo experiments utilizing canine WJMSCs and micellar nanoparticles.

INTRODUÇÃO: As células-tronco mesenquimais são capazes de regenerar diferentes tipos de tecidos, no entanto, a maioria dos métodos para sua obtenção são invasivos. Recentemente, foi descoberta a existência destas células no sangue menstrual. OBJETIVO: Padronizar as técnicas de coleta e transporte do fluido menstrual, bem como a caracterização, isolamento, expansão e criopreservação de células-tronco do fluido menstrual e avaliar a disponibilidade de células tronco mesenquimais no fluido menstrual. MÉTODOS: No período de agosto de 2011 a março de 2012 foram selecionadas 20 voluntárias com ciclo menstrual regular, sem doença ginecológica. O fluido menstrual foi coletado no dia de maior fluxo e submetido a imunofenotipagem e cultivo celular. Foram realizadas duas passagens em meio de cultura até atingir semi-confluência das células-tronco, as quais foram, em seguida, criopreservadas. RESULTADOS: Os parâmetros analisados apresentaram os seguintes valores médios: volume de fluido menstrual 6,90±5,60mL; tempo de transporte 17,20±5,50h; número de células totais 3,95 x106±3,88 x106 com 76,05%±24,57 de células viáveis. Após a cultura, as células mesenquimais aumentaram de 0,14%±0,26 para 96,19%±2,14. Na primeira passagem de cultura, após 15 a 21 dias, as colônias formaram grupos que atingiram a confluência, que a partir da segunda passagem ocorreu em cerca de 3 dias. As células-tronco mesenquimais criopreservadas eram viáveis. CONCLUSÃO: As células-tronco do fluido menstrual podem ser obtidas sem métodos invasivos. O fluido menstrual pode ser transportado em condições ideais de temperatura até 24 horas após a coleta. As células tronco mesenquimais podem ser caracterizadas por imunofenotipagem, isoladas, cultivadas e expandidas e, em seguida, criopreservadas. O fluido menstrual contém células tronco mesenquimais viáveis e apropriadas para cultivo / INTRODUCTION: Mesenchymal stem cells may renovate different tissues, but techniques to obtain these cells are invasive. Recently, those cells were detected in menstrual blood. OBJECTIVE: Patterning techniques of collection, transportation, characterization, isolation, expansion and cryopreservation of stem cells in menstrual fluid. METHODS: From August 2011 to March 2012 twenty volunteers were selected with regular menstrual cycle without gynecological diseases. They collected menstrual fluid on the most intense flux day to analysis by immunophenotyping and cellular culture. Culture was made in 2 stages until reached semi-confluence of stem cells and these cells were cryopreserved. RESULTS: Average of menstrual fluid volume was 6,90±5,60mL, transportation time was 17,20±5,50h, and total number of cells was 3,95 x106±3,88 x106 witch 76,05%±24,57 were viables. After culture, mesenchymal stem cells increased from 0,14%±0,26 to 96,19%±2,14. After 15 to 21 days of culture in first passage, colonies formed clusters that reached confluence. In second passage, it happens after 3 days of culture and stem cells were cryopreserved. CONCLUSION: Stem cells of menstrual fluid may be easily obtained without invasive methods. Menstrual fluid can be transported in good conditions of temperature up to 24 hours of collection. Mesenchymal stem cells of menstrual fluid may be characterized by immunophenotyping, as well as it is possible to isolated, cultivate and cryopreserved them. Menstrual fluid has viable and proper for culture mesenchymal stem cells

A heterogeneidade entre as células tumorais e o suporte a elas proporcionado pelos componentes do microambiente tumoral (TME) são os dois principais responsáveis pela progressão do câncer e por tornar essas doenças essencialmente incuráveis. Assim, identificar as principais dependências das células malignas, sejam elas internas ou advindas do meio extracelular, é fundamental para entender seu comportamento e propor terapias mais eficientes. Nesta tese, abordamos aspectos destas duas questões separadamente. Em um primeiro trabalho, investigamos as interações de células tumorais com células-tronco mesenquimais (MSCs), um dos principais componentes do TME. MSCs participam ativamente do nicho tumoral, especialmente por serem capazes de liberar uma vasta gama de moléculas que, via sinalização parácrina, podem modular as células ao seu redor. No entanto, os principais mediadores e respectivos efeitos do secretoma dessas células nos tumores ainda precisam ser melhor elucidados. Ao investigar esses efeitos em glioblastomas (GBM), um dos tumores primários mais agressivos em adultos, mostramos que o secretoma de células-tronco mesenquimais derivadas de tecido adiposo humano (hADSCs) foi capaz de bloquear a autofagia das células malignas. Nossos dados revelaram que o secretoma de hADSCs ativou a via de sinalização de mTORC1 e reduziu a translocação nuclear de TFEB, um fator de transcrição chave que regula a autofagia e a a função lisossomal, nas células de GBM, impedindo que o fluxo autofágico fosse completado. Já em um segundo trabalho, no contexto da heterogeneidade celular em tumores, propusemos uma abordagem para análise de dados de céulas únicas focada em outliers. Minorias celulares com níveis anormalmente elevados, ou reduzidos, de expressão de determinados genes ou proteínas são em muitos casos responsáveis por resistir aos tratamentos e levar à recidiva da doença, ao mesmo tempo que, por serem outliers, são muitas vezes ignoradas ou excluídas das análises de dados. Assim, decidimos utilizar métodos estatísticos em dados de expressão de células únicas para detectar e analisar células outliers, comparando o seu comportamento com as demais células não-outliers. Denominamos essa abordagem de Single Cell OUTlier analysis (SCOUT) e a testamos em dados de células tumorais avaliadas por citometria de massas e por sequenciamento de RNA de células únicas (sc-RNA-seq). Como resultado, pudemos confirmar que, especialmente diante de determinados tratamentos, células outliers podem se comportar de maneira distinta de não-outliers, revelando informações potencialmente relevantes ao desenvolvimento de estretégias terapêuticas. Por fim, desenvolvemos uma ferramenta para automatizar a detecção e seleção de outliers em dados de célula única a fim de facilitar o estudo dessas células em diversos aspectos na pesquisa do câncer. / Intratumoral heterogeneity and the support provided by components of the tumor microenvironment (TME) to malignant cells are major contributors to cancer progression, and the two main factors that make this disease essentially incurable. Thus, identifying malignant cells dependencies, either in the intra- or extracellular environment, is fundamental to understand their behavior and propose more efficient therapies. In this thesis, we approached aspects of these two issues separately. In a first work, we investigated interactions between tumors and mesenchymal stem cells (MSCs), one of the main components in the TME. MSCs actively participate in the tumor niche, especially due to their capacity of releasing a wide range of molecules that can modulate cells in their surroundings. However, little is known about the effects of MSCs-derived molecules in tumor cells behavior. In investigating these effects on glioblastomas (GBM), one of the most aggressive primary tumors in adults, we found out that the secretome of human adipose-derived stromal cells (hADSCs) was able to block autophagy in malignant cells. Our data revealed that hADSCs secretome activated mTORC1 signaling pathway and reduced nuclear translocation of TFEB, a master transcription factor that regulates autophagy and lysosomal function, in GBM cells, preventing autophagic flux from being completed. In a second work, we addressed intratumoral heterogeneity by proposing an approach to analyze outliers in single cell data. Cellular minorities with abnormally high, or low, expression levels of certain genes or proteins are in many cases responsible for resisting treatments and lead to disease relapse, while for being outliers they are also frequently ignored or excluded from data analysis. Thus, we decided to apply statistical methods on single cell expression data to detect outliers and analyze them, comparing their behavior with the remaining non-outlier cells. We called this approach Single Cell OUTlier analysis (SCOUT) and tested it on tumor cell datasets obtained from mass cytometry and single cell RNA sequencing (scRNA-seq) experiments. Using SCOUT we were able to confirm that, especially upon specific treatments, outlier cells may behave differently from non-outliers, revealing potentially relevant information to aid in the development of novel therapeutic strategies. Finally, we developed a tool to automate detection and selection of outliers in single cell data with the aim to facilitate the study of these cells under different contexts in cancer research.