Identificação do perfil imunofenotípico e da atividade proliferativa das células mononucleares da medula óssea em ratas ooforectomizadas e/ou tireoidectomizadas

Orientador: Katherine Athayde Teixeira de CarvalhoMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências BiológicasResumo : Hipotireoidismo é uma das patologias mais freqüentes em mulheres de meia idade, sendo que nesse período se dá o início do climatério. Com o objetivo de analisar as diferenças no aumento ou na diminuição da porcentagem de células da linhagem mononuclear da medula óssea, devido a falta de hormônios ovarianos e/ou tireoidianos, foi feita a ooforectomia e/ou tireoidectomia em ratas da linhagem Wistar adultas. Para a confirmação do estado de hipotireoidismo foi feito a dosagem de TSH, também foi feita a coloração de Papanicolau para comprovação do estado de diestro nas ratas ooforectomizadas. Para a obtenção das células mononucleares foi feita a punção e aspiração da medula-óssea da crista ilíaca e em seguida o isolamento das células mononucleares através do gradiente de densidade FicollHypaque. Foram usados marcadores específicos para proteínas de membrana de células endoteliais, células precursoras B/pró-B e células-tronco mesenquimais. Para a identificação e quantificação da porcentagem dessas diferentes populações, foi utilizado o citometro de fluxo. Foi comprovado que os grupos tireoidectomizados estavam com hipotireoidismo e os grupos ooforectomizados estavam no período diestro do cliclo estral. Os resultados mostram que os grupos com hipotireoidismo obtiveram um aumento na porcentagem de células endoteliais. Com a falta dos hormônios ovarianos houve um aumento nas células precursoras B/pró-B. Também houve diminuição da porcentagem de células-tronco mesenquimais nos grupos ooforectomizados. Foi visto que os hormônios ovarianos estão diretamente ligados na lifopoiése das células da linhagem B, juntamente com regulação da produção das células-tronco mesenquimais. No hipotireoidismo há um aumento das células endoteliais na medula óssea. Sendo assim, tanto os hormônios ovarianos e tireoidianos possuem funções fundamentais na regulação dessas três populações de células da medula óssea

Natural Membrane Differentiates Human Adipose-Derived Mesenchymal Stem Cells to Neurospheres by Mechanotransduction Related to YAP and AMOT Proteins

Adipose tissue-derived mesenchymal stem cells (ADMSCs) are promising candidates for regenerative medicine, as they have good cell yield and can differentiate into several cell lines. When induced to the neuronal differentiation, they form neurospheres composed of neural precursors (NPs) that can be an alternative in treating neurodegenerative diseases. This study aimed to characterize NPs from neurospheres obtained after seeding ADMSCs on a natural polyisoprene-based membrane. The ADMSCs were isolated from adipose tissue by enzymatic dissociation, were subjected to trilineage differentiation, and were characterized by flow cytometry for specific ADMSC surface markers. For neuronal differentiation, the cells were seeded on polystyrene flasks coated with the membrane and were characterized by immunocytochemistry and RT-PCR. The results demonstrated that the isolated cells showed characteristics of ADMSCs. At 15 to 25 days, ADMSCs seeded on the natural membrane developed neurospheres. Then, after dissociation, the cells demonstrated characteristic neuronal markers expressed on NPs: nestin, ß-III tubulin, GFAP, NeuN, and the YAP1/AMOT in the cytoplasm. In conclusion, it was demonstrated that this membrane differentiates the ADMSCs to NPs without any induction factors, and suggests that their differentiation mechanisms are related to mechanotransduction regulated by the YAP and AMOT proteins

The role of mitochondrial fusion and fission in the process of cardiac oxidative stress

Summary. Mitochondria are the energy suppliers in the cell and undergo constant fusion and fission to meet metabolic demand during the cell life cycle. Well- balanced mitochondrial dynamics are extremely important and necessary for cell survival as well as for tissue homeostasis. Cardiomyocytes contain large numbers of mitochondria to satisfy the high energy demand. It has been established that deregulated processes of mitochondrial dynamics play a major role in myocardial cell death. Currently, cardiac mitochondrial cell death pathways attract great attention in the cell biology and regenerative medicine fields. Importantly, mitochondrial dynamics are tightly linked to oxidative stress-induced cardiac damage. This review summarizes molecular mechanisms of mitochondrial fusion and fission processes and their potential roles in myocardial cell death triggered by oxidative stress. Advances in understanding the effect of both normal and abnormal mitochondrial dynamics on heart protection will lead to significant improvement of therapeutic discoverie

Laser Ablated Albumin Functionalized Spherical Gold Nanoparticles Indicated for Stem Cell Tracking

Cell tracking in cell-based therapy applications helps distinguish cell participation among paracrine effect, neovascularization, and matrix deposition. This preliminary study examined the cellular uptake of gold nanoparticles (AuNPs), observing cytotoxicity and uptake of different sizes and AuNPs concentrations in Adipose-derived stromal cells (ASCs). ASCs were incubated for 24 h with Laser ablated Albumin functionalized spherical AuNPs (LA-AuNPs), with average sizes of 2 nm and 53 nm in diameter, in four concentrations, 127 µM, 84 µM, 42 µM, and 23 µM. Cytotoxicity was examined by Live/Dead assay, and erythrocyte hemolysis, and the effect on the cytoskeleton was investigated by immunocytochemistry for β-actin. The LA-AuNPs were internalized by the ASCs in a size and concentration-dependent manner. Clusters were observed as dispersed small ones in the cytosol, and as a sizeable perinuclear cluster, without significant harmful effects on the cells for up to 2 weeks. The Live/Dead and hemolysis percentage results complemented the observations that the larger 53 nm LA-AuNPs in the highest concentrated solution significantly lowered cell viability. The demonstrated safety, cellular uptake, and labelling persistency with LA-AuNPs, synthesized without the combination of chemical solutions, support their use for cell tracking in tissue engineering applications

Adipose-Derived Stromal Cells and Mineralized Extracellular Matrix Delivery by a Human Decellularized Amniotic Membrane in Periodontal Tissue Engineering

Periodontitis is a prevalent disease characterized by the loss of periodontal supporting tissues, bone, periodontal ligament, and cementum. The application of a bone tissue engineering strategy with Decellularized Human Amniotic Membrane (DAM) with adipose-derived stromal cells (ASCs) has shown to be convenient and valuable. This study aims to investigate the treatments of a rat periodontal furcation defect model with DAM, ASCs, and a mineralized extracellular matrix (ECM). Rat ASCs were expanded, cultivated on DAM, and with a bone differentiation medium for four weeks, deposited ECM on DAM. Periodontal healing for four weeks was evaluated by micro-computed tomography and histological analysis after treatments with DAM, ASCs, and ECM and compared to untreated defects on five consecutive horizontal levels, from gingival to apical. The results demonstrate that DAM preserves its structure during cultivation and healing periods, supporting cell attachment, permeation, bone deposition on DAM, and periodontal regeneration. DAM and DAM+ASCs enhance bone healing compared to the control on the gingival level. In conclusion, DAM with ASC or without cells and the ECM ensures bone tissue healing. The membrane supported neovascularization and promoted osteoconduction

Demyelination Lesions Do Not Correlate with Clinical Manifestations by Bordetella pertussis Toxin Concentrations

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized as an inflammatory demyelinating disease. Given the need for improvements in MS treatment, many studies are mainly conducted through preclinical models such as experimental allergic encephalomyelitis (EAE). This study analyzes the relationships between histopathological and clinical score findings at EAE. Twenty-three female Rattus norvegicus Lewis rats from 6 to 8 weeks were induced to EAE. Nineteen rats underwent EAE induction distributed in six groups to establish the evolution of clinical signs, and four animals were in the control group. Bordetella pertussis toxin (PTX) doses were 200, 250, 300, 350 and 400 ng. The clinical scores of the animals were analyzed daily, from seven to 24 days after induction. The brains and spinal cords were collected for histopathological analyses. The results demonstrated that the dose of 250 ng of PTX induced a higher clinical score and reduction in weight. All induced groups demonstrated leukocyte infiltration, activation of microglia and astrocytes, and demyelinated plaques in the brains in histopathology. It was concluded that the dose of 250 ng and 350 ng of PTX were the best choices to trigger the brain and spinal cord demyelination lesions and did not correlate with clinical scores

The Neural Multilineage Differentiation Capacity of Human Neural Precursors from the Umbilical Cord—Ready to Bench for Clinical Trials

Mesenchymal stem cells (MSC) are promising for regenerative medicine as they have a vast differentiation capacity, immunomodulatory properties and can be isolated from different tissues. Among them, the umbilical cord is considered a good source of MSC, as its collection poses no risk to donors and is unrelated to ethical issues. Furthermore, umbilical cord mesenchymal stem cells (UC-MSC) can differentiate into several cell lines, including neural lineages that, in the future, may become an alternative in the treatment of neurodegenerative diseases. This study used a natural functional biopolymer matrix (NFBX) as a membrane to differentiate UC-MSC into neurospheres and their Neural precursors without using neurogenic growth factors or gene transfection. Through the characterization of Neural precursors and differentiated cells, it was possible to demonstrate the broad potential for the differentiation of cells obtained through cultivation on this membrane. To demonstrate these Neural precursors’ potential for future studies in neurodegenerative diseases, the Neural precursors from Wharton’s jelly were differentiated into Schwann cells, oligodendrocytes, cholinergic-, dopaminergic- and GABAergic-like neurons

Human Mesenchymal Stem Cells Seeded on the Natural Membrane to Neurospheres for Cholinergic-like Neurons

This study aimed to differentiate human mesenchymal stem cells (hMSCs) from the human umbilical cord in cholinergic-like neurons using a natural membrane. The isolation of hMSCs from Wharton’s jelly (WJ) was carried out using “explant” and mononuclear cells by the density gradient from umbilical blood and characterized by flow cytometry. hMSCs were seeded in a natural functional biopolymer membrane to produce neurospheres. RT-PCR was performed on hMSCs and neurospheres derived from the umbilical cord. Neural precursor cells were subjected to a standard cholinergic-like neuron differentiation protocol. Dissociated neurospheres, neural precursor cells, and cholinergic-like neurons were characterized by immunocytochemistry. hMSCs were CD73+, CD90+, CD105+, CD34- and CD45- and demonstrated the trilineage differentiation. Neurospheres and their isolated cells were nestin-positive and expressed NESTIN, MAP2, ßIII-TUBULIN, GFAP genes. Neural precursor cells that were differentiated in cholinergic-like neurons expressed ßIII-TUBULIN protein and choline acetyltransferase enzyme. hMSCs seeded on the natural membrane can differentiate into neurospheres, obtaining neural precursor cells without growth factors or gene transfection before cholinergic phenotype differentiation