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.

Doctor of Philosophy / Department of Animal Sciences and Industry / Duane Davis / Umbilical cords matrix stem cells (UCs) have been characterized most thoroughly in humans (HUCs) and are considered to have great promise for regenerative medicine and cell-based therapy. Although UCs were first identified in pigs the description of porcine UCs (PUCs) is limited. Here we reported some standard mesenchymal stem cell characteristics for PUCs. Development of knowledge about PUCs is useful because the pig is a valuable biomedical model for humans and the species is an important human food source. PUCs were isolated from Wharton’s jelly using an explant technique. They attached on the plastic and showed fibroblast-like morphology. Immunophenotype analysis showed they are positive for CD44, CD90 and CD105 and negative for CD31, CD45 and SLA-DR. Under specific in vitro conditions, PUCs were differentiated to adipocytes, chondrocytes and osteocytes. The growth curve of PUCs exhibited a lag phase, log phase and doubling time of 24, 60 and 13.8 hour respectively. Engraftment potential of allogeneic PUCs administered orally and intraperitoneally (IP) was evaluated. Newborn, 1-day, 1-week, 2-week and 3-week old pigs were administered a dose of fluorescently labeled PUCs (1.1x107 cells/kg body weight) and their tissue incorporation were evaluated using confocal microscopy with confirmation by PCR to detect SRY gene, the Y-chromosome gene of male PUCs in female recipients. One week after PUCs administration, they were found mostly in the gastrointestinal tract and abdominal organs after either oral or intraperitoneal transplantation. The intestinal mucosa layer around the base of villi and intestinal crypts was the main location. PUCs were also detected in thoracic organs, muscle and bone marrow. Additionally, PKH26-labeled fibroblasts labeled were detected in recipient intestine 1 week after IP injection. Donor cells were not found in blood at one week post transplantation. When recipients were sacrificed at 6 h after IP injection PKH26-labeled PUCs were found mostly in omentum and diaphragm by PCR. It is likely these are the primary sites for donor cells in the peritoneal cavity to enter the circulation. Fluorescent in situ hybridization (FISH), using an SRY probe and PCR, demonstrated the PUCs isolated from recipient intestines by enzymatic digestion. Therefore, transplanted PUCs were recovered from the intestinal mucosa and were viable and able to proliferate in vitro.

Diabetes mellitus (DM) designa um conjunto de patologias devidas à falta ou ação deficiente da insulina. O DM1 é causado por ataque auto-imune às células β-pancreáticas secretoras de insulina, enquanto DM2 é relacionado com idade e obesidade. Alotransplante de pâncreas ou de ilhotas são alternativas terapêuticas, mas escassez de órgãos e necessidade de imunossupressão são obstáculos importantes. Células-tronco, isoladas da massa interna de blastocistos e de tecidos adultos, são capazes de auto-renovação e diferenciação, podendo ser utilizadas para o tratamento de doenças. Os objetivos deste trabalho foram: a) buscar novos genes envolvidos no processo de diferenciação de células tronco em células secretoras de insulina e b) isolar e caracterizar células-tronco mesenquimais humanas. Células-tronco embrionárias murinas foram induzidas a se diferenciar em clusters similares a ilhotas. O RNA foi utilizado para sondar microarrays de DNA (CodeLink) e os genes diferencialmente expressos foram confirmados por Q-PCR, sendo possível identificar 16 novos genes associados à diferenciação. Dois tipos diferentes de células-tronco mesenquimais foram isoladas da veia (MSC) e do sangue (UCB) de cordão umbilical humano. Imunofenotipagem e caracterização molecular por Q-PCR, apontaram para a existência de dois tipos diferentes de progenitoras mesenquimais adultas no cordão umbilical humano. / Diabetes mellitus (DM) defines a number of pathologies caused by the lack or deficient action of the insulin hormone. DM1 is caused by an auto-immune attack to insulin secreting β-pancreatic cells, while DM2 is related to ageing and obesity. Pancreas and islet allo-transplantation constitute therapeutic alternatives, but severe organ shortage and the absolute requirement for immunossupression still constitute important obstacles. Stem cells isolated from the blastocist inner cell mass and from adult tissues are capable of self-renewal and differentiation, and may be utilized for treatment of several diseases. The objectives of this work were: a) to search for new genes involved in the process of differentiation of stem cells into insulin-secreting cells and b) to isolate and characterize mesenchymal stem cells. Murine embryonic stem cells were induced to differentiate in islet-like clusters. Total RNA was utilized to probe DNA microarrays (CodeLink) and the differentially expressed genes were confirmed by Q-PCR, with 16 new genes being identified as associated with differentiation. Two different types of mesenchymal stem cells were isolated from human umbilical cord vein (MSC) and blood (UCB). Immunophenotyping and molecular characterization by Q-PCR, pointed to the existence of two different types of progenitor mesenchymal stem cells in human umbilical cord.

Objetivo: Calcular os valores preditivos da ultrassonografia de primeiro trimestre gestacional para a detecção da artéria umbilical única. Avaliar a relação dos marcadores ultrassonográficos de cromossomopatias do primeiro trimestre (translucência nucal, osso nasal e ducto venoso), além da idade gestacional do exame, CCN, sexo fetal, medida da bexiga fetal, alterações de morfologia e IMC da gestante, com a acurácia do diagnóstico no primeiro trimestre. Métodos: Estudo longitudinal prospectivo envolvendo 1.564 gestantes, que foram submetidas à ultrassonografia com avaliação do cordão umbilical entre 11 e 13 semanas e 6 dias, no período de novembro de 2007 a setembro de 2009. Posteriormente, realizaram a avaliação do cordão umbilical em ultrassonografia realizada no segundo ou terceiro trimestres. Foi verificada a concordância do diagnóstico de AUU no primeiro trimestre com o diagnóstico no segundo trimestre, calculando-se o coeficiente Kappa. Os testes qui-quadrado e exato de Fisher foram utilizados para verificar a existência de associação entre a acurácia da ultrassonografia de primeiro trimestre e as variáveis da ultrassonografia e da gestante (translucência nucal, osso nasal, ducto venoso, idade gestacional do exame, CCN, sexo fetal, medida da bexiga fetal, alterações de morfologia e IMC da gestante). Resultados: A concordância dos diagnósticos de AUU no primeiro e segundo trimestres foi moderada (Kappa = 0,609), sendo que a sensibilidade da ultrassonografia de primeiro trimestre em relação à ultrassonografia de segundo trimestre foi de 76%, a especificidade foi de 99%, o valor preditivo positivo foi de 51,6% e o valor preditivo negativo foi de 99,6%. A acurácia foi de 98,7%. Dentre as variáveis analisadas, que poderiam ter influenciado na acurácia da ultrassonografia de primeiro trimestre na detecção de AUU, a única que se mostrou estatisticamente significante foi o sexo fetal. Conclusão: A sensibilidade da ultrassonografia de primeiro trimestre na detecção da AUU é de 76%, o que é menor do que a observada no segundo ou terceiro trimestres. / Objective: To calculate the predictive values of first gestational trimester ultrasonography for detection of single umbilical artery. Assess the relation of ultrasound markers of chromosomal disease in the first trimester (nuchal translucency, nasal bone and ductus venosus) in addition to gestational age at exam, CRL, fetal gender, measurement of fetal bladder, morphological alterations and BMI of a pregnant woman, with accuracy of diagnosis in the first trimester. Methods: A prospective longitudinal study was conducted from November 2007 to September 2009 encompassing 1564 pregnant women submitted to ultrasound imaging for umbilical cord assessment between the 11 and 13 weeks and six days. Later they underwent evaluation of the umbilical cord by ultrasound performed in the second or third trimesters. Consistency of SUA diagnosis in the first trimester was verified with that of the second trimester by calculating the Kaplan coefficient. The Chi-square and Fisher\'s exact tests were used to verify if there was an association between accuracy of ultrasonography of the first trimester and the variables of ultrasonography and those of the pregnant woman (nuchal translucency, nasal bone, ductus venosus, gestational age at exam, CRL, fetal gender, measurement of fetal bladder and morphological alterations as well as pregnant woman\'s BMI). Results: SUA diagnoses in the first and second trimester disclosed moderate consistency (Kaplan=0.609) while sensitivity of first trimester ultrasound in relation to that of the second trimester was of 76% and specificity was of 99%, positive predictive value was of 51.6% and negative predictive value was of 99.6%. Accuracy was of 98.7%. Among the analyzed variables, fetal gender was the only one with a statistical significance that might bear influence on first trimester ultrasound accuracy for detection of SUA. Conclusion: Sensitivity of the first trimester ultrasound for detection of SUA is of 76%, that is to say, lower than that observed in the second or third trimesters

O Magnésio é o segundo cátion intracelular mais comum e desempenha importante papel na modulação de funções de transporte e receptores, atividades enzimáticas, metabolismo energético, síntese de proteínas e ácidos nucleicos e proteção de membranas biológicas. Apesar de sua importância, o conhecimento de sua homeostase não é completo, principalmente por dificuldade de acesso a seus estoques intracelulares e da ausência de métodos laboratoriais confiáveis para medida da fração iônica. O desenvolvimento recente de um eletrodo íon-seletivo permitiu a determinação das concentrações de Mg iônico(Mgi), em pequenas amostras de sangue, o que possibilitou a realização de estudos para determinação desta fração no período neonatal. A presença de alguns distúrbios, como o Crescimento Intra-uterino Restrito(CIUR) e a Asfixia Perinatal, poderiam potencialmente levar a desvios da homeostase do Mg, ainda não totalmente esclarecidos. O objetivo deste estudo foi descrever, em Recém-nascidos de termo(RNT) sem CIUR, os níveis de Mgi e total (MgT) em sangue de cordão umbilical, 3o e 7o dias de vida e comparar os valores obtidos entre os RNT, com e sem CIUR e asfixia perinatal. Realizou-se um estudo prospectivo, no qual foram incluídos 95 RNT, divididos em dois grupos de estudo: Grupo I - sem CIUR(50 RN - 52,6%) e Grupo II - com CIUR(45 RN - 47,4%). A presença de CIUR foi determinada por um peso de nascimento abaixo do percentil 10 para a curva de Ramos(1983), associado a uma relação P/P50 < 0,85. Cada um desses grupos foi subdividido em 2 subgrupos : Grupo Ia - 30 RN (31,6%), sem CIUR e sem asfixia perinatal; Grupo Ib - 20 RN(21,0%), sem CIUR e com asfixia perinatal; Grupo IIa - 40 RN(42,1%), com CIUR e sem asfixia perinatal; Grupo IIb - 5 RN(5,3%), com CIUR e asfixia perinatal. A presença de asfixia perinatal foi indicada por um Apgar de 5o minuto < 6 associada a presença de um dos seguintes critérios: pH de sangue de cordão umbilical < 7,2 , disfunção de um ou mais órgãos, sequelas neurológicas no período neonatal imediato. Foram realizadas determinações de Mgi, Cálcio iônico(Cai), Uréia(U), pH, MgT, Fósforo(P) e Creatinina(Cr), em sangue de cordão umbilical, no 3o e no 7o dias de vida. Verificou-se que nos RNT sem CIUR(Grupo Ia), as concentrações médias de MgT, ao nascimento, foram menores do que as de RN com CIUR e elevaram-se, de forma significante, até o 7o dia de vida, enquanto as de Mgi mantiveram-se. As concentrações de Mgi neste grupo, foram significativamente menores do que as de RN com CIUR(Grupo IIa) durante a 1a semana de vida e do que as de RN com asfixia perinatal(Grupo Ib) no 3o e 7o dias de vida. Concluiu-se que, em RNT sem CIUR, há um aumento dos níveis de MgT durante a 1a semana de vida, sem alteração das concentrações de Mgi. A presença de CIUR, bem como a asfixia perinatal, podem influenciar as concentrações neonatais de Mg, através de seus efeitos de modulação da homeostase deste íon, durante os períodos fetal e neonatal / Magnesium is the second most abundant intracellular cation and plays an important role in regulation of transporting and receptors functions, enzymatic activities, energy metabolism, protein and nucleic acid synthesis and biologic membranes protection. In spite of this, the knowledge of its homeostasis is still limited, mainly due to inacessibility of its intracellular stores and the absence of a reliable methodology to measuring the ionized fraction. The recent development of an ion-selective electrode has allowed not only the determination of ionized magnesium(iMg) concentrations in a small blood sample volume, but also an increasing number of researches as to this fraction in neonatal period. The presence of some disorders,i.e. like Intrauterine Growth Restriction (IUGR) and Perinatal Asphyxia, could lead to an unclear imbalance of magnesium homeostasis, in a way not yet clear. The aim of this study was to describe, in term newborns without IUGR, iMg and Total Mg (TMg) concentrations in umbilical cord blood, third and seventh days of life and to compare the results among term newborns with and without IUGR and perinatal asphyxia. Ninety-five term newborn infants were enrolled in a prospective study and were divided into two study groups: Group I : without IUGR(50RN - 52.6%) and Group II - with IUGR(45RN - 47.4%). Intrauterine growth restriction was defined as a birth weight below the 10th percentil for Ramos Curve(1983) besides to a birth weight ratio <0,85. Each one of these groups were divided in two subgroups: Group Ia :30 RN (31,6%), without IUGR or perinatal asphyxia; Group Ib : 20 RN (21,0%), without IUGR, with perinatal asphyxia ; Group IIa : 40 RN (42,1%), with IUGR, without perinatal asphyxia; Group IIb: 5 RN(5,3%), with perinatal asphyxia and IUGR. Perinatal asphyxia was defined as a 5 minutes Apgar score < 6 besides to one of the following: umbilical cord blood pH < 7,2, disfunction of one or more organs, neonatal neurologic manifestations. iMg, TMg, ionized calcium, urea, pH, phosphorus and creatinine concentrations were determined in umbilical cord blood, third and seventh days of life. We observed that in term newborns without IUGR (Group Ia), TMg concentrations increased significantly during the first week of life, while iMg concentrations remained unchanged. iMg levels in this group, were significantly lower than in the group with IUGR (Group IIa) from birth to 7th day of life and than in the group without IUGR, with perinatal asphyxia (Group Ib) in the third and seventh days of life. We concluded that in term newborns without IUGR, TMg levels increased during the first week of life, while iMg levels remained unchanged. The presence of IUGR, as well as, perinatal asphyxia, may influence neonatal levels of magnesium, through their effect on the modulation of this ion homeostasis, during fetal and neonatal periods

Les cellules souches suscitent de grands espoirs pour la thérapie cellulaire et l'ingénierie tissulaire. Les CSM du tissus foetaux (sang placentaire et gelée de Wharton du cordon ombilical), à l'origine d'épiblaste embryonnaire, sont considérées comme plus primitives que les CSM provenant de sources adultes. Les conditions de culture ayant un impact sur le comportement des cellules, dans notre étude, nous avons exploré l'effet de la concentration de l'oxygène sur l'expansion, l'immunophénotypage et la différenciation de ces cellules. L'objectif de ce travail est d'identifier la méthode optimale d'isolation des CSM issues de tissus foetaux. Compte tenu du faible taux de succès dans l'isolement des CSM extraites du sang placentaire, nous nous sommes dirigés vers les CSM-GW. Nous y avons déterminé in situ, les marqueurs spécifiques exprimés dans la gelée de Wharton et à la périphérie. Des études sur la morphologie, la cinétique de croissance, et sur l'expression phénotypique des marqueurs de surface, des CSM-GW, ont été effectuées sur une longue durée (7 passages) à différentes conditions de culture. Nous avons montré que la GW est composée d'une abondante matrice extracellulaire riche en collagènes et glycosaminoglycannes et que les cellules possèdent un phénotype variable selon leur localisation dans la gelée. Ce tissu est capable de fournir une quantité importante de CSM (6,7x105 Cs/cm de cordon) qui gardent une morphologie constante. Enfin, quel que soit le passage, la concentration de l'oxygène ne semble pas avoir d'effet sur le phénotype des cellules. En revanche, une faible teneur en oxygène durant l'expansion semble diminuer le temps de doublement des cellules, favoriser la chondrogénèse et inhiber la différenciation ostéogénique. Enfin, quelles que soient les conditions de culture, la différenciation adipogénique des CSM-GW semble difficile à obtenir / Stem cells are the hopes for cell therapy and tissue engineering. MSCs from fetal tissue (umbilical cord blood and WJ), which are a source of embryonic epiblast grow relatively faster comparing to other adult sources. The culture condition can affect cell behavior. In our study, we explored the effect of oxygen concentration on the expansion, immunophenotyping, and differentiation of these cells. The aim of this work is to identify the optimal method for isolation of MSCs derived from fetal tissue. Given the low rate of success in the isolation of MSCs from cord blood, we headed to WJ-MSCs. We have determined in siu, the specific markers expressed in the WJ and in the perivascular region. Studies on the morphology growth kinetics, and phenotypic expression of surface makers of MSCs isolated from WJ were made over a long period (7 passages) in different culture conditions. We have shown that WJ is composed of an abundant extracellular matrix rich in collagen and glycasominoglycans and have variable phenotype depending from their localization in the jelly. This tissue is able to provide a large amount of MSCs (6.7x105 Cs/cm of cord) that maintain a constant morphology. Finally, regardless of the passage, the oxygen concentration does not effect on the phenotype of the cells. In contrast, a low oxygen concentration during expansion appears to decrease the doubling time of MSCs, promote chondrogenesis and inhibit osteogenic differentiation. Finally, whatever the culture conditions, adipogenic differentiation of WJ-MSC seems difficult to obtain

RESUMO BALDISSERA, J.L.C. Avaliação das diferentes metodologias de realização do ensaio clonogênico e validação do método de criopreservação e ressuspensão do sangue de cordão umbilical e placentário criopreservado. 2015, 78 f. Dissertação. Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2015. O sangue de cordão umbilical e placentário (SCUP) tem sido utilizado como fonte de célulastronco hematopoéticas (CTHs) para transplante. A qualidade desse produto pode ser afetada durante as várias etapas do seu processamento. Neste estudo, foi avaliada a melhor metodologia de preparo da amostra para a realização do ensaio clonogênico (pura, diluída ou lavada) e validado o método de criopreservação e de ressuspensão das bolsas de SCUP. Foi avaliada também a funcionalidade da enzima aldeído desidrogenase (ALDH) como método para determinar a função das CTHs do SCUP, em 15 unidades criopreservadas pelo Laboratório de Criobiologia e Terapia Celular do Centro de Hematologia e Hemoterapia de Santa Catarina (HEMOSC). As unidades foram descongeladas em quatro etapas. O conteúdo dos segmentos e da bolsa foi coletado e ressuspenso com solução de albumina 5%, ACD 5% e solução fisiológica. A suspensão celular obtida foi utilizada para realização do ensaio clonogênico, avaliação da viabilidade celular, quantificação das células nucleadas (CN), CD34+ e das ALDH br . Os parâmetros tempo, custo e o resultado do ensaio clonogênico, utilizados para avaliar a metodologia, indicaram que a suspensão celular diluída é o melhor método a ser utilizado para a realização do ensaio clonogênico. A quantificação das CN e das células CD34+ totais pré-criopreservação e pós-criopreservação/descongelamento foi 8,3 (±1,9) x 10 8 e 8,2 (±2,0) x 10 8 (p = 0,3388) e 3,3 (±2,7) x 10 6 e 3,2 (±2,1) x10 6 (p = 0,4455), respectivamente. A quantificação das CN e das células CD34+ viáveis pré-criopreservação e pós-criopreservação/descongelamento foi 8,1 (±1,9) e 6,3 (±1,7) x 10 8 (p < 0,0001) e 3,27 (±2,0) x 10 6 e 2,8 (±1,8) x 10 6 (p = 0,0063), respectivamente. A porcentagem de células nucleadas e CD34+ viáveis no segmento proximal e na bolsa de 20 mL foi, respectivamente, 66,3 (±11,8) e 75 (35-93); 76,5 (±11,6) e 89 (75-100). No ensaio clonogênico foi observado crescimento médio de 31,8 (±7,6) unidades formadoras de colônias granulócito-macrófago (CFU-GM) x 10 5 CN plaqueadas obtidas da bolsa pós-criopreservação/descongelamento. Não foi encontrada correlação entre as células ALDH br /CD45 + viáveis e a quantificação das CFUGM ou das células CD34+ viáveis da bolsa pós-criopreservação/descongelamento. O coeficiente de correlação entre as células nucleadas e as células ALDEFLUOR bright da bolsa e do segmento pós-criopreservação/descongelamento foi (r) = 0,9399 com p < 0,0001 e (r) = 0,5478 com p = 0,0426, respectivamente. Foi encontrada correlação entre quantificação das células CD34+ e das CFU-GM da bolsa e do segmento pós-criopreservação/descongelamento. Esses dados indicam que o método utilizado para a realização da criopreservação e o descongelamento das unidades de SCUP encontra-se validado, e que o segmento pode ser utilizado como uma ferramenta de controle de qualidade para a seleção da unidade de SCUP para transplante. Palavras-chave: Validação. Sangue de cordão umbilical e placentário. Aldefluor. Célulastronco hematopoéticas. / ABSTRACT BALDISSERA, J.L.C. Evaluation of different methods of performing clonogenic assay and validation of the method of cryopreservation and resuspension of cryopreserved umbilical cord and placental blood. 2015. 78 f. Master Dissertation. Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2015. Umbilical cord and placental blood (UCPB) has been used as a source of hematopoietic stem cells (HSCs) for transplant. The quality of this product may be affected during the various stages of processing it. In this study, the author reviewed the best preparation methodology for performing the clonogenic assay (pure, diluted or washed) and validated the method of cryopreservation and resuspension of UCPB bags. The author also evaluated the functionality of the aldehyde dehydrogenase enzyme (ALDH) as a method to determine the function of umbilical cord and placental blood HSCs, in 15 cryopreserved units by the Laboratory of Cryobiology and Cell Therapy of the Center for Hematology and Hemotherapy of Santa Catarina (HEMOSC). The units were thawed in four steps. The content of the segments and the bag was collected and resuspended in a 5% albumin solution, 5% acid ci trate dextrose and saline solution. The cell suspension obtained was used to conduct the clonogenic assay, the assessment of cell viability, the quantification of nucleated cells (NC), CD34 + and ALDH br . The parameters of time, cost and the result of the clonogenic assay, used to evaluate the methodology, indicated that the diluted cell suspension is the best method to be used when performing a clonogenic assay. The quantification of the nucleated cells (NC) and the total CD34+ cells pre-cryopreservation and post-cryopreservation/thawing was 8,3 (±1,9) x 10 8 and 8,2 (±2,0) x 10 8 (p = 0,3388) and 3,3 (±2,7) x 10 6 and 3,2 (±2,1) x10 6 (p = 0,4455), respectively. The quantification of the NC and CD34+ viable cells pre-cryopreservation and post-cryopreservation/thawing was 8,1 (±1,9) and 6,3 (±1,7) x 10 8 (p < 0,0001) and 3,27 (±2,0) x 10 6 and 2,8 (±1,8) x 10 6 (p = 0,0063), respectively. The percentage of viable nucleated cells and CD34+ viable cells in the proximal segment and in the 20mL bag was 66,3 (±11,8) and 75 (35-93); 76,5 (±11,6) and 89 (75-100), respectively. In the clonogenic assay an average growth of 31,8 (± 7,6) colony-forming granulocyte-macrophage units (CFUGM) x 10 5 NC plated, obtained from the post-cryopreservation/thawing bag was observed. No correlation between the ALDH br /CD45 + viable cells and the quantification of CFU-GM or CD34+ viable cells obtained from the bag post cryopreservation was found. The coefficient of correlation between nucleated cells and ALDEFLUOR bright cells from the bag and segment after cryopreservation were (r) = 0, 9399 with p < 0, 0001 and (r) = 0, 5478 with p = 0,0426, respectively. A correlation between quantification of CD34+ cells and CFU-GM bag and segment cells after cryopreservation/thawing was found. This data indicates that the method used to perform the cryopreservation and thawing of the UCPB unit has been validated, and that the segment can be used as a tool for quality control when making the selection of UCPB for transplant. Keywords: Validation. Umbilical cord and placental blood. Aldefluor. Hematopoietic stem cells.

Introdução: A célula NK possui uma importante função no sistema imune inato de defesa primária contra vírus e patógenos e também realiza a imunovigilâcia tumoral. Muitos estudos clínicos tem avaliado o uso dessas células na imunoterapia adotiva. A expansão e a ativação da célula NK requer sinais e estímulos para manter a sua sobrevivência. Atualmente existem muitos protocolos para a expansão e ativação da célula NK, porém não existe uma definição do melhor método para uso clínico. Objetivo: O estudo tem como objetivo avaliar a melhor forma para expansão das células NK isoladas de células mononucleares do sangue de cordão umbilical e placentário.Método: Foram avaliadas cinco diferentes condições para expansão de células NK de mononucleares isoladas do sangue do cordão umbilical e placentário. Foram testados protocolos utilizando as interleucinas (IL), IL-2, IL-3, IL-15; com ou sem a presença do co-cultivo com células-tronco mesenquimais do cordão umbilical (CTM-CU) e, também o co-cultivo com células apresentadoras de antígeno artificiais ligadas a IL-21 à membrana (mbIL21 APC). Resultados: Os protocolos utilizando co-cultivo com APC mbIL21 foram superiores aos demais quanto à capacidade de expansão de células NK (CD3-, CD56+, CD16+). O protocolo de co-cultura de APC, CTM-CU e estímulo com IL-2 apresentaram um aumento significativo de NK (CD3-, CD56+, CD16+) quando comparado ao protocolo de APC/IL-2 sem CTM-CU (p<0,05). Conclusão: A expansão ex vivo de células NK na presença das APC e CTM-CU apresentaram uma proporção estatisticamente superior de célula NK CD16+ quando comparada com condições de cultivo com apenas a APC, tendo essas células NK potencial para utilização na imunoterapia adotiva associada com anticorpos monoclonais ou anticorpos bi-específicos. / Background: Natural killer (NK) cells play a major role in innate immunity, especially against viral pathogens, and are also a part of the immune surveillance of tumors. Several clinical trials have evaluated the use of these cells for adoptive cell immunotherapy. Ex vivo expansion of NK cells, however, is a complex process which requires multiple cell signals to ensure cell survival, proliferation, and activation. There are many protocols used for NK cell expansion and activation, however, there is a lack of evidence regarding which method is the most effective for clinical grade NK cells expansion. Objective: The main purpose of this study is to evaluate an optimal protocol for the ex vivo expansion of NK cells isolated from umbilical cord blood mononuclear cells (CB-MNC). Methods: Five different conditions for the expansion of umbilical cord-derived NK cells were evaluated. Each protocol was a different combination of interleukins (IL-2, IL-3, and IL-15) with or without the presence of feeder cells or artificial antigen presenting cells (aAPCs). Feeder cells utilized were umbilical cord-derived mesenchymal stem cells (UC-MSC), and aAPCs were membrane-bound IL-21 artificial APCs (mbIL21 aAPCs). Results: Protocols employing mbIL21 aAPCs demonstrated greater expansion of natural killer cells (CD3- CD56+) than the other protocols. The protocol employing aAPCs, IL-2 and UC-MSC feeder cells had a statistically significant higher proportion of CD16+ NK cells when compared to the protocol without the MSC feeder cells, but there was no significant difference in the expansion of total natural killer cells concerning these two protocols. Conclusion: Ex vivo expansion of NK cells in the presence of aAPCs and UC-MSC feeder cells yielded a significant higher proportion of CD16+ NK when compared to the aAPCs only culture condition, and could be a better product for NK adoptive immunotherapy in conjunction with monoclonal or bi-specific antibodies.

A injúria renal aguda representa um estado de senescência precoce induzida por estresse, e as células-tronco mesenquimais podem ser uma alternativa para seu tratamento. Células-tronco jovens reduzem o fenótipo de envelhecimento em rins quando comparadas a células idosas. O objetivo deste estudo foi avaliar se o tratamento com jovens células-tronco mesenquimais derivadas de cordão umbilical humano podem interferir na senescência renal induzida por lesão de isquemia-reperfusão em ratos. Ratos machos foram submetidos ao modelo de isquemia de artérias renais bilateralmente por 45 minutos, com reperfusão após, e alguns animais receberam 1 X 106 células por via intraperitoneal após 6 horas da indução da lesão. Os animais foram eutanasiados no segundo ou no sétimo dia pós-isquêmico. No segundo dia após a lesão de isquemia-reperfusão, o tratamento com as células melhorou a filtração glomerular e a função tubular, melhorou a expressão renal de aquaporina-2 e reduziu a infiltração de macrófagos nos rins. Proteínas relacionadas à senescência (-galactosidase, p21, p16 e fator de transformação do crescimento ) e microRNAs (mir-29a e miR-34a) estiveram com a expressão aumentada após a isquemia-reperfusão, e houve redução nesses parâmetros com o tratamento. A redução na expressão de Klotho e o estado pró-oxidativo gerados pela isquemia-reperfusão também foram revertidos pelo tratamento. A senescência induzida pela injúria renal aguda é um processo independente de telômeros. Ao sétimo dia pós-lesão, os ratos isquêmicos mantinham defeito de concentração urinária, que foi revertido nos animais tratados. Além disso, o tratamento reduziu o índice de necrose tubular aguda em tecido renal e reduziu o infiltrado macrofágico túbulo-intersticial. O marcador pró-senescência p16 foi completamente restabelecido nos animais tratados. Nossos dados demonstram que o tratamento com jovens células-tronco mesenquimais derivadas de cordão umbilical humano atenua a resposta inflamatória e de estresse oxidativo que ocorre na injúria renal aguda, e reduz a expressão de proteínas e microRNAs relacionados à senescência. Nossos achados expandem as perspecivas para o tratamento da injúria renal aguda / Acute kidney injury represents a status of premature stress-induced senescence, and mesenchymal stem cells are an alternative for treatment. Young stem cells reduce aging phenotype in kidneys when compared to old cells. The objective of this study was to evaluate if treatment with young human umbilical cord mesenchymal stem cells could interfere in kidney senescence induced by renal ischemia-reperfusion in rats. Male rats were induced to ischemia-reperfusion injury by 45-minutes clamping of both renal arteries; some rats received 1X106 cells intraperitonally six hours later. Rats were euthanatized on post-renal ischemia reperfusion days two and seven. At day 2 after ischemia-reperfusion injury, treatment with cells improved glomerular filtration, tubular function, improved renal expression of aquaporin 2 and decreased macrophage kidney infiltration. Senescence-related proteins (?-galactosidase, p21, p16 and transforming growth factor ?) and microRNAs (miR-29a and miRNA-34a) were overexpressed after ischemia-reperfusion, and reversed by the treatment. The Klotho reduced expression and the pro-oxidative status induced by ischemia-reperfusion were reversed by the treatment. Senescence induced acute kidney injury is a telomere-independent process. At day 7, ischemic rats maintained urinary concentrating defect, which is reversed in treated animals. Moreover, treatment decreased the index of acute tubular necrosis in kidney tissue and decreased macrophage kidney infiltration. Senescence marker p16 was completely restored in treated animals. Our data demonstrate that young human umbilical mesenchymal stem cells treatment attenuates the inflammatory and oxidative stress response occurring in acute kidney injury, and reduces the protein and microRNA expression related to senescence. Our findings broaden the perspectives for the treatment of AKI

Objetivo: Calcular os valores preditivos da ultrassonografia de primeiro trimestre gestacional para a detecção da artéria umbilical única. Avaliar a relação dos marcadores ultrassonográficos de cromossomopatias do primeiro trimestre (translucência nucal, osso nasal e ducto venoso), além da idade gestacional do exame, CCN, sexo fetal, medida da bexiga fetal, alterações de morfologia e IMC da gestante, com a acurácia do diagnóstico no primeiro trimestre. Métodos: Estudo longitudinal prospectivo envolvendo 1.564 gestantes, que foram submetidas à ultrassonografia com avaliação do cordão umbilical entre 11 e 13 semanas e 6 dias, no período de novembro de 2007 a setembro de 2009. Posteriormente, realizaram a avaliação do cordão umbilical em ultrassonografia realizada no segundo ou terceiro trimestres. Foi verificada a concordância do diagnóstico de AUU no primeiro trimestre com o diagnóstico no segundo trimestre, calculando-se o coeficiente Kappa. Os testes qui-quadrado e exato de Fisher foram utilizados para verificar a existência de associação entre a acurácia da ultrassonografia de primeiro trimestre e as variáveis da ultrassonografia e da gestante (translucência nucal, osso nasal, ducto venoso, idade gestacional do exame, CCN, sexo fetal, medida da bexiga fetal, alterações de morfologia e IMC da gestante). Resultados: A concordância dos diagnósticos de AUU no primeiro e segundo trimestres foi moderada (Kappa = 0,609), sendo que a sensibilidade da ultrassonografia de primeiro trimestre em relação à ultrassonografia de segundo trimestre foi de 76%, a especificidade foi de 99%, o valor preditivo positivo foi de 51,6% e o valor preditivo negativo foi de 99,6%. A acurácia foi de 98,7%. Dentre as variáveis analisadas, que poderiam ter influenciado na acurácia da ultrassonografia de primeiro trimestre na detecção de AUU, a única que se mostrou estatisticamente significante foi o sexo fetal. Conclusão: A sensibilidade da ultrassonografia de primeiro trimestre na detecção da AUU é de 76%, o que é menor do que a observada no segundo ou terceiro trimestres. / Objective: To calculate the predictive values of first gestational trimester ultrasonography for detection of single umbilical artery. Assess the relation of ultrasound markers of chromosomal disease in the first trimester (nuchal translucency, nasal bone and ductus venosus) in addition to gestational age at exam, CRL, fetal gender, measurement of fetal bladder, morphological alterations and BMI of a pregnant woman, with accuracy of diagnosis in the first trimester. Methods: A prospective longitudinal study was conducted from November 2007 to September 2009 encompassing 1564 pregnant women submitted to ultrasound imaging for umbilical cord assessment between the 11 and 13 weeks and six days. Later they underwent evaluation of the umbilical cord by ultrasound performed in the second or third trimesters. Consistency of SUA diagnosis in the first trimester was verified with that of the second trimester by calculating the Kaplan coefficient. The Chi-square and Fisher\'s exact tests were used to verify if there was an association between accuracy of ultrasonography of the first trimester and the variables of ultrasonography and those of the pregnant woman (nuchal translucency, nasal bone, ductus venosus, gestational age at exam, CRL, fetal gender, measurement of fetal bladder and morphological alterations as well as pregnant woman\'s BMI). Results: SUA diagnoses in the first and second trimester disclosed moderate consistency (Kaplan=0.609) while sensitivity of first trimester ultrasound in relation to that of the second trimester was of 76% and specificity was of 99%, positive predictive value was of 51.6% and negative predictive value was of 99.6%. Accuracy was of 98.7%. Among the analyzed variables, fetal gender was the only one with a statistical significance that might bear influence on first trimester ultrasound accuracy for detection of SUA. Conclusion: Sensitivity of the first trimester ultrasound for detection of SUA is of 76%, that is to say, lower than that observed in the second or third trimesters