Mesenchymal stem cells in dogs with demyelinating leukoencephalitis as an experimental model of multiple sclerosis.

Researchers have used dogs with neurological sequelae caused by distemper as an experimental model for multiple sclerosis, owing to the similarities of the neuropathological changes between distemper virus-induced demyelinating leukoencephalitis and multiple sclerosis in humans. However, little is known about the role of mesenchymal stem cells in treating such clinical conditions. Therefore, we investigated the use of mesenchymal stem cells in four dogs with neurological lesions caused by the distemper virus. During the first year after cellular therapy, the animals did not demonstrate significant changes in their locomotive abilities. However, the intense (Grade V) myoclonus in three animals was reduced to a moderate (Grade IV) level. At one year after the mesenchymal stem cell infusions, three animals regained functional ambulation (Grade I), and all four dogs started to move independently (Grades I and II). In two animals, the myoclonic severity had become mild (Grade III). It was concluded that the use of mesenchymal stem cells could improve the quality of life of dogs with neurological sequelae caused by canine distemper, thus presenting hope for similar positive results in human patients with multiple sclerosis.

Modeling the Interplay Between Neurons and Astrocytes in Autism Using Human Induced Pluripotent Stem Cells.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with unclear etiology and imprecise genetic causes. The main goal of this work was to investigate neuronal connectivity and the interplay between neurons and astrocytes from individuals with nonsyndromic ASD using induced pluripotent stem cells. METHODS: Induced pluripotent stem cells were derived from a clinically well-characterized cohort of three individuals with nonsyndromic ASD sharing common behaviors and three control subjects, two clones each. We generated mixed neural cultures analyzing synaptogenesis and neuronal activity using a multielectrode array platform. Furthermore, using an enriched astrocyte population, we investigated their role in neuronal maintenance. RESULTS: ASD-derived neurons had a significant decrease in synaptic gene expression and protein levels, glutamate neurotransmitter release, and, consequently, reduced spontaneous firing rate. Based on co-culture experiments, we observed that ASD-derived astrocytes interfered with proper neuronal development. In contrast, control-derived astrocytes rescued the morphological neuronal phenotype and synaptogenesis defects from ASD neuronal co-cultures. Furthermore, after identifying interleukin-6 secretion from astrocytes in individuals with ASD as a possible culprit for neural defects, we were able to increase synaptogenesis by blocking interleukin-6 levels. CONCLUSIONS: Our findings reveal the contribution of astrocytes to neuronal phenotype and confirm previous studies linking interleukin-6 and autism, suggesting potential novel therapeutic pathways for a subtype of individuals with ASD. This is the first report demonstrating that glial dysfunctions could contribute to nonsyndromic autism pathophysiology using induced pluripotent stem cells modeling disease technology.

Transplantation of human immature dental pulp stem cell in dogs with chronic spinal cord injury.

PURPOSE:: To investigate the therapeutic potential of human immature dental pulp stem cells in the treatment of chronic spinal cord injury in dogs. METHODS:: Three dogs of different breeds with chronic SCI were presented as animal clinical cases. Human immature dental pulp stem cells were injected at three points into the spinal cord, and the animals were evaluated by limb function and magnetic resonance imaging (MRI) pre and post-operative. RESULTS:: There was significant improvement from the limb function evaluated by Olby Scale, though it was not supported by the imaging data provided by MRI and clinical sign and evaluation. CONCLUSION:: Human dental pulp stem cell therapy presents promising clinical results in dogs with chronic spinal cord injuries, if used in association with physical therapy.

Transplantation of human immature dental pulp stem cell in dogs with chronic spinal cord injury

Abstract Purpose: To investigate the therapeutic potential of human immature dental pulp stem cells in the treatment of chronic spinal cord injury in dogs. Methods: Three dogs of different breeds with chronic SCI were presented as animal clinical cases. Human immature dental pulp stem cells were injected at three points into the spinal cord, and the animals were evaluated by limb function and magnetic resonance imaging (MRI) pre and post-operative. Results: There was significant improvement from the limb function evaluated by Olby Scale, though it was not supported by the imaging data provided by MRI and clinical sign and evaluation. Conclusion: Human dental pulp stem cell therapy presents promising clinical results in dogs with chronic spinal cord injuries, if used in association with physical therapy.

Macroscopic and microscopic analysis of 2 embryos and 1 foetus derived from a sheep (Ovis aries) without breed / Análise macroscópica e microscópica de 2 embriões e 1 feto derivados de ovelha (Ovis aries) sem raça

The interest in embryology, the science of the development of a zygote into a completely developed foetus, has increased greatly in recent years due to a number of studies involving embryonic and induced pluripotent stem cells. In addition, the development of techniques such as cloning has aided to understand the critical events that occur during embryonic development. In this study, we describe the morphology of two sheep embryos and one foetus using macroscopic and microscopic techniques. We investigated sheep without defined breed on days 24, 32, and 50 of gestation (estimated by crown-rump length [CR]). Macroscopically, we observed the development of E1 (24 days), with visible optic vesicle, but without retinal pigmentation and the forelimbs bud in development. In the E2 (32 days), we noticed the presence of optic retinal pigmentation and forelimbs more developed in comparison with E1. As expected, F1 revealed an eyeball already covered and the forelimbs developed. Meanwhile, microscopic analysis revealed somite, ventricle, atrium, and oral cavity in development in E1. However, in F1 we were able to identify more complex structures, such as ossification in the spine, ventricle, atrium, intraventricular septum, pericardial sac, and oral cavity with tongue. This work brings more precise and detailed data on the morphological characteristics of the major organ systems (nervous, circulatory, respiratory, digestive, and urinary) at each embryonic and foetal stage analysed.(AU)

O interesse em Embriologia, a ciência do desenvolvimento de um zigoto em um feto completamente desenvolvido, tem aumentado consideravelmente nos últimos anos devido a uma série de estudos envolvendo células-tronco pluripotentes embrionárias e induzidas. Além disso, o desenvolvimento de técnicas como a clonagem tem ajudado a compreender os eventos críticos que ocorrem durante o desenvolvimento embrionário. Neste estudo, descrevemos a morfologia de dois embriões de ovinos e um feto utilizando técnicas macroscópicas e microscópicas. Obtivemos ovelhas sem raça definida com 24, 32 e 50 dias de gestação (estimado pelo método de Crown-Rump, CR). Os conceptos foram mensurados, pesados e caracterizados a olho nu. Macroscopicamente, observamos o desenvolvimento dos embriões E1 (24 dias), apresentando globo ocular sem pigmentação de retina e broto do membro torácico e pélvico. Já o E2 (32 dias), apresentava globo ocular com pigmentação na retina e os membros torácicos e pélvicos mais desenvolvidos. O F1 apresentou olhos cobertos com uma membrana e membros torácicos e pélvicos mais desenvolvidos. Enquanto isso, microscopicamente observamos no E1 somitos, ventrículo, átrio e cavidade oral ainda em desenvolvimento. Porém, no F1 já era possível observar ossificação da coluna espinhal, coração com estruturas mais complexas, como ventrículo, átrio, septo interventricular e saco pericárdio. Além disso, na cavidade oral observamos a formação da língua. Este trabalho fornece informações precisas e detalhadas sobre as características morfológicas dos principais órgãos dos sistemas (nervoso, circulatório, respiratório, digestivo e urinário) em cada fase embrionária e fetal analisadas.(AU)

Aquapuncture Using Stem Cell Therapy to Treat Mdx Mice.

Duchenne muscular dystrophy (DMD) occurs due to genetic mutations that lead to absence or decrease of dystrophin protein generating progressive muscle degeneration. Cell therapy using mesenchymal stem cell (MSC) has been described as a treatment to DMD. In this work, MSC derived from deciduous teeth, called stem cells from human exfoliated deciduous teeth (SHED), were injected in acupoint as an alternative therapy to minimize muscle degeneration in twenty-two mdx mice. The treatment occurred three times with intervals of 21 days, and animals were analyzed four times: seven days prior treatment (T-7); 10 days after first treatment (T10); 10 days after second treatment (T31); and 10 days after third treatment (T52). Animals were evaluated by wire test for estimate strength and blood was collected to perform a creatinine phosphokinase analysis. After euthanasia, cranial tibial muscles were collected and submitted to histological and immunohistochemistry analyses. Treated groups presented improvement of strength and reduced creatinine phosphokinase levels. Also, a slight dystrophin increase was observed in tibial cranial muscle when aquapuncture was associated SHED. All therapies have minimized muscle degeneration, but the association of aquapuncture with SHED appears to have better effect, reducing muscle damage, suggesting a therapeutic value.

Mesenchymal stem cells: emphasis in adipose tissue

The study of stem cells has evolved rapidly in recent decades. The importance is given to the concept that these cells are potentially able to become any cell type and have the power of self-renewal throughout the life of the organism. Mesenchymal stem cells (MSCs) can be isolated from various organs of the body such as bone marrow, adipose tissue, synovium, muscle and dermis, deciduous teeth, umbilical cord, placenta, liver, spleen and thymus. After their isolation in vitro, mesenchymal stem cells have the capacity to differentiate into various mesenchymal lineages and various tissues after the use of appropriate cultures. Studies have reported that mesenchymal stem cells from adipose tissue have the potential to differentiate themselves, like the cells commonly studied bone marrow. Adipose tissue is attractive due to its easy access, rapid expansion in vitro and only one collects the large amount of tissue. This review intends to show the protocols for isolation, cell culture and means of commercial cellular differentiation most widely used with emphasis on adipose tissue.

Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow.

During fetal development, mesenchymal progenitor (MP) cells are co-localized in major hematopoietic territories, such as yolk sac (YS), bone marrow (BM), liver (LV), and others. Studies using mouse and human MP cells isolated from fetus have shown that these cells are very similar but not identical to adult mesenchymal stem cells (MSC). Their differentiation potential is usually restricted to production of highly committed osteogenic and chondrogenic precursors. Such properties of fetal MP cells can be very useful for tissue regeneration, when a great number of committed precursors are required. The objectives of this study were to isolate and characterize MP cells from canine YS, BM, and LV in early and late stages of fetal development. Gestational stage was identified, and cell culture conditions were evaluated for efficient isolation of canine MP cells. All canine fetal MP cells expressed vimentin, nestin, and CD44 proteins. Cytokeratin 18 expression was observed in BM- and LV-MP cells, and vascular endothelial (VE)-cadherin expression was observed only in YS-MP cells. A small number of MP cells (5%) from LV and YS expressed Oct3/4 protein. The differentiation potential of canine fetal MP cells varied significantly: YS- and BM-MP cells differentiated into bone and cartilage, whereas LV-MP cells differentiation was limited to osteogenic fate. None of the canine fetal MP cells were able to differentiate into adipose cells. Our data suggest that canine fetal MP cells are an appropriate in vitro model to study MP biology from hematopoietic territories and they are a source of committed osteogenic and chondrogenic precursors for regenerative medicine.

Carcinoembryonic antigen (CEA) mimicry by an anti-idiotypic scFv isolated from anti-Id 6.C4 hybridoma.

Since carcinoembryonic antigen (CEA) is expressed during embryonic life, it is not immunogenic in humans. The use of anti-idiotypic (Id) antibodies as a surrogate of antigen in the immunization has been considered a promising strategy for breaking tolerance to some tumor associated antigens. We have described an anti-Id monoclonal antibody (MAb), designated 6.C4, which is able to mimic CEA functionally. The anti-Id MAb 6.C4 was shown to elicit antibodies that recognized CEA in vitro and in vivo. In the present study, we sought to verify whether a single chain (scFv) antibody obtained, the scFv 6.C4, would retain the ability to mimic CEA. Two scFv containing the variable heavy and light chain domains of 6.C4 were constructed with a 15-amino acid linker: one with and another without signal peptide. DNA immunization of mice with both forms of scFv individually elicited antibodies able to recognize CEA.

Efeito de mutações sítio-dirigidas nas hélices I, V e VI sobre a maturação, dimerização e interação com o agonista do receptor AT1 de angiotensina II / Effect of site-directed mutations in helices I, V and VI on the maturation, dimerization and interaction with agonist of angiotensin II AT1 receptor

Objetivos: Analisar a participação de resíduos aromáticos e alifáticos específicos encontrados na face mais externa das hélices I, V e VI do receptor AT1 na maturação, tráfego, formação do complexo agonista/receptor e dimerização. Métodos: Construção do vetor contendo o receptor A T 1 alinhado com a proteína fluorescente verde (EGFP) e realização de mutações pontuais nos resíduos V29 e 137 da hélice I, L195, L197, 1201, L202, L205 e F2o6 da hélice V e 1258, F259, F261, L262, L265 e 1266 da hélice VI para ácido glutâmico e alanina. Células HEK293 transfectadas com estas construções de forma transiente ou estável foram utilizadas para a realização dos experimentos de microscopia de fluorescência, ligação e afinidade à Ang II, BRET, análise do trafego subcelular, dimerização do receptor e experimentos funcionais através de ensaio de gene repórter. Resultados: O estudo da distribuição subcelular do receptor AT1 mostrou que o receptor selvagem encontra-se na membrana e que a substituição de resíduos para alanina, em todos os casos, não afetou sua localização, exceto no mutante L205A que foi encontrado no núcleo. Por outro lado, as mutações para ácido glutâmico, na maioria das vezes, afetaram o tráfego do receptor. Embora muitos dos mutantes da hélice V contendo ácido glutâmico tenham sido encontrados na membrana, todos os da hélice VI ficaram retidos no retículo endoplasmático. Da mesma forma, a afinidade pela Ang II nos mutantes para alanina não foi afetada, enquanto que as mutações para resultaram em diminuição da afinidade. A homodimerização do receptor A T 1 selvagem foi observada, sendo que a mutação dos resíduos em questão, que acarreta efeitos diversos não interferiu com a dimerização na maioria dos casos, como observado nos experimentos de BRET. Ensaios com o gene repórter para luciferase confirmaram a funcionalidade de alguns dos receptores mutantes do AT1 estudados aqui. É interessante que alguns mutantes para ácido glutâmico tiveram aumento na atividade da luciferase mesmo não sendo expressos na membrana plasmática como é o caso do mutante L 197E. Conclusões: Os resíduos alifáticos e aromáticos da hélice V do receptor AT1 são responsáveis por manter a configuração do sítio de ligação para Ang II. Além disso, os resultados do presente trabalho permitem sugerir que a face externa da hélice VI seja necessária para o correto dobramento do receptor AT1. Finalmente, nossos dados corroboram evidências anteriores que o receptor AT1 seja um homodímero.