Although it is known that the regenerative function of neural stem/progenitor cells (NSPCs) declines with age, causal mechanisms underlying this phenomenon are not understood. Here, we systematically analyze subventricular zone (SVZ) NSPCs, in various groups of rats across the aging spectrum, using in vitro and in vivo histological and behavioral techniques. These studies indicate that although NSPC function continuously declines with advancing age, there is a critical time period during middle age (13-15 months) when a striking reduction in NSPC survival and regeneration (proliferation and neuronal differentiation) occurs. The studies also indicate that this specific temporal pattern of NSPC deterioration is functionally relevant at a behavioral level and correlates with the decreasing expression of the redox-sensitive transcription factor, Nrf2, in the NSPCs. When Nrf2 expression was suppressed in 'young' NSPCs, using short interfering RNAs, the survival and regeneration of the NSPCs was significantly compromised and mirrored 'old' NSPCs. Conversely, Nrf2 overexpression in 'old' NSPCs rendered them similar to 'young' NSPCs, and they showed increased survival and regeneration. Furthermore, examination of newborn Nrf2 knockout (Nrf2-/-) mice revealed a lower number of SVZ NSPCs in these animals, when compared to wild-type controls. In addition, the proliferative and neurogenic potential of the NSPCs was also compromised in the Nrf2-/- mice. These results identify a novel regulatory role for Nrf2 in NSPC function during aging and have important implications for developing NSPC-based strategies to support healthy aging and to treat age-related neurodegenerative disorders.

La réussite de la thérapie cellulaire à l'aide des cellules souches embryonnaires, nécessite une bonne compréhension des mécanismes moléculaires qui contrôlent leur différenciation. Une des voies de signalisation, impliquée dans le contrôle de la différenciation des cellules souches, est la voie p38MAPK. Dans le but de comprendre les mécanismes moléculaires impliqués dans le contrôle de la différenciation précoce des cellules ES, nous avons réalisé un criblage sur puces à ADN. Les résultats du criblage ont montré que certains gènes régulés différentiellement, comme le gène Bcl2, sont des cibles communes de p38MAPK et de p53. En plus de son rôle de répresseur de tumeur, p53 a été impliqué dans le développement embryonnaire et dans la biologie des cellules ES. Ainsi, il est connu que p53 agit comme un répresseur du gène de pluripotence, Nanog. Il est également connu que p53 interfère avec le processus de reprogrammation des cellules adultes ; et que son activité transcriptionnelle augmente avec l'entrée des cellules ES en différenciation. En revanche, son rôle dans la différenciation et dans la formation des différents lignages issus des cellules ES, reste inconnu. Nous avons trouvé que le traitement des cellules ES sauvages avec l'inhibiteur spécifique de p53, la pifithrine-α, durant le processus de différenciation, inhibe les lignages mésodermiques, et à l'inverse, stimule la neurogenèse. De plus, la transfection transitoire des cellules ES avec des siRNAs spécifiques, dirigés contre p53 ainsi que l'utilisation des cellules ES déficientes pour le gène p53, montrent que l'absence de p53, affecte les lignages cardiaques, du muscle lisse et du muscle squelettique. / Embryonic stem cells (ESCs) differentiate in vitro into all cell lineages. We previously found that p38MAPK controls two independent successive steps during the early mesodermal commitment of ESCs. The first one is Brachyury dependent, a master gene of mesoderm formation whereas the second one is not. In order to understand the molecular mechanism implicated in the second step, we treated ESCs with the p38 specific Inhibitor PD169316 and performed microarray experiments on mRNAs extracted from treated versus untreated cells. Our results show that many regulated genes are common targets of p38MAPK and p53 transcription factor. In addition to its role as a tumor suppressor and cell cycle checkpoint control, p53 has been involved in embryonic development, but its role in ESC differentiation is still unknown. We found that treatment of wild type ESCs with the p53 specific inhibitor pifithrin α during the differentiation process inhibits mesodermal lineages and, by contrast, stimulates neurogenesis. Likewise, ESCs Transfected with p53 siRNAs and p53 KO ESCs show an inhibition of cardiac, endothelial, smooth muscle and skeletal muscle lineage formation. Furthermore, p38MAPK inhibition by PD169316 for 24h induces a strong decrease of p53 protein level. Our results suggest that p53 mediates the p38MAPK control of the commitment of ESCs towards mesodermal lineages. The involvement of the various p53 isoforms in this process will be discussed.

A terapia celular baseada na utilização de células-tronco mesenquimais (CTMs) tem sido considerada uma estratégia promissora para regenerar o tecido ósseo. Apesar da medula óssea constituir a principal fonte de CTMs, o tecido adiposo tem sido investigado como uma fonte alternativa, devido a maior disponibilidade e facilidade de obtenção dessas células. Porém, as CTMs obtidas de fontes distintas podem exibir diferenças a nível molecular, que ainda não estão esclarecidas pela literatura e que poderiam influenciar o potencial regenerativo dessas células. Assim sendo, o objetivo desse estudo foi comparar os perfis de expressão gênica em larga escala de CTMs isoladas da medula óssea (CTMs-MO) e do tecido adiposo (CTMs-TA) de ratos, e de osteoblastos (OBs) diferenciados a partir dessas células (OBs-MO e OBs-TA, respectivamente). As células foram cultivadas em meio de crescimento para manutenção das características de CTMs ou em meio osteogênico para indução da diferenciação osteoblástica. O RNA total das células foi extraído e a análise genômica foi realizada pela tecnologia de microarray utilizando lâminas Agilent 4x44k. Os perfis de expressão gênica foram analisados pelo software GeneSpring 14.8 GX, considerando o tecido de origem (CTMs-TA vs CTMsMO e OBs-TA vs OBs-MO) e a diferenciação celular (OBs-MO vs CTMs-MO e OBs-TA vs CTMs-TA) como parâmetros de comparação entre os grupos (fold change 2.0; p 0.05). Os dados do microarray foram confirmados por PCR em tempo real para todos os genes de interesse avaliados. Os perfis de expressão gênica entre as CTMs obtidas de fontes distintas (CTMs-TA vs CTMs-MO) e entre os OBs diferenciados a partir destas células (OBs-TA vs OBs-MO) mostraram que as células provenientes da medula óssea apresentaram uma sobreexpressão de genes relacionados à osteogênese enquanto que, as células provenientes do tecido adiposo, uma sobre-expressão de genes relacionados à angiogênese, independente da diferenciação celular. Adicionalmente, as células provenientes do tecido adiposo (CTMs e OBs) apresentaram uma sobre-expressão de genes relacionados à diferenciação adipogênica. Esses resultados, em conjunto, mostraram que as células preservaram características do tecido de origem. As análises comparativas entre as células indiferenciadas e diferenciadas obtidas da mesma fonte (OBs-MO vs CTMs-MO e OBs-TA vs CTMs-TA) mostraram que as CTMs, independente do tecido de origem, apresentaram uma sobre-expressão de genes relacionados com os processos de angiogênese, diferenciação osteoblástica e morfogênese do osso, o que sugere que as CTMs podem estar mais envolvidas com os eventos iniciais que regem o processo regenerativo que os OBs. A análise do dendrograma mostrou que os OBs-TA apresentaram um padrão de expressão gênica mais próximo ao das CTMs, em termos de similaridade e, os OBsMO, um padrão mais distante, o que sugere que os OBs-MO possam ter atingido um estágio de diferenciação celular mais avançado que os OBs-TA. Os resultados mostraram diferenças moleculares entre as populações de células que poderiam implicar em utilizações terapêuticas específicas, sugerindo que as células provenientes do tecido adiposo seriam mais indicadas para terapias angiogênicas enquanto que, as células provenientes da medula óssea, mais indicadas para terapias osteogênicas / Cell therapy based on the use of mesenchymal stem cells (MSCs) has been considered a promising strategy to regenerate bone tissue. Although bone marrow represents the main source of MSCs, adipose tissue has been investigated as an alternative source due to the greater availability and ease of isolation of these cells. However, MSCs obtained from different sources may show differences at the molecular level, which are not yet established in the literature and that could influence the regenerative potential of these cells. Thus, the aim of this study was to compare the large-scale gene expression profiles of MSCs isolated from bone marrow (BMMSCs) and adipose tissue (AT-MSCs) of rats, and of the osteoblasts differentiated from these cells (BM-OBs and AT-OBs, respectively). Cells were cultured in growth medium to maintain the characteristics of MSCs or in osteogenic medium to induction of osteoblastic differentiation. Total RNA was extracted and genomic analysis was performed by microarray technology using Agilent 4x44k slides. The gene expression profiles were analyzed by GeneSpring 14.8 GX software, considering the source (AT-MSCs vs BM-MSCs and AT-OBs vs BM-OBs) and cell differentiation (BM-OBs vs BM-MSCs and AT-OBs vs AT-MSCs) as parameters of comparison between the groups (fold change 2.0; p 0.05). The microarray data were confirmed by real-time PCR for all the interest genes evaluated. The gene expression profiles between the MSCs obtained from different sources (AT-MSCs vs BM-MSCs) and between the OBs differentiated from these cells (AT-OBs vs BM-OBs) showed that the cells from the bone marrow presented an overexpression of genes related to osteogenesis whereas the cells from adipose tissue showed an overexpression of genes related to angiogenesis, independent of cell differentiation. In addition, cells from adipose tissue (MSCs and OBs) showed an overexpression of genes related to adipogenic differentiation. These results, together, showed that the cells preserved characteristics of the source. Comparative analyses between the undifferentiated and differentiated cells obtained from the same source (BM-OBs vs BM-MSCs and AT-OBs vs AT-MSCs) showed that MSCs, regardless of source, showed an overexpression of genes related to the angiogenesis, osteoblastic differentiation and bone morphogenesis process, suggesting that MSCs may be more involved with the early events that govern the regenerative process than OBs. Dendrogram analysis showed that AT-OBs had a gene expression profile closer to MSCs in terms of similarity and the BM-OBs had a more distant profile, suggesting that BM-OBs may have reached a stage of cell differentiation more advanced than AT-OBs. The results showed molecular differences between cell populations that could imply in specific therapeutic uses, suggesting that cells derived from adipose tissue would be better suited for angiogenic therapies, whereas bone marrow cells are more suitable for osteogenic therapies

The Gangliosidoses, to include GM1 gangliosidosis and Sandhoff disease are a class of incurable lysosomal storage disorders characterized by an abnormal accumulation of gangliosides leading to progressive neurodegeneration and eventually death. GM1 gangliosidosis is caused by a genetic defect in the lysosomal-specific acid β-galactosidase, which results in the massive accumulation of ganglioside GM1 primarily in the central nervous system (CNS). Sandhoff disease (SD) results from a defect in the β-subunit of β- Hexosaminidase A and leads to the accumulation of ganglioside GM2 and its asialo derivative (GA2). As there are no effective therapies for these glycosphingolipid (GSL) storage disorders, I studied substrate reduction therapy (SRT), stem cell therapy, and adeno-associated viral (AAV) gene therapy in neonatal mice as early intervention therapies and were effective in reducing CNS GSL storage. In addition, AAV gene therapy was also evaluated in the adult GM1 gangliosidosis mice. Furthermore, analysis of the brain lipids in mice, cats, and humans with Sandhoff disease revealed that the SD cat model is intermediate between the SD mouse and the SD patient with respect to GM2 and GA2 accumulation. These findings are the first to compare the different therapies and provide valuable information for the translation of mouse studies to clinical trials in patients. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The rapid development of regenerative medicine and the urgent need for cells which are able to modulate the immune system, or even differentiate into variable cell types, have led to the research of mesenchymal stem cells. The Sertoli cells, which are essential for the proper development of sperm in the testis, have a strikingly similar character. In the previous research, a cell culture expressing markers of mesenchymal stem cells and Sertoli cells from juvenile male testes of X. tropicalis was established. At the same time, the cells were modified by the introduction of gene for the red fluorescent protein (RFP) in their genome. The aim of this diploma thesis was to clarify their characteristics after microinjection to X. tropicalis tadpoles (allogeneic transplantation). For these experiments, it was necessary to develop a reliable technique for the preparation of sections, which won't be harmfull for the samples. Using the vibratome sectioning method along with immunohistochemical labeling, the cell culture has been found to contain precursors of Sertoli and peritubulare myoid cells.

As células tronco possuem a capacidade de auto renovação ilimitada e de se manterem indiferenciadas durante um período prolongado de tempo, até se diferenciarem em uma linhagem celular específica. Devido as dificuldades encontradas na utilização de células embrionárias por questões éticas, de segurança e histocompatibilidade e a limitada plasticidade das células tronco adultas, as células tronco fetais, derivadas de tecidos extraembrionários, aparecem no cenário terapêutico como uma alternativa promissora. Uma vez que apresentam alta capacidade de proliferação e expansão in vitro, além de sua característica imunogênica, por se encontrarem na interface materno fetal. Por conta destas características, a membrana amniótica surgiu como uma nova e importante fonte de células tronco em diferentes espécies. Neste trabalho, foram utilizados 8 fetos de coelhos para a coleta da membrana amniótica. A membrana foi isolada pela técnica de explante, as amostras foram cultivadas em meio DMEM-HIGH, e posteriormente criopreservadas nas passagens P4 e P8 para realizar os experimentos. Em cultivo, essas células apresentaram aderência a placa, alta capacidade de proliferação e morfologia semelhante à fibroblastos. Na caracterização imunofenotípica, houve expressão de marcadores de citoesqueleto, de células mesenquimais, proliferação, pluripotência e para precursores de células hematopoiéticas, embora não tenha ocorrido a expressão de marcadores de células hematopoiéticas (CD-45) e de células precursoras neuronais. Quando induzidas a diferenciação in vitro, as células amnióticas tiveram capacidade de se diferenciarem nas linhagens osteogênica, adipogênica e condrogênica. Além disso, quando injetadas em camundongos nude, não foi verificada a formação de tumor. Nossos resultados demostram que células de membrana amniótica de coelho podem ser consideradas células tronco mesenquimais com possibilidade de serem utilizadas no futuro na terapia celular / Stem cells are capable of unlimited self renewal and to remain undifferentiated for a prolonged period of time up to differentiate into a specific cell lineage. Due to the difficulties in the use of embryonic cells due ethical reasons, security and histocompatibility, and in the other hands, the limited plasticity of adult stem cells, fetal stem cells, derived from extraembryonic tissues, appear in the therapeutic setting as a promising alternative. They have high capacity of proliferation and in vitro expansion, as well as immunogenic properties, since they are at the maternal-fetal interface. Due to these characteristics, the amniotic membrane has emerged as an important new source of stem cells in different species. In this work, we used 8 fetuses of rabbits to collect amniotic membrane. The membrane was isolated by the explant technique. Samples were cultured in DMEM-HIGH, and cryopreserved in the passages P4 and P8 in order to perform the experiments. In culture, these cells exhibited adhesion to the dishes, high proliferative capacity and fibroblast-like morphology. In immunophenotypic characterization, there was expression of markers related to cell cytoskeleton, mesenchymal stem cells, proliferation, pluripotency, and hematopoietic precursor cells. And there was no expression of hematopoietic cell markers (CD-45) and neuronal precursor cells. When induced to in vitro differentiation, the amniotic cells were able to differentiate in osteogenic, adipogenic and chondrogenic lineages. Furthermore, when injected into nude mice, tumor formation has not been verified. Our results demonstrate that rabbit amniotic membrane cells can be considered mesenchymal stem cells with possibility to be used in the future in cell therapy

University of Macau / Institute of Chinese Medical Sciences

Objetivos: Avaliar o potencial terapêutico e a segurança do uso intravítreo de (FMMO) contendo células CD34+ em pacientes portadores de degeneração macular relacionada com a idade na forma atrófica. Casuística e Métodos: Foram avaliados 10 pacientes com degeneração macular relacionada à idade (DMRI) seca e acuidade visual no pior olho <=20/100. Foi obtido aspirado da medula óssea de todos os pacientes, e após o processamento do material no hemocentro, foi injetado 0,1 ml da suspensão de FMMO intravítreo no olho com pior acuidade. Os pacientes foram avaliados no baseline, 1, 3, 6, 9 e 12 meses após a injeção. Todos realizaram medida da melhor acuidade visual corrigida (MAVC), microperimetria, eletrorretinografia multifocal (ERGmf), autofluorescência, Tomografia de coerência óptica (OCT) e responderam o questionário VFQ-25 em todos seguimentos. Também foi realizada angiografia fluoresceínica antes da injeção, seis e doze meses após. Resultados: Todos os pacientes completaram o seguimento de seis meses, e seis finalizaram os doze meses. Antes da injeção, a média da MAVC foi de 1,18 logMAR (20/320-1); variando de 20/125 a 20/640-2. Aos doze meses, a média foi de 1,0 logMAR (20/200), com melhora significativa em todos os meses de seguimento. A média do limiar de sensibilidade na microperimetria mostrou melhora significativa a partir do sexto mês (p=0,009). Ao se dividirem os pacientes com área de atrofia maior e menor, por meio da medida da hipoautofluorescência, observou-se que a melhora foi significativa apenas no grupo de menor atrofia. Não houve diferença significativa na ERGmf. A angiofluoresceinografia não apresentou crescimento de neovasos ou tumores. O questionário de qualidade de vida mostrou diferença significativa na saúde mental (p=0,003) e na visão de cores (p=0,005) e forte tendência à significância na análise que abordou a visão geral (p=0,05) e dependência (p=0,067). Houve declínio em relação à saúde geral (p=0,77). Conclusões: Os resultados indicam que o uso de FMMO intravítreo em pacientes com DMRI é seguro e está associado à melhora da acuidade visual e microperimetria. Pacientes com área menor de atrofia têm melhor resposta. Não é possível afirmar, mas acredita-se no resgate funcional das células em sofrimento, que ainda não se degeneraram. / Objectives: To assess the therapeutic potential and safety of the intraviteral use of a bone marrow mononuclear fraction (BMMF) containing CD 34+ cells in patients with the atrophic form of age-related macular degeneration (AMD). Casuistic and Methods: The study was conducted on 10 patients with the atrophic form of ARMD with worse eye visual acuity <=20/100. Bone marrow was aspirated from each patient under local anesthesia. After processing and separation of mononuclear cells, 0.1 ml of the suspension was injected intravitreally into the eye of lower acuity. The patients were evaluated at baseline and 1, 3, 6, 9 and 12 months after the injection. All were submitted to measurement of best corrected visual acuity (BCVA), microperimetry, multifocal electroretinography (ERGmf), autofluorescence, and optical coherence tomography (OCT) and all responded to the VFQ-25 questionnaire at all follow-up visits. Fluorescein angiography was performed before the injection and six and 12 months later. Results: All patients completed the six month follow-up and six completed the 12 month follow-up after the injection. Before the injection, mean BCVA was 1.18 logMAR (20/320-1), ranging from 20/125 to 20/640-2 and at 12 months it was 1.0 logMAR (20/200), with a significant improvement over all followup months. Mean perimetric threshold sensitivity improved significantly starting at the sixth month (p=0.009). When the patients with a larger area of atrophy measured by hypoautofluorescence were considered separately, a significant improvement was observed only in the group with lower atrophy. There was no significant difference in electroretinography. Angiofluoresceinography did not reveal neovessel or tumor growth. The quality of life questionnaire showed a significant difference in mental health (p=0.003) and color vision (p=0.005), and a strong tendency in the analysis of general vision (p=0.05) and dependence (p=0.067). There was a decline in general health (p=0.77). Conclusions: The results indicate that the use of intravitreal BMMF in patients with AMD is safe and is associated with improved visual acuity and microperimetry. Patients with a smaller atrophy area obtained a better response. The paracrine effect of these cells may explain the functional improvement observed in the present study; however, a larger series should be study to confirm these clinical findings.

The liver is a multi-functional and highly regenerative organ. While resilient, the liver is susceptible to organ damage and failure. In both the acute and chronic settings liver disease has dire consequences for health. A common cause of liver damage is adverse reactions to drugs which can lead to drug induced liver injury (DILI). This creates major problems for patients, clinicians, the pharmaceutical industry and regulatory authorities. In the context of drug overdose or serious adverse reactions, liver failure can be acute and life threatening, and in some cases require orthotopic liver transplantation. While transplantation is highly successful, such an approach has limitations and justifies basic science attempts to develop better human models to study liver injury and to develop scalable intervention strategies. With this in mind, we have studied the importance of microRNAs (miRs) in regulating human drug metabolism in pluripotent stem cell – derived hepatocytes and their potential to reduce liver toxicity in response to toxic levels of paracetamol. miRs are small non-coding RNAs that are approximately 20 - 24 nucleotides long and their major function is to fine tune gene expression of their target genes. Recently, it has been demonstrated that microRNAs play a role in regulating the first phase of drug metabolism however the second phase of drug metabolism, drug conjugation, has not been studied in detail. Drug conjugation is a crucial stage in human drug metabolism, and any alterations in this process can lead to changes in compound pharmacology, including therapeutic dose and clearance from the body. To test the importance of miRs in regulating phase II drug metabolism we opted to study the metabolism of a common used analgesic, paracetamol. When taken in the appropriate amounts paracetamol is modified by sulfotransferases (SULTs) and UDP - glucuronosyltransferases (UGTs) and removed from the body without organ damage. However, when paracetamol is taken above the recommended dose it is metabolised by phase I enzymes to generate a toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI), which if untreated can lead to massive hepatocyte cell death and liver failure, placing the patient in a life threatening situation. In order to promote non-toxic drug metabolism, in the context of drug overdose, we employed candidate miRs to regulate different parts of the paracetamol metabolism pathway. In summary, we have focused on studying human drug metabolism in the major metabolic cell type of the liver, the hepatocyte. We have identified a novel microRNA (called miR-324-5p) which regulates phase II drug metabolism and reduces cell cytotoxicity. Additionally, a supportive role of anti-microRNA- 324 in response to fulminant plasma collected from paracetamol overdose patients is also observed. The findings of this project are novel, provide proof of concept and exemplify the power of stem cell based models to identify new approaches to treating human liver damage.

A maioria das perdas auditivas adquiridas ou congênitas decorre de dano ou perda das células ciliares da cóclea ou dos seus neurônios associados. A irreversibilidade da surdez em mamíferos ocorre devido à incapacidade de substituição das células perdidas, seja por divisão celular ou por regeneração de células endógenas no epitélio da orelha interna. Com isso o objetivo deste trabalho foi obtenção de linhagens de células progenitoras do epitélio coclear de fetos de cães com 40 dias de gestação, colaborando com futuras pesquisas relacionadas a trabalhos de tratamento para surdez neurossensorial. Foram utilizados oito fetos caninos com idade compreendidos a 40 dias de gestação, nos quais, foi realizada uma dissecação no crânio, expondo a cóclea para a retirada do epitélio coclear, visando sua analise morfológica, e obtenção de suas células. Para analise morfológica do tecido colear realizou-se as técnicas macroscópica, microscópica e de imunohistoquímica. As células obtidas da cóclea foram fotodocumentadas, e submetidas às analises de método colorimétrico MTT (3-(4,5-Dimethylthiazol-2-&#1091;l)-2,5-Diphenyltetrazolium Bromide), análise do ciclo celular, análise da imunofenotipagem e da diferenciação celular. Em cultivo as células apresentaram formato fibroblastóide. Na caracterização imunofenotipica apresentaram marcação positiva para marcadores de células-tronco mesenquimais e de pluripotência e marcação negativa para células hematopoiéticas. Apresentaram ainda a capacidade de diferenciação para linhagens celulares osteogênicas, adipogênicas e condrogênicas. Essas análises sugeriram resultados satisfatórios na obtenção, quantificação e caracterização dessas células, as quais foram adquiridas a partir de células do epitélio coclear de feto de cão, as quais poderão constituir fontes de células a serem utilizadas na terapia celular da espécie canina destinada ao tratamento de surdez causada por lesões ou danos do epitélio coclear / Most acquired or congenital hearing loss results from damage or loss of hair cells of the cochlea or their associated neurons. The irreversibility of deafness in mammals is due to the lost cells replacement inability, either by cell division or by regeneration of endogenous cells in the epithelium of the inner ear. Therefore, the objective of this work was to increase knowledge about getting progenitor cell lines of the cochlear epithelium from dogs&rsquo; fetuses with 40 days of gestation, collaborating with future research related to treatment work for sensorineural deafness. Eight canine fetuses were used aged as mentioned above, in which, a dissection was performed in the skull, exposing the cochlea to the withdrawal of the cochlear epithelium, for their morphological analysis and cells obtainment. For morphological analysis of the cochlear tissue was held the macroscopic techniques, microscopy and immunohistochemistry. The cochlea cells obtained were photo documented and analyzed for the colorimetric method MTT ( 3- ( 4,5- dimethylthiazol -2- &#1091;l ) -2,5 - Diphenyltetrazolium Bromide), cell cycle analysis, immunophenotyping analysis and cell differentiation. In culture, the cells showed fibroblast format. In immunophenotype characterization, they presented positive staining for mesenchymal stem cell markers and pluripotency and negative marking for hematopoietic cells. They also exhibit the differentiation capacity for cell osteogenic lineages, adipogenic and chondrogenic. These analyzes suggested satisfactory results in obtainment, quantification and characterization of these cells, which were acquired from the dog fetal cells&rsquo; cochlear epithelium, which may be cells of fonts to be used in cellular therapy of canine species for the treatment of deafness caused by injury or damage to the cochlear epithelium