Mesenchymal stromal cells derived from exfoliated deciduous teeth express neuronal markers before differentiation induction

Abstract Objective: This study aimed to evaluate neuronal markers in stromal cells from human exfoliated deciduous teeth (SHED) and standardize the isolation and characterization of those cells. Methodology: Healthy primary teeth were collected from children. The cells were isolated by enzymatic digestion with collagenase. By following the International Society for Cell and Gene Therapy (ISCT) guidelines, SHED were characterized by flow cytometry and differentiated into osteogenic, adipogenic, and chondrogenic lineages. Colony-forming unit-fibroblasts (CFU-F) were performed to assess these cells' potential and efficiency. To clarify the neuronal potential of SHED, the expression of nestin and βIII-tubulin were examined by immunofluorescence and SOX1, SOX2, GFAP, and doublecortin (DCX), nestin, CD56, and CD146 by flow cytometry. Results: SHED showed mesenchymal stromal cells characteristics, such as adhesion to plastic, positive immunophenotypic profile for CD29, CD44, CD73, CD90, CD105, and CD166 markers, reduced expression for CD14, CD19, CD34, CD45, HLA-DR, and differentiation in three lineages confirmed by staining and gene expression for adipogenic differentiation. The average efficiency of colony formation was 16.69%. SHED expressed the neuronal markers nestin and βIII-tubulin; the fluorescent signal intensity was significantly higher in βIII-tubulin (p<0.0001) compared to nestin. Moreover, SHED expressed DCX, GFAP, nestin, SOX1, SOX2, CD56, CD146, and CD271. Therefore, SHED had a potential for neuronal lineage even without induction with culture medium and specific factors. Conclusion: SHEDs may be a new therapeutic strategy for regenerating and repairing neuronal cells and tissues.

Management of Airway Remodeling in a Mouse Model of Allergic Airways Inflammation Using Extracellular Vesicles from Human Bone Marrow-Derived Mesenchymal Stromal Cells

Abstract: Asthma is a chronic respiratory disease affecting 300 million people worldwide. It results in several structural changes in the airways, which are minimally accessible in clinical practice. Cell therapy using mesenchymal stromal cells (MSCs) is a promising strategy for treating asthma due to the paracrine activity of MSCs, which influences tissue regeneration and modulates the immune response. Studies using extracellular vesicles (EV) released by MSCs have demonstrated their regenerative properties in animal models. The aim of this study was to evaluate the potential of EVs isolated from human bone marrow MSCs (hBM-MSCs) to control lung tissue remodeling in ovalbumin-induced allergic asthma in Balb/c mice. We isolated hBM-MSCs from a single donor, expanded and characterized them, and then isolated EVs. Asthma was induced in 43 male Balb/c mice, divided into four groups: control, asthmatic (AS), asthmatic plus systemic EVs (EV-S), and asthmatic plus intratracheal EVs (EV-IT). Upon completion of asthma induction, animals were treated with EVs either locally (EV-IT) or intravenously (EV-S). Seven days after, we performed bronchoalveolar lavage (BAL) and the total nuclear cells were counted. The animals were euthanized, and the lungs were collected for histopathological analysis of the airways. The EV-S group showed improvement in only the total BAL cell count compared with the AS group, while the EV-IT group showed significant improvement in almost all evaluated criteria. Therefore, we demonstrate that the local application of EVs derived from hBM-MSCs may be a potential treatment in controlling asthma.

Adipose tissue-derived stromal/stem cells + cholecalciferol: a pilot study in recent-onset type 1 diabetes patients

ABSTRACT Objective: Adipose tissue-derived stromal/stem cells (ASCs) and vitamin D have immunomodulatory actions that could be useful for type 1 diabetes (T1D). We aimed in this study to investigate the safety and efficacy of ASCs + daily cholecalciferol (VIT D) for 6 months in patients with recent-onset T1D. Materials and methods: In this prospective, dual-center, open trial, patients with recent onset T1D received one dose of allogenic ASC (1 x 106 cells/kg) and cholecalciferol 2,000 UI/day for 6 months (group 1). They were compared to patients who received chol-ecalciferol (group 2) and standard treatment (group 3). Adverse events were recorded; C-peptide (CP), insulin dose and HbA1c were measured at baseline (T0), after 3 (T3) and 6 months (T6). Results: In group 1 (n = 7), adverse events included transient headache (all), mild local reactions (all), tachycardia (n = 4), abdominal cramps (n = 1), thrombophlebitis (n = 4), scotomas (n = 2), and central retinal vein occlusion at T3 (n = 1, resolution at T6). Group 1 had an increase in basal CP (p = 0.018; mean: 40.41+/-40.79 %), without changes in stimulated CP after mixed meal (p = 0.62), from T0 to T6. Basal CP remained stable in groups 2 and 3 (p = 0.58 and p = 0.116, respectively). Group 1 had small insulin requirements (0.31+/- 0.26 UI/kg) without changes at T6 (p = 0.44) and HbA1c decline (p = 0.01). At T6, all patients (100%; n = 7) in group 1 were in honeymoon vs 75% (n = 3/4) and 50% (n = 3/6) in groups 2 and 3, p = 0.01. Conclusions: Allogenic ASC + VIT D without immunosuppression was safe and might have a role in the preservation of β-cells in patients with recent-onset T1D. ClinicalTrials.gov: NCT03920397.

Padronização de queimaduras térmicas em ratos por contato direto com a superfície cutânea / Standardization of thermal burns in rats by direct contact with the skin surface / Estandarización de quemaduras térmicas en ratas por contacto directo con la superficie cutánea

Introdução: Queimaduras cutâneas são lesões geralmente causadas pela energia térmica da transferência de calor, que determina um processo cicatricial desordenado. Para entender o processo e para avaliar possibilidades terapêuticas, são necessários modelos animais, assim como a padronização da elaboração da queimadura experimental. Objetivo: Detalhar uma técnica que promove queimaduras padronizadas de espessura total da pele em ratos Wistar. Método: Realizaram-se queimaduras padronizadas em 22 ratos Wistar divididos aleatoriamente em GI e GII. Acoplou-se à extremidade de um ferro de solda uma peça cerâmica de 484mm2 de superfície, que em contato com a pele durante 30 segundos, aquecida a 100° Celsius com uma pressão de 54 gramas, exercida pela própria massa do ferro da solda, determinou as queimaduras. No quarto dia, avaliou-se a coloração, umidade e elasticidade para caracterizar macroscopicamente as queimaduras e mensurou-se a área das queimaduras dos grupos, nos quais aplicou-se o teste t de Student para comparar os resultados. Resultados: Evidenciaram-se, em todos os animais, queimaduras com coloração branca cerosa, seca e inelástica. Constatou-se uma área média das queimaduras 403,5±71,48mm2 no GI e 403,7±71,19mm2 no GII (p=0,995). Conclusão: O contato da peça cerâmica de 484mm2 com pressão de massa de 54 gramas a 100° Celsius durante 30 segundos determinou queimaduras de espessura total de pele com área média semelhante entre os grupos, ao quarto dia de evolução, permitindo comparações seguras entre GI e GII ao longo do tempo cicatricial.

Introduction: Skin burns are lesions usually caused by energy of heat transfer that determine a disordered cicatricial process, to understand it and to evaluate therapeutic possibilities are needed animal models, as well as the standardization of experimental burn. Objective: To describe a technique that promotes standardized burns of total skin thickness for experimental study in Wistar rats. Methods: Standardized burns were performed on 22 Wistar rats randomly divided into GI and GII. A ceramic piece of 484 mm2 was attached to the end of a soldering iron, which in contact with the skin for 30 seconds at 100° Celsius at a pressure of 54 grams. On the fourth day, the staining, moisture and elasticity were evaluated to characterize the burns and the burn area of the groups was measured, in which Students t-test. Results: Burns characterized by waxy, dry and inelastic white color were observed in all the animals. It was found an average area of burns to 403.5±71.48mm2 in GI and 403.7±71.19mm2 in GII (p=0.995). Conclusion: The contact of the ceramic piece of 484mm2 with mass pressure of 54 grams at 100° Celsius for 30 seconds, determined total skin thickness burns with similar mean area groups, on the fourth day, allowing the safe comparison between the groups over the cicatricial time

Introducción: Las quemaduras cutáneas son lesiones en general causadas por la transferencia de calor que determinan una cicatrización desordenado, para entenderlo y evaluar posibilidades terapéuticas, se necesitan modelos animales, así como la estandarización de la quemadura experimental. Objetivo: Describir una técnica que promueve quemaduras estandarizadas del grosor total de la piel para el estudio experimental en ratas Wistar. Métodos: Se realizaron quemaduras en veintidós ratas Wistar divididas aleatoriamente en GI y GII. Se colocó una pieza cerámica de 484mm2 en un soldador en contacto con la piel durante 30 segundos a 100° Celsius a una presión de 54 gramos determinó la quemaduras. En el cuarto día, la tinción, la humedad y la elasticidad se evaluaron para caracterizar las quemaduras y se midió el área de las quemadura y se aplicó la prueba t de Student. Resultados: Se observaron quemaduras caracterizadas por un color blanco ceroso, seco e inelástico en todos los animales. Se encontró un área media de quemaduras 403.5±71.48mm2 en GI y 403.7±71.19 mm2 en GII (p=0.995). Conclusión: El contacto de la pieza de cerámica de 484 mm2 con una presión de masa de 54 gramos a 100° Celsius durante 30 segundos, determinó quemaduras de espesor total de la piel con área media similares entre los grupos en el cuarto día, lo que permite la comparación segura entre GI y el GII a lo largo del tiempo cicatricial

A complexidade cicatricial em queimaduras e a possibilidade da terapia com células-tronco derivadas do tecido adiposo: revisão / The complexity of burn healing and the possibility of adipose tissue-derived stem cell therapy: review / La complejidad de la cicatrización de las quemaduras y la posibilidad de la terapia con células madre derivadas del tejido adiposo: revisión

OBJETIVO: Verificar as especificidades do processo cicatricial em queimaduras e identificar se as células-tronco derivadas do tecido adiposo (CTDA) podem se tornar aliadas em sua reparação. MÉTODO: Realizou-se uma revisão da literatura com consultas a Biblioteca Virtual de Saúde, Google Acadêmico, PubMed, Scientific Electronic Library Online, Mendeley catalog of academic literature, artigos e bibliografia especilizada em cicatrização, queimaduras, células-tronco derivadas do tecido adiposo publicados entre 2012 e 2016. RESULTADOS: As queimaduras se diferenciam de outras lesões pela intensidade da inflamação sistêmica e pela sobreposição das fases cicatriciais desorganizando-as. As CTDA são encontradas no estroma vascular do tecido adiposo e podem ser obtidas por lipoaspiração. Estas células podem interferir nas fases cicatriciais pela secreção de citocinas de crescimento, substâncias imunomoduladoras e anti-inflamatórias, assim como pela capacidade de diferenciação celular e homing. CONCLUSÃO: As queimaduras ocorrem na população mundial de forma preocupante, com características de morbidade e mortalidade. Os problemas sistêmicos e locais da reparação tecidual parecem ser atenuados pelas CTDA, sua abundante disponibilidade para o cultivo celular e suas habilidades as apontam como aliadas na recuperação das queimaduras.(AU)

OBJECTIVE: To evaluate the distinctions of the healing process in burns and identify how the adipose-tissue derived stem cells (ATSC) may become allies in this repair process. METHODS: Review of the literature was carried out with consultations at Virtual Health Library, Google Scholar, PubMed, Scientific Electronic Library Online and Mendeley catalog of academic literature and specialized bibliography in healing, burns and ATSC published between 2012 and 2016. RESULTS: Burns differentiate themselves from other lesions by the intensity of systemic inflammation and by the overlapping of the healing wound phases disorganizing them. The ATSC are found in the vascular stroma of adipose tissue and can be obtained by liposuction. These cells can interfere in the healing wound phases by the secretion of growth cytokines, immunomodulatory and anti-inflammatory substances and the ability of cell differentiation and homing. CONCLUSION: Burns occur in the world population in a worrisome way with characteristics of morbimortality. The problems of tissue repair seem to be attenuated by ATSC, their abundant availability for cell culture and their abilities point them as promising allies in healing of burn wound.(AU)

Objetivo: Verificar las especificidades del proceso cicatricial en quemaduras y identificar si las células-tronco derivadas del tejido adiposo (CTDA) pueden tornarse aliadas en su reparación. Método: Se realizó una revisión del literatura con consultas en Biblioteca Virtual de Salud, Google Académico, PubMed, Scientific Electronic Library online online, Mendeley catalog of academic literature, artículos y bibliografía especilizada, quemaduras, CTDA, publicados entre 2012 y 2016. Resultados: Las quemaduras se diferencian de otras lesiones por la intesidad de la inflamación sistémica y por la sobreposición de las fases cicatriciales desorganizándolas. Las CTDA son encontradas en el estroma vascular del tejido adiposo y pueden ser obtenidas por lipoaspiracion. Estas celulas pueden interferir en las fases cicatriciales por secrecion de citoquinas del crecimiento, sustancias inmunomoduladoras, anti inflamatorias y por la capacidad de diferenciacion celular e homing. Conclusión: Las quemaduras ocurren en la población mundial de forma preocupante con caracteristicas de morbimortalidad. Los problemas de la reparacion tecidual parecen ser atenuados por las CTDA, su abundante disponibilidad para el cultivo celular y sus habilidades las apuntan como aliadas en la recuperacion de las quemaduras.(AU)

Autologous Transplantation of Bone Marrow Adult Stem Cells for the Treatment of Idiopathic Dilated Cardiomyopathy / Transplante Autólogo de Células-Tronco Adultas para Tratamento da Cardiomiopatia Dilatada Idiopática

Background: Morbimortality in patients with dilated idiopathic cardiomyopathy is high, even under optimal medical treatment. Autologous infusion of bone marrow adult stem cells has shown promising preliminary results in these patients. Objective: Determine the effectiveness of autologous transplantation of bone marrow adult stem cells on systolic and diastolic left ventricular function, and on the degree of mitral regurgitation in patients with dilated idiopathic cardiomyopathy in functional classes NYHA II and III. Methods: We administered 4,54 x 108 ± 0,89 x 108 bone marrow adult stem cells into the coronary arteries of 24 patients with dilated idiopathic cardiomyopathy in functional classes NYHA II and III. Changes in functional class, systolic and diastolic left ventricular function and degree of mitral regurgitation were assessed after 3 months, 6 months and 1 year. Results: During follow-up, six patients (25%) improved functional class and eight (33.3%) kept stable. Left ventricular ejection fraction improved 8.9%, 9.7% e 13.6%, after 3, 6 and 12 months (p = 0.024; 0.017 and 0.018), respectively. There were no significant changes neither in diastolic left ventricular function nor in mitral regurgitation degree. A combined cardiac resynchronization and implantable cardioversion defibrillation was implanted in two patients (8.3%). Four patients (16.6%) had sudden death and four patients died due to terminal cardiac failure. Average survival of these eight patients was 2.6 years. Conclusion: Intracoronary infusion of bone marrow adult stem cells was associated with an improvement or stabilization of functional class and an improvement in left ventricular ejection fraction, suggesting the efficacy of this intervention. There were no significant changes neither in left ventricular diastolic function nor in the degree of mitral regurgitation. .

Fundamento: Pacientes portadores de cardiomiopatia dilatada idiopática apresentam alta morbimortalidade, mesmo em tratamento clínico otimizado. A infusão autóloga de células-tronco adultas da medula óssea mostrou resultados clínicos preliminares promissores nesses pacientes. Objetivo: Determinar a eficácia do transplante autólogo de células-tronco adultas da medula óssea sobre as funções sistólica e diastólica, e o grau de insuficiência mitral em pacientes portadores de cardiomiopatia dilatada idiopática em classes funcionais NYHA II e III. Métodos: Infundiram-se 4,54 x 108 ± 0,89 x 108 células-tronco adultas da medula óssea nas artérias coronárias de 24 pacientes com cardiomiopatia dilatada idiopática em classes funcionais NYHA II e III. Após 3 meses, 6 meses e 1 ano, avaliaram-se as mudanças de classe funcional, das funções ventricular esquerda sistólica e diastólica, e do grau da insuficiência mitral. Resultados: No seguimento, seis (25%) pacientes melhoraram sua classe funcional e oito (33,3%) mantiveram sua classe funcional inicial. A fração de ejeção ventricular esquerda aumentou 8,9%, 9,7% e 13,6%, após 3 e 6 meses e 1 ano (p = 0,024; p = 0,017 e p = 0,018), respectivamente. A função diastólica ventricular esquerda e o grau de insuficiência mitral não demonstraram mudanças significativas. Dois pacientes (8,3%) receberam cardioversor e ressincronizador implantável. Ocorreram quatro (16,6%) mortes súbitas e quatro (16,6%) mortes por insuficiência cardíaca terminal. A sobrevida média desses oitos pacientes foi de 2,6 anos. Conclusão: A infusão intracoronariana de células-tronco adultas da medula óssea em pacientes com cardiomiopatia dilatada idiopática promoveu melhora ...

Comparison of two surgical techniques for creating an acute myocardial infarct in rats / Comparação de duas técnicas cirúrgicas para criar um infarto agudo do miocárdio em ratos

Objective: To perform a comparative assessment of two surgical techniques that are used creating an acute myocardial infarc by occluding the left anterior descending coronary artery in order to generate rats with a left ventricular ejection fraction of less than 40%. Methods: The study was completely randomized and comprised 89 halothane-anaesthetised rats, which were divided into three groups. The control group (SHAM) comprised fourteen rats, whose left anterior descending coronary artery was not occluded. Group 1 (G1): comprised by 35 endotracheally intubated and mechanically ventilated rats, whose left anterior descending coronary artery was occluded. Group 2 (G2): comprised 40 rats being manually ventilated using a nasal respirator whose left anterior descending coronary artery was occluded. Other differences between the two techniques include the method of performing the thoracotomy and removing the pericardium in order to expose the heart, and the use of different methods and suture types for closing the thorax. Seven days after surgery, the cardiac function of all surviving rats was determined by echocardiography. Results: No rats SHAM group had progressed to death or had left ventricular ejection fraction less than 40%. Nine of the 16 surviving G1 rats (56.3%) and six of the 20 surviving G2 rats (30%) had a left ventricular ejection fraction of less than 40%. Conclusion: The results indicate a tendency of the technique used in G1 to be better than in G2. This improvement is probably due to the greater duration of the open thorax, which reduces the pressure over time from the surgeon, allowing occlusion of left anterior descending coronary artery with higher accuracy. .

Objetivo: Realizar uma avaliação comparativa de duas técnicas cirúrgicas que são usadas para criar um infarto agudo do miocárdio pela oclusão da artéria coronária descendente anterior esquerda, a fim de gerar ratos com uma fração de ejeção ventricular esquerda inferior a 40%. Métodos: O estudo foi completamente randomizado e composto por 89 ratos anestesiados com halotano, que foram divididos dentro de três grupos. O grupo controle (SHAM) composto por 14 ratos, cuja artéria coronária descendente anterior esquerda não foi ocluída. Grupo 1 (G1): composto por 35 ratos intubados endotraquealmente e ventilados mecanicamente, cuja artéria coronária descendente anterior esquerda foi ocluída. Grupo 2 (G2): constituído por 40 ratos sendo ventilados manualmente utilizando um respirador nasal, cuja artéria coronária descendente anterior esquerda foi ocluída. Outras diferenças entre as duas técnicas incluem o método de realizar a toracotomia e remover o pericárdio, a fim de expor o coração, e o uso de diferentes métodos e tipos de sutura para fechar o tórax. Sete dias após a cirurgia, a função cardíaca de todos os ratos sobreviventes foi determinada por ecocardiografia. Resultados: Nenhum rato do grupo SHAM foi a óbito ou teve fração de ejeção ventricular esquerda menor que 40%. Nove dos 16 ratos sobreviventes do G1 (56,3%) e seis dos 20 ratos sobreviventes do G2 (30%) tiveram uma fração de ejeção ventricular esquerda inferior a 40%. Conclusão: Os resultados indicam uma tendência da técnica utilizada no G1 ser melhor do que a do G2. Esta melhora deve-se provavelmente à maior duração do tórax aberto, o que reduz a pressão de tempo sobre o cirurgião, permitindo uma oclusão da artéria coronária descendente anterior esquerda com maior acurácia. .

Genetic evaluation of mesenchymal stem cells by G-banded karyotyping in a Cell Technology Center

OBJECTIVE: To present the initial results of first three years of implementation of a genetic evaluation test for bone marrow-derived mesenchymal stem cells in a Cell Technology Center. METHODS: A retrospective study was carried out of 21 candidates for cell therapy. After the isolation of bone marrow mononuclear cells by density gradient, mesenchymal stem cells were cultivated and expanded at least until the second passage. Cytogenetic analyses were performed before and after cell expansion (62 samples) using G-banded karyotyping. RESULTS: All the samples analyzed, before and after cell expansion, had normal karyotypes, showing no clonal chromosomal changes. Signs of chromosomal instability were observed in 11 out of 21 patients (52%). From a total of 910 analyzed metaphases, five chromatid gaps, six chromatid breaks and 14 tetraploid cells were detected giving as total of 25 metaphases with chromosome damage (2.75%). CONCLUSION: The absence of clonal chromosomal aberrations in our results for G-banded karyotyping shows the maintenance of chromosomal stability of bone marrow-derived mesenchymal stem cells until the second passage; however, signs of chromosomal instability such as chromatid gaps, chromosome breaks and tetraploidy indicate that the long-term cultivation of these cells can provide an intermediate step for tumorigenesis. .

Brazilian minipig as a large-animal model for basic research and stem cell-based tissue engineering. Characterization and in vitro differentiation of bone marrow-derived mesenchymal stem cells

Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources. Objective: This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering. Material and Methods: Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR. Results: MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (p<0.001, p<0.001, p=0.031; respectively). Conclusions: The isolation, cultivation, and differentiation of BM-MSCs from BR1 makes this animal eligible as a useful large-animal model for stem cell-based studies in Brazil. .

Formação in vitro de túbulos capilares a partir de células de sangue de cordão umbilical humano com perspectivas para aplicação terapêutica / In vitro formation of capillary tubules from human umbilical cord blood cells with perspectives for therapeutic application

OBJETIVO: As células progenitoras endoteliais (CPE), caracterizadas pelo marcador CD133+, contribuem para a neovascularização, e o aumento no número dessas células pode ser uma ferramenta terapêutica promissora. O sangue de cordão umbilical humano contém um número significante de CPE, sugerindo a possibilidade do uso destas células para a revascularização de tecidos isquêmicos. O objetivo desse trabalho foi analisar a funcionalidade das células CD133+ diferenciadas in vitro. MÉTODOS: As células diferenciadas foram caracterizadas por citometria de fluxo; a expressão do mRNA de VEGF foi avaliada por RT-PCR e a funcionalidade, por meio de ensaios de formação de túbulos capilares. RESULTADOS: As células diferenciadas perderam os marcadores de CPE, mantiveram em níveis baixos os marcadores das linhagens hematopoética e monocíticas e aumentaram a expressão dos marcadores de células endoteliais adultas. As células diferenciadas apresentaram transcritos no mRNA de VEGF e mostraram-se capazes de formar túbulos capilares in vitro. CONCLUSÃO: As células CD133+ diferenciadas in vitro em células endoteliais demonstraram serem funcionalmente ativas, abrindo perspectiva para seu uso futuro em aplicações terapêuticas.

OBJECTIVE: Endothelial progenitor cells (EPC) caracterized by the CD133+ marker, contribute to the neovascularization. Increasing EPC number in vitro could be a promising therapeutic tool. Human umbilical cord blood maintains a significant number of EPC, suggesting the possibility to use these cells to induce the revascularization of ischemic tissues. The aim of this study was to analize the in vitro function of differentiated CD133+ cells. METHODS: Cells were characterized by flow cytometry, VEGF mRNA expression was evaluated by the RT-PCR analysis and the functionally by essays of capillary tubes formation. RESULTS: Differentiated cells lost EPC markers, maintained low levels of markers for hematopoietic and monocytic cell lines and increased the expression of adult endothelial cell markers. Differentiated cells expressed VEGF mRNA and were capable to induce in vitro capillary tubules formation. CONCLUSION: CD133+ cells differentiated into endothelial cells in vitro are functionally active initiating the possibility of their use in future therapeutic applications.