Mesenchymal stem cells (MSC) represent a heterogenous population of nonhematopoietic stem cells with multipotent differential potential. MSC can be isolated from various tissues of organism, the most common tissue are bone marrow or adipose tissue. MSC are good candidates for treatment of autoimmune diseases and possess the ability to prevent graft rejection or graft versus host disease. The immunosuppressive drugs are currently used for inhibition of unwanted immune reaction but they exhibit serious side effects. The use of MSC in therapy can reduce doses of immunosuppressive drugs and eliminate side effects. The study of MSC and immunosuppressant interactions should be detected before MSC can be used for clinical application. We aimed to analyze the interaction between MSC and immunosuppressive drugs and their effect on immune cells. Cyclosporine A and mycophenolate mofetil were used in our research. We demonstrated changes in the expression of surface molecules, production of interleukin 6 and in metabolic activity of MSC after treatment with immunosuppressive drugs. MSC are in organism, in cooperation with the number of other cells. Therefore we studied MSC cocultured with splenocytes in the presence of immunosuppressive drugs. Our results show the effect of MSC and immunosuppressive drugs on different...

Mesenchymal stem cells (MSCs) represent a heterogeneous population of stromal cells with a pluripotent differentiation potential. They can be isolated from multiple tissues of mesodermal origin, such as bone marrow, adipose tissue, umbilical cord blood and peripheral blood and afterwards externally expanded according their adherence to the plastic surfaces. These cells show remarkable immunomodulatory properties, suppressing T-, B- and NK-cell functions, and also modulating dendritic cell activities and influencing immune responses during tissue repair and recovery. MSCs have been shown to possess ability to migrate to sites of inflammation and tissue injury. All these properties make MSCs a promising tool for clinical application. Our primary goal was to identify processes that may influence immunoregulatory effects of MSCs. In order to promote immunossupressive qualities of MSCs we established the scheme comprising MSCs precultivated with various cytokines and Toll-like receptors (TLR) ligands in vitro, with the final aim to improve the therapeutic effect of MSCs on wound healing in vivo. We studied modulation of MSCs properties and consequently the effect of influenced MSCs on cells of the immune system. The immunosuppression is mainly mediated through secreted factors that MSCs produced after...

Electrospinning is widely used technique to produce nanoscale constructs for tissue engineering. This technique can be used to spin wide range of polymers. One of them is polyvinyl alcohol (PVA), which has very good properties for use in this field. PVA is nontoxic, has good mechanical strength and it's degradable and biocompatible. Electrospun PVA nanofibers have limitations because of their -OH side groups. These groups cause solubility of PVA in water. The solubility can be adjusted with crosslinking techniques, but PVA still remains very hydrophilic, which is causing low adhesion of cells. In recent research we decided to reduce the hydrophilicity of PVA using plasma modification. Polymer modification with cold plasma is an economic and quite simple process to change the surface chemistry without side effects that come with conventional chemical treatment. With radical, formed by discharge, we have deposited hydrocarbons on the PVA surface and we rapidly increased hydrophobicity of the polymer surface. The change of surface chemistry has only a little effect on the fiber morphology. The increase of hydrophobicity allowed better adhesion of mesenchymal stem cells on plasma modified PVA as compared to non-modified PVA and a huge change in cell morphology was observed. These changes suggest that we...

Regenerative medicine is an emerging technology and with hundreds of cell therapies currently in clinical trials there is a need to expand the limited knowledge related to their storage, shipment and preservation. The most widely used medium for human cell cryopreservation is 10%wt dimethyl sulfoxide (DMSO) in serum. However given its potential toxicity, DMSO usage is a key issue in cryopreservation. Methods specify the need to reduce cell exposure time to DMSO above 0°C as much as possible but the maximum amount of time cells can be exposed to DMSO to prevent a detrimental effect needs to be clarified. There are also regulatory issues and concerns with the xenotoxicity, ethics and supply of the other core component in the standard cryomedia formulation: Foetal Bovine Serum (FBS). Developing a viable alternative to FBS is crucial. In cryobiology literature thawing appears poorly understood. A stable process is as vital as freezing to prevent injury to cells. Protocols are currently too vague for cell therapy regulation and need improvement. The time dependent DMSO cytotoxicity was evaluated by overexposing cells to DMSO during and/or after cryopreservation. A broad investigation found that after 1 hour overexposure post thaw viability of human mesenchymal stem cells (hMSCs) was reduced from 96.3±0.6% to 74.1±4.0% and the co-expression of five key hMSC markers was changed from 97.9±1.3% to 68.3±2.6%. This significant change could cause indicate a change in product efficacy and affect patient health, to prevent this, DMSO exposure must be kept to below 1 hour. A range of alternative vehicle solutions were screened and human platelet lysate (hPL) investigated as an alternative. In depth experimentation with hPL as a cryopreservation vehicle solution and culture supplement (in place of FBS) found it to be a worthy, statistically similar alternative. With no xenological or ethical concerns, lower costs than other serum-free alternatives hPL could allow for a move away from xenological components. A heat transfer model was developed and determined that 720J is required to thaw a vial. Using the heat transfer model and additional factors such as pre-thaw stabilisation and on thaw dilution, a two-stage experiment found that the current standard process (warming in a 37°C waterbath) within the current paradigm of a 1.8mL cryovial is optimal but further work is required to define the process for scaled-up product.

Recent advances in the clinical diagnosis and treatment of diseases using cell transplantation have emphasized the urgent need to cryopreserve many types of cells. In transfusion medicine, red blood cell (RBC) transfusions are used to treat anemia and inherited blood disorders, replace blood lost during or after surgery and treat accident victims and mass casualty events. In regenerative medicine, mesenchymal stem cell (MSC) therapy offers promising treatment for tissue injury and immune disorders. Current cryoprotective agents (CPAs) utilized for RBCs and MSCs are 40% glycerol and 10% dimethyl sulfoxide (DMSO), respectively. Although glycerol is required for successful cryopreservation of RBCs, it must be removed from RBCs post-thaw using costly and time-consuming deglycerolization procedures to avoid intravascular hemolysis. Unfortunately, while DMSO prevents cell damage and increases post-thaw MSC viability and recovery, recent reports have suggested that MSCs cryopreserved in DMSO display compromised function post-thaw. As a result, improvements to the current cryopreservation protocols such as reducing post-thaw RBC processing times and improving MSC function post-thaw are necessary in order to meet the increasing demands of emerging cellular therapies. Ice recrystallization has been identified as a significant contributor to cellular injury and death during cryopreservation. Consequently, the ability to inhibit ice recrystallization is a very desirable property for an effective CPA, unlike the conventional CPAs such as DMSO and glycerol that function via a different mechanism and do not control or inhibit ice recrystallization. Over the past few years, our laboratory has reported several different classes of small molecules capable of inhibiting ice recrystallization such as lysine-based surfactants, non-ionic carbohydrate-based amphiphiles (alkyl and aryl aldonamides) and O-linked alkyl and aryl glycosides. The use of these small molecule ice recrystallization inhibitors (IRIs) as novel CPAs has become an important strategy to improve cell viability and function post-thaw. With the overall goal to identify highly effective inhibitors of ice recrystallization, the first part of this thesis examines the IRI activity of three diverse classes of small molecules including carbohydrate-based surfactants bearing an azobenzene moiety, fluorinated aryl glycosides and phosphate sugars. While the majority of the carbohydrate-based surfactants and fluorinated aryl glycosides were not effective inhibitors of ice recrystallization, this work revealed that monosaccharides possessing a phosphate group could be effective IRIs. Our laboratory has previously demonstrated that small molecule IRIs β-PMP-Glc and β-pBrPh-Glc can protect human RBCs from cellular injury during freezing using reduced concentrations of glycerol (15% w/v). This was significant as reducing the concentration of glycerol can drastically decrease deglycerolization times. Consequently, structure- function studies were conducted on β-PMP-Glc and β-pBrPh-Glc to elucidate key structural features that further enhance their IRI activity and may increase their cryoprotective ability. In particular, the influence of an azido moiety on the IRI activity of β-PMP-Glc and β-pBrPh-Glc was investigated and it was determined that the position of the azide substituent on the pyranose ring is crucial for effective inhibition of ice recrystallization. Furthermore, the presence of an azido group at C-3 was found to increase the IRI activity of β-PMP-Glc and β-pBrPh-Glc. Despite the discovery that β-PMP-Glc and β-pBrPh-Glc are beneficial additives for the freezing of RBCs, a significant amount of cellular damage occurred during deglycerolization, resulting in very low cell recoveries. Thus, IRI active azido aryl glucosides were explored for their cryopreservation potential in RBCs to determine whether they could function as effective additives that reduce cellular damage post-thaw and improve cell recovery. One of the most significant results of this thesis is the discovery that azido aryl glucosides can successfully cryopreserve RBCs in the presence of 15% glycerol with significantly improved cell recovery. This thesis also explores the use of small molecule IRIs to improve the cryopreservation of MSCs. In particular, the addition of an N-aryl-aldonamide (2FA) to the standard 10% DMSO solution was found to enhance the proliferative capacity of MSCs post-thaw. Lastly, the ability of small molecule IRIs to cross the cell membrane and behave as permeating CPAs was evaluated in two different cell models, RBCs and human umbilical vein endothelial cells (HUVECs). These studies demonstrated that small molecule IRIs are capable of permeating the cell membrane and controlling intracellular ice recrystallization.

Orientador: Elenice Deffune / Banca: Sérgio Luis Felisbino / Banca: João Tadeu Ribeiro Paes / Resumo: Levando-se em consideração avanços tecnológicos na área médica e o impacto dos programas de saúde que determinaram curvas de longevidade cada vez maiores e taxas de natalidade cada vez menores, o novo desafio do gestor público são as conseqüências: o envelhecimento e a vida como uma "doença crônica". Entre os principais desafios está a abordagem das doenças crônico-degenerativas que determinam o aumento de lesões cartilaginosas articulares. A débil capacidade de regeneração e as limitações das alternativas de tratamento fazem as técnicas derivadas da biotecnologia, como o transplante autólogo de condrócitos (TAC) e o uso de células-tronco, o foco das investigações. O TAC requer coleta de material cartilaginoso de área sadia, podendo causar nova lesão; no entanto, pode-se evitar este perigo com o uso de células-tronco. As células-tronco mesenquimais, adultas, podem se diferenciar em condrócitos mediante o uso de meio de cultura específico em consonância a um arcabouço 3D, mas muitos problemas como evitar a calcificação e estimular a condrogênese em meio favorável constituem o desafio para os pesquisadores na atualidade. 1) produzir anticorpo monoclonal específico a CTMs de coelho para monitorá-las, 2) determinar o volume ideal de coleta de medula óssea para microencapsulação e condrogênese, 3) realizar a microencapsulação das CTMs em novos arcabouços: BIOGEL3D e BACTCELL3D, comparando seu desempenho com o modelo clássico em alginato. Foram utilizados 25 coelhos Nova Zelândia sendo divididos em diferentes grupos em função do volume de MO coletado: G1 = 6mL, G2 = 9mL, G3 = 12mL, G4 = 15mL e G5 = volume ideal de coleta determinado pelos indicadores dos outros grupos. O material coletado foi diluído 1:2 em RPMI 1640 com 3.000U de heparina sódica. Após a contagem celular, as amostras foram submetidas a separação em gradiente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Technological advances in the medical area combined with the impact of health programs that enhance longevity, together with lower natality rates created new challenges to the public manager such as aging and life as a "chronic disease". Among the major problems are the chronic degenerative diseases that increase articular lesions. The limited regeneration capabilities and the limitations of actual treatment alternatives made biotechnology derived techniques the focus of investigations. The autologous chondrocyte transplantation (ACT) requires a small biopsy of health cartilage, which can lead to a new lesion. However, the use of stem cells can avoid this possibility. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes by using a specific culture medium together with a 3D scaffold, but some questions such as the risks of calcification remain as key factors to researchers. 1) to produce a monoclonal antibody that recognizes MSCs in order to characterize them, 2) to determine the optimal bone marrow collection volume for cell microencapsulation and chondrogenesis and 3) to microencapsulate MSCs in two novel scaffolds: BIOGEL3D and BACTCELL3D, comparing them with sodium alginate. 25 New Zealand rabbits were divided into 5 groups related to bone marrow collection volume: G1 = 6mL, G2 = 9mL, G3 = 12mL, G4 = 15mL and G5 = optimal volume determined by the study. The collected material was diluted in RPMI1640 medium 1:2, with 3000U sodium heparin. After cell count and viability assessment the samples were submitted to density gradient centrifugation in order to isolate the lymphomononuclear (LMN) fraction. These cells were seeded to obtain and expand the MSCs in DMEM Knockout® (InvitrogenTM) supplemented with antibiotic/antimycotic, Lglutamine, essential aminoacids, non essential aminoacids and fetal bovine serum (all from InvitrogenTM). The cells were cultivated in 5% CO2 ...(Complete abstract click electronic access below) / Mestre

Neste trabalho tentamos traçar quais seriam os efeitos funcionais e de expressão gênica de mutações patogênicas no gene TCOF1 em células não embrionárias. A partir do estabelecimento de culturas celulares oriundas de periósteo facial de quatro pacientes portadores da síndrome de Treacher Collins (STC), obtivemos populações celulares com mais de 95% de marcação positiva para antígenos que caracterizam células de origem mesenquimal. Demonstramos que as células-tronco mesenquimais e fibroblastos provenientes de pacientes com STC apresentaram redução da expressão de TCOF1 de aproximadamente 31% quando comparados aos controles. Tal redução se mostrou compatível com a diminuição da expressão dos transcritos portadores de mutação patogênica observada no seqüenciamento do cDNA dos pacientes. Portanto, estes resultados sugerem que há degradação do transcrito mutado, que muito possivelmente deve ser regulada pelo mecanismo de non-sense mediated mRNA decay (NMD) e corroboram o modelo de haploinsuficiência da Treacle. Como o pico de expressão do gene Tcof1 é detectado durante o estágio embrionário de formação das células da crista neural e embriões de camundongos Tcof1+/- apresentam redução da proliferação das células neuroepiteliais, testamos se haveria alteração da capacidade proliferativa de células mesenquimais adultas com mutações no TCOF1. A análise das taxas de crescimento das culturas celulares provenientes de pacientes com STC se mostrou semelhante aos controles normais, indicando que menores níveis de transcritos do gene não interferem na capacidade proliferativa celular. Além disso, avaliamos o potencial de diferenciação óssea in vitro, mostrando que menores níveis de TCOF1 também não parecem influenciar a capacidade de diferenciação celular. Apesar de ainda não termos identificado um fenótipo celular, a deficiência de TCOF1 em células não embrionárias resulta na alteração da expressão gênica, já que a análise da expressão genômica das culturas celulares identificou uma série de genes diferencialmente expressos. Ademais, genes de parada do ciclo celular e apoptose com expressão aumentada em células embrionárias de camundongo Tcof1+/- não apresentaram expressão aumentada nas células humanas com mutações patogênicas no TCOF1, sugerindo que a via da p53 não está ativa nestas células não embrionárias. Dentre os genes diferencialmente expressos encontrados, o aumento dos transcritos de DAXX nas células-tronco mesenquimais com mutações patogênicas no TCOF1 poderia modular as funções da p53 nessas células, constituindo um bom alvo de investigações futuras para a elucidação da função do TCOF1 em células não embrionárias. Os resultados obtidos sugerem que deficiência de TCOF1 em células mesenquimais leva à ativação de outras vias de sinalização, cujo efeito funcional é ainda desconhecido. Quais seriam as funções e em quais vias celulares o TCOF1 agiria, são questões a serem elucidadas a respeito do papel do gene na fase adulta. / In this work we tried to outline the effects of functional and gene expression of pathogenic mutations in the gene TCOF1 in non-embryonic cells. From the establishment of facial periosteum derived cell culture of four patients with Treacher Collins syndrome (TCS), we obtained cell populations that are more than 95% positive for mesenchymal origin characteristic antigens. We demonstrated that mesenchymal stem cells and fibroblasts from patients with TCS showed reduction of approximately 31% in the TCOF1 expression when compared to controls. This reduction was consistent with the decrease in the expression of transcripts carrying the pathogenic mutations observed when sequencing the cDNA of the patients. Therefore, these results suggest that degradation of the mutated transcript occurs and it may possibly be governed by the mechanism of non-sense mediated mRNA decay (NMD), thus corroborating the Treacle haploinsufficiency model. As the peak of TCOF1 gene expression is detected during the embryonic stage of the formation of neural crest cells and Tcof1+/- mice embryos had shown reduction of neuroepithelium cells proliferation rate, we tested if there is a change in proliferative capacity of adult mesenchymal cells with mutations in TCOF1. The analysis of the growth rate of TCS patients cells was similar to normal controls, indicating that lower levels of transcripts of the gene does not interfere with cellular proliferative capacity. Furthermore, we evaluated the bone differentiation potential of these cells in vitro, showing that lower levels of TCOF1 also seem does not influence cell differentiation ability. Although a cellular phenotype has not yet been identified, deficiency of TCOF1 in non-embryonic cells results in gene expression change, since genomic analysis of the expression of cell cultures identified a number of differentially expressed genes. Furthermore, arresting cell cycle and apoptosis related genes with increased expression in embryonic cells of Tcof1+/- mice showed no increased expression in human cells with pathogenic mutations in TCOF1 suggesting that the p53 pathway is not active in these non-embryonic cells. Among the differentially expressed genes found, the increase of DAXX transcripts in mesenchymal stem cells with pathogenic mutations in TCOF1 could modulate the function of p53 in these cells, providing a good target for future investigations to elucidate the function of TCOF1 in non-embryonic cells. The results suggest that deficiency of TCOF1 in mesenchymal cells leads to activation of other signaling pathways, whose functional effects are still unknown. The functions and cellular pathways in which the TCOF1 act, are issues yet to be elucidated concerning the role of the gene during adulthood.

Células-tronco mesenquimais (CTM) apresentam tropismo a tumores, sendo importantes componentes do estroma tumoral. No cérebro, o nicho perivascular é uma importante fonte de CTM, as quais podem contribuir direta e/ou indiretamente para o desenvolvimento de tumores, embora os mecanismos envolvidos sejam pouco conhecidos. No presente trabalho, investigou-se a influência de CTM sobre a proliferação, capacidade invasiva e tumorigenicidade de células de Glioblastoma (GBM) humano. Sabe-se que CTM produzem TGFB1, uma citocina multifuncional envolvida em imunomodulação, proliferação, migração e transição epitelial-mesenquimal de células tumorais. Experimentos in vitro, realizados com meios condicionados de CTM de cordão umbilical humano com silenciamento permanente do gene TGFB1, demonstraram que o TGFB1 secretado por CTM é capaz de aumentar significativamente a proliferação e viabilidade de células de GBM humano da linhagem U87FP635. Esses resultados revelam uma importante ação parácrina dessa citocina regulatória, quando produzida por outros tipos celulares contidos no microambiente tumoral. Entretanto, sob condições experimentais que melhor mimetizam o microambiente tumoral, detectou-se que CTM também afetam o comportamento de células tumorais por um mecanismo alternativo, dependente de contato celular, mas independente dos níveis de TGFB1 secretados pelas CTM. Sob condições de cocultivo celular, envolvendo contato físico entre CTM e células de GBM U87FP635, detectou-se um aumento significativo na quantidade de células tumorais viáveis. Quando cultivadas na forma de esferoides tumorais, o contato com CTM aumentou a capacidade invasiva das células U87FP635. Finalmente, em modelo in vivo ectópico de GBM, células U87FP635 geraram tumores mais desenvolvidos quando coinjetadas com CTM. Esses efeitos pró-tumorigênicos foram observados tanto em contato com CTM controles, quanto com CTM contendo o gene TGFB1 permanentemente silenciado. Assim, esses achados indicam que CTM podem exercer efeitos pró-tumorigênicos por dois mecanismos alternativos e independentes: ação parácrina de TGFB1 secretado por CTM e ação mediada por contato célula-célula. Nas condições experimentais testadas, o mecanismo dependente de contato célula-célula demonstrou ser predominante. O estudo proteômico do secretoma dessas células identificou 126 proteínas diferencialmente expressas além de 10 proteínas exclusivamente detectadas em meios condicionados de cocultivos de CTM com células de GBM U87FP635. Cerca de 80% dessas proteínas exclusivamente secretadas pelo contato célula-célula são componentes de exossomos e estão envolvidas em proliferação celular e desenvolvimento tecidual. Esses resultados apontam uma interação dinâmica de comunicação entre CTM e células tumorais, e revelam algumas proteínas interessantes potencialmente envolvidas em uma ação pró-tumorigênica de CTM mediada por contato celular / Mesenchymal stem cells (MSC) display tropism to tumors, being recruited to its microenvironment where they comprise the tumor stroma. In brain, perivascular niche is a substantial source of MSC. Although mechanisms involved are poorly understood, MSC may directly and/or indirectly contribute to tumor development. Herein, the influence of MSC on the proliferation, invasiveness and tumorigenicity of human glioblastoma cells (GBM) was investigated. Moreover, since MSC releases TGFB1, a multifunctional cytokine with roles in immunomodulation, proliferation, migration and epithelial-mesenchymal transition of tumor cells, we analyzed if MSC-secreted TGFB1 affects GBM behavior. In vitro studies performed in the presence of conditioned media from human umbilical cord MSC with a stable TGFB1 gene expression knockdown showed that MSC-secreted TGFB1 is able to significantly increase the proliferation and viability of a GBM cell line (U87FP635). These results revealed an important paracrine effect of this regulatory cytokine when secreted by other cell types in tumor microenvironment. However, under experimental conditions that better mimic the tumor microenvironment, it was found that MSC also affect tumor cell behavior by an alternative mechanism dependent on cell-cell contact, but independent of TGFB1 levels secreted by MSC. The cell-cell contact between MSC and GBM U87FP635 significantly enhaced tumor viable cells. Additionally, the spheroid tumor cell culture with MSC cell contact increased the invasiveness of U87FP635 cells. Finally, in vivo ectopic implantation model showed more developed tumors when GBM U87FP635 cells were coinjected with MSC. These pro-tumorigenic effects were found both in cell-cell contact with control MSC, as with MSC containing TGFB1 gene expression knockdown. Thus, these findings indicate that MSC can exert pro-tumorigenic effects by two alternative and independent mechanisms: paracrine action of TGFB1 secreted by MSC and action mediated by cell-cell contact. In the present experimental conditions, the cell-cell contact-dependent mechanism was predominant. The secretome proteomic study of those cells identified 126 differentially expressed proteins as well as 10 proteins exclusively detected in conditioned media from GBM U87FP635 cell cocultures with MSC. About 80% of proteins uniquely secreted by cell-cell contact are exosomes components and are involved in cell proliferation and tissue development. These results indicate a dynamic interaction of communication between MSC and tumor cells and reveal some interesting proteins potentially involved in a MSC pro-tumorigenic action mediated by cell contact

A fissura lábio-palatina não-sindrômica (FLP NS) é uma doença multifatorial, determinada pela interação entre fatores genéticos e ambientais e sua incidência é estimada entre 0,34 e 2,29 a cada 1000 nascimentos. Trata-se de uma embriopatia causada por erros durante a morfogênese orofacial, a qual depende de uma fina regulação de mecanismos como proliferação celular, remodelagem de matriz extracelular, e transição epitélio-mesenquimal. Apesar de intensos esforços para se determinar fatores genéticos e ambientais de susceptibilidade, a etiologia desta malformação permanece pouco compreendida. Vários loci associados às FLP NS vêm sendo identificados por meio de estudos de mapeamento gênico convencionais, entretanto, a grande maioria dos resultados não se replica em diferentes estudos, e não há clareza acerca do efeito funcional das variantes detectadas. Neste contexto, uma abordagem interessante para investigar a etiologia da doença é a análise de expressão gênica, que pode ser utilizada para identificar alterações de vias biológicas que convergem na manifestação do quadro clínico. Em vista disso, neste trabalho nós utilizamos a análise do transcriptoma de células-tronco de polpa dental de pacientes portadores de FLP NS, com o intuito de identificar padrões de expressão relacionados a mecanismos biológicos relevantes para a embriopatogênese da doença. Obtivemos padrões de expressão que sugerem desregulação de mecanismos associados à remodelagem de matriz extracelular e à transição epitélio-mesenquimal. Além disso, ao utilizarmos diferentes condições de cultura celular, verificamos em uma nova amostra de pacientes a desregulação de vias biológicas relacionadas ao reparo de DNA e checkpoint do ciclo celular. Nossos dados revelam a aplicabilidade das células-tronco de polpa dental para este tipo de abordagem, e indicam que tais perfis de expressão podem levar ao acometimento da morfogênese lábio-palatina. Além disso, mostramos pela primeira vez uma conexão entre desregulação de expressão gênica e a documentada maior incidência de formas esporádicas de câncer em famílias segregando a FLP NS. Nossos resultados abrem novas possibilidades para a investigação da etiologia das FLP NS, e ajudarão na compreensão dos eventos embrionários que predispõem a essa malformação. / Non-syndromic cleft lip and palate (NSCL/P) is a multifactorial disease determined by the interplay between genetic and environmental factors, with a variable incidence of 0.34-2.29:1000 births. This malformation arises from errors during lip and palate morphogenesis, which requires tight regulation of biological mechanisms such as cellular proliferation, extracellular matrix remodelling, and epithelial-mesenchymal transition. Albeit much effort has been put into determining the genetic and environmental factors underlying disease susceptibility, the aetiology of NSCL/P remains obscure. Many candidate loci have been identified through conventional gene mapping strategies, however, there is a general lack of reproducibility across studies, and there is no consensus with regard to the functional implications of the identified genetic variants. In this context, an alternative approach resides in assessing differential expression patterns to identify alterations in biological networks that could lead to phenotype manifestation. Here, we analysed the transcriptome of dental pulp stem cells from NSCL/P patients in order to pinpoint dysregulated pathways involved in the embryopathogenesis of the disease. We encountered expression patterns related to dysregulation of extracellular matrix remodelling and epithelial mesenchymal transition. Moreover, by subjecting a novel NSCL/P sample to differential cell culture conditions, we observed abnormal transcription of genes partaking in DNA repair and cell cycle checkpoint pathways. Our results show the applicability of dental pulp stem cells to this strategy and suggest that the observed expression patterns could lead to impairment of lip and palate morphogenesis. Moreover, we described for the first time a connection between abnormal gene expression in these individuals and the elevated occurrence of sporadic cancer types in NSCL/P families. Our results open new possibilities to investigate the aetiology of NSCL/P and provide further insight into the ontogenetic events underlying disease predisposition.

As células mesenquimais estromais multipotentes (CMM) são na atualidade uma fonte atrativa para aplicações na engenharia de tecidos e na terapia celular. Devido à baixa disponibilidade nos tecidos (0,01%-0,0005%) e às elevadas doses necessárias para uma infusão (aproximadamente 106 células/Kg paciente) tornou-se necessário o desenvolvimento de tecnologias de expansão in vitro, eficientes e de custo reduzido, que permitam a obtenção de CMM com manutenção das características funcionais (diferenciação e inibição da proliferação de linfócitos), imunofenotípicas e citogenéticas. As CMM são células aderentes, ou seja, necessitam de um substrato sólido para se aderir e proliferar. O procedimento convencional de expansão em garrafas estáticas, geralmente envolve um processo laborioso em que não há correto controle e monitoramento dos parâmetros de cultivo e possui uma maior susceptibilidade à contaminação devido à excessiva manipulação para atingir o número ideal de células. Além disso, este tipo de cultivo não permite uma produção em larga escala. Em função disso, o presente trabalho foi proposto com o objetivo de desenvolver um bioprocesso escalonável, economicamente viável e eficiente para expansão de CMM derivadas da medula óssea em microcarregadores. Para isso, as células foram cultivadas em microcarregador Cyotdex 3, em frasco spinner com o meio -MEM suplementado com 15% de SFB. Foram avaliadas neste trabalho, a adesão celular aos microcarregadores, crescimento, metabolismo, recuperação celular final e avaliação das propriedades funcionais e imunofenotípicas pré e pós cultivo, comparando ao cultivo já estabelecido em garrafas estáticas. De maneira geral, os resultados obtidos mostraram que foi possível expandir CMM utilizando a tecnologia de microcarregadores. A análise do metabolismo celular mostrou que não houve exaustão de nutrientes importantes como glicose e glutamina durante o cultivo, tampouco formação dos subprodutos lactato e amônia em concentrações inibitórias. As células recuperadas após a expansão mantiveram as características imunofenotípicas e funcionais. A produção média (n=10) foi de aproximadamente 4,9x105 cel/mL. Como o sistema utilizado permite o escalonamento, se utilizássemos um biorreator de 1L, seria possível a produção de aproximadamente 5x108 células que seriam suficientes para tratar mais de 3 pacientes de até 70Kg na dose de 2x106 células/Kg. Para expansão da mesma quantidade de células na forma tradicional seriam necessárias 135 garrafas de 175 cm2 com um custo total de expansão duas vezes superior à estimativa do custo de expansão utilizando microcarregadores. / Multipotent mesenchymal stromal cells are currently an attractive source for applications in tissue engineering and cell therapy. Due to the low availability in tissues (0,01%-0,0005%) and the high doses necessary for an infusion (about 106 cells/Kg patient), it has become necessary the development of effective and low cost technologies for in vitro expansion that enable to obtain MSC with maintenance of functional (differentiation and inhibition of lymphocytes proliferation), immunophenotypic and cytogenetics characteristics. MSC are adherent cells, i.e., they need a solid substrate to adhere and proliferate. The conventional procedure for expansion in static flasks normally involves a laborious process in which there is no suitable control and monitoring of the cultivation parameters besides presenting a higher susceptibility to contamination due to excessive manipulation to reach the ideal amount of cells. Moreover, this kind of cultivation does not allow a large scale production. For this reason, this work was proposed with the objective to develop a low cost, effective and scalable bioprocess for expansion of bone marrow-derived MSC in microcarriers. Cells grew on microcarriers Cyotdex 3, in spinner flasks with the -MEM medium supplemented with 15% FBS. We evaluated the cell adhesion to microcarriers, growth, metabolism, final cell recovery, and the functional and immunophenotypic properties before and after cultivation, comparing them with the cultivation already established in static flasks. In general, the results obtained showed that it was possible to expand MSC using microcarriers technology. The analysis of the cell metabolism showed that there was no depletion of important nutrients such as glucose and glutamine during cultivation, neither formation of lactate and ammonia subproducts in inhibitory concentrations. The cells recovered after the expansion kept the immunophenotypic and functional characteristics. The mean production (n=10) was about 4,9x105 cel/mL. As the system used allows the scale-up, if we had used a bioreactor of 1L it would had been possible to produce approximately 5x108 cells that would be enough to treat more than three patients of up to 70kg with a dose of 2x106 cells/kg. For the expansion of the same amount of cells in the traditional way, it would be necessary 135 T-flasks of 175 cm2 with total cost twice higher than the estimate cost of expansion using microcarriers.