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- ItemAcesso aberto (Open Access)Avaliação molecular e imunohistoquímica da uretra de ratas após trauma induzido e terapia intravenosa com células-tronco derivadas de músculo(Universidade Federal de São Paulo (UNIFESP), 2017-02-22) Bilhar, Andreisa Paiva Monteiro [UNIFESP]; Castro, Rodrigo de Aquino [UNIFESP]; Bortolini, Maria Augusta Tezelli [UNIFESP]; http://lattes.cnpq.br/1150368284144393; http://lattes.cnpq.br/6590913930590292; http://lattes.cnpq.br/8795441347109037; Universidade Federal de São Paulo (UNIFESP)Objective: To identify the migration to the urethra of muscle derived stem cells (MDSCs) after intravenous (IV) injection in rats submitted to pelvic floor trauma due to vaginal distension (DV) and to analyze the effects of MDSC in the urethra of rats after seven and thirty days of the trauma in regards to: (1) mRNA expression of collagens, vascular endothelial growth factor (VEGF), nerve growth factor (NGF), Ki67 cell proliferation marker, and the expression of genes related to smooth and striated muscle apparatus; (2) expression of smooth and striated muscle proteins. Methods: MDSCs expressing green fluorescent protein (GFP) were injected into the tail vein of the rats at day three after DV. The urethras of five groups were analyzed: Control, Trauma 7 days (animals submitted to DV, sacrificed 7 days), MDSC 7 days (animals submitted to DV receiving IV therapy with MDSC, sacrificed 7 days), Trauma 30 days (animals submitted to DV, sacrificed at 30 days), MDSC 30 days (animals submitted to DV receiving IV therapy with MDSC, sacrificed at 30 days). Location of GFP cells was verified at 2 hours and 7 days after IV injection. RT-qPCR was used for gene expression and immunohistochemistry for protein identification and quantification. Results: MDSCs were early identified in the urethra, but only deformed cells after 7 days. After seven days of trauma, the MYH11 gene was overexpressed in the Trauma group in relation to Control (p=0.01). Expression of the mKi67 gene was increased in the MDSC group relative to Trauma (p=0.03) and Control (p=0.02). Expression of the COL1A1 and COL3A1 genes increased in the MDSC group relative to the Control (p=0.001 and p=0.002). The gene expression of NGF was decreased in the MDSC group in relation to Trauma (p=0.001) and there was no difference in expression of the MYH2, Desmin and VEGF gene. In the same period, immunohistochemical expression of MHY11, MYH2 and Desmin was increased in the MDSC group in relation to Trauma (p=0.01, p=0.005 and p=0.02) and decreased in the Trauma group in relation to the Control (p =0.01, p=0.008 and p=0.03). After 30 days of trauma, MYH11 and MYH2 gene expression was increased in the Trauma group compared to Control (p=0.03 and p=0.04). Expression of the mKi67 gene was increased in the MDSC group relative to Trauma (p=0.02) and Control (p=0.01). Expression of the COL1A1 gene increased in the MDSC group compared to the Control (p=0.008) and Trauma (p=0.03) and COL3A1 gene expression increased in the MDSC group compared to Control (p=0.03). However, the gene expression of NGF was decreased in the MDSC group in relation to Trauma (p=0.002) and there was no difference in the gene expression of Desmin and VEGF. In the same period, immunohistochemical expression of MYH11, MYH2 and Desmin was increased in the MDSC group in relation to Trauma (p=0.01, p=0.002 and p=0.01) and MYH2 gene expression decreased in the Trauma group compared to Control (p=0.04). Conclusion: MDSCs migrated early to the traumatized urethra, but did not integrate into the tissue. IV administration of MDSC alters the expression of genes related to cell proliferation, neural growth factor and extracellular matrix and the expression of smooth and striated muscle proteins in traumatized rat urethra.
- ItemAcesso aberto (Open Access)Caracterização in vitro de neuroesferas e seu potencial de regeneração na doença de alzheimer e lesão por stab wound(Universidade Federal de São Paulo (UNIFESP), 2016-03-06) Paiva, Daisylea de Souza [UNIFESP]; Monteiro, Beatriz de Oliveira [UNIFESP]; http://lattes.cnpq.br/0245964878412260; http://lattes.cnpq.br/3693514872706694; Universidade Federal de São Paulo (UNIFESP)Objetctive: verify the role of neural stem cells (NSC) in three different aspects: in vitro behaviour, use in memory repair in transgenic animals for Alzheimer's Disease (DA, APPswe/PS1dE9) following transplantation, and expression of proteins related to injury response. Methods: We used in vitro techniques for cell culture of neuroespheres, genome-wide techniques as well as transplantation and behavioral tests. Results: We verified that the APPswe/PS1dE9 transgene affect neurospheres growth rate as well as promoting cellular death. Furthermore, is important to note that differentiated astrocytes from AD animals neurospheres presented hypertrophic morphology, alike the astrocytes from WT animals. Therefore, in this model is possible to identify alterations in NSC as well as soluble amyloid precursor protein secretion during the embrionic stage that can promote proliferation without interfere in the normal development of the animal. When transplanted in transfenic animals for DA, NSCs were able to contribute to neural repair by increasing neurogenesis and secretion of BDNF neurotrophin. Nevertheless there was no effect on memory and habituation in transplanted AD animals. Therefore we demonstrated that even without improving memory at behavioural level, NSC transpantation increased the neurotrophic support in hippocampus and can be responsible for other modifications at molecular level that should be better investigated. From the results of in vitro experiments and to better understand the behaviour of astrocytes in AD we decide to compare the expression pattern of proteins overexpressed at the lesion site of the SW (stab wound). To perform this analysis, we used the genome-wide data from Magdalena Götz's lab to select candidates that could play a role in the stab wound lesion and in AD. Among the top upregulated genes in reactive macroglia (astrocytes and NG2-glia), most of them were related to inflammation and, among these genes we selected four candidates (Osteopontin, CD68 and Galectin-1 and -3). We verified that although they were overexpressed in reactive macroglia after SW injury, the same cells do not express these proteins in AD animals. Whereby SW is an accute injury that is healed within few weeks, we assumed that the downregulation of these proteins could be related to the disease chronicity. Conclusion: By evaluating the in vitro features and role in neurodegeneration processes, we verified that when transplanted, NSC secrete factors that increase the neurotrophic support and during development can play a role in compensating cell death in AD animals. However, in old AD animals macroglial cells do not express proteins that could assist lesion resolution. This work offers insights about the role of NSC and macroglia in Alzheimer's disease.
- ItemAcesso aberto (Open Access)Comparação das características in vitro e perfil de expressão gênica entre células-tronco mesenquimais derivadas de pólipos nasais e da medula óssea(Universidade Federal de São Paulo (UNIFESP), 2018-11-07) Oliveira, Pedro Wey Barbosa De [UNIFESP]; Gregorio, Luis Carlos [UNIFESP]; http://lattes.cnpq.br/0512614520137100; http://lattes.cnpq.br/6030505570897126; Universidade Federal de São Paulo (UNIFESP)Introduction: Chronic rhinosinusitis (CRS) is clinically defined as persistent inflammation of the nasal mucosa and paranasal sinuses lasting at least 12 weeks;; Chronic rhinosinusitis with nasal polyposis is a disease with its pathophysiological mechanism not yet fully known;; it is believed that nasal polyps are formed by an imbalance in the inflammatory response and an imbalance of tissue remodeling. Classically, we observe a decrease in TGF beta and Treg determining a low immune response and an intense inflammatory response, especially an eosinophilic TH2 response. At the same time, we have an altered tissue repair mechanism with decreased fibrosis formation and intense mucosal edema, resulting in an altered extracellular matrix with elevation of hydrostatic pressure and possibly the formation of polyps. Mesenchymal stem cells (MSCs) are progenitor adult stem cells with the primary goal of supporting the tissue function in which they are in, but they also exhibit immunomodulation and tissue repair characteristics in the healing process. Therefore, we believe that it is fundamental to understand the behavior of the MSCs, especially the the MSC of the nasal polyp, in an attempt to elucidate the mechanism of its formation. Objective: To compare the in vitro characteristics and gene expression profile of mesenchymal stem cells of the nasal polyp (PO-MSC) with the mesenchymal stem cells of the bone marrow (BM-MSC). Materials and methods: We isolated the PO-MSC and BM-MSC and submitted the two groups to in vitro cell differentiation, immunophenotyping, proliferation and co-culture with lymphocytes and finally gene expression profile analysis of the two groups. Results and Discussion: The two groups showed fibroblastoid morphology and a similar potential for osteogenic / adipogenic differentiation, immunophenotyping showed that CTM-PN had a lack of molecules associated with the immune system. Co-cultures of peripheral blood and CTMs also showed a lower ability of CTM-PN to modulate the immune response. We detected in CTM-PN a distinct gene expression profile compared to CTM-MO. CTM- PN expressed higher levels of specific markers of progenitor stem cells (eg, CD133 and ABCB1), whereas the CTM-MO showed high expression of cytokines and growth factors (eg, FGF10, KDR and GDF6). Gene ontology analysis showed that the most expressed genes in the CTM-PN were related to the matrix remodeling process, hexose and glucose transport. CTM-MO showed highly expressed genes related to a distinct biological behavior such as angiogenesis, blood vessel morphogenesis, cell-cell signaling, and regulation of response to external stimuli. Conclusion: CTM-PN and CTM-MO are different sub-populations of CTMs, with distinct transcription profiles that reflect specific biological properties, compatible with the anatomical location of the tissue from which they were extracted. CTM-PN clearly has a lower immunomodulation capacity when compared to CTM-MO;; resulting in a reduced ability to control the local inflammatory process. The genes most significantly expressed in the CTM-PN are partially related to the process of tissue remodeling, extracellular matrix metabolism and tissue regeneration process;; physiopathological basis of RSCcPN. CTM-PN plays an important role in the development of nasal polyps.
- ItemAcesso aberto (Open Access)Comparação entre membrana amniótica com e sem epitélio como substrato para cultura de células epiteliais do limbo ex vivo(Conselho Brasileiro de Oftalmologia, 2011-04-01) Covre, Joyce Luciana [UNIFESP]; Loureiro, Renata Ruoco [UNIFESP]; Cristovam, Priscila Cardoso [UNIFESP]; Ricardo, José Reinaldo da Silva [UNIFESP]; Freymüller-Haapalainen, Edna [UNIFESP]; Gomes, José Álvaro Pereira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)PURPOSE: To evaluate the efficacy and ultrastructural aspects of human limbal epithelial cells cultured on amniotic membrane (AM) with and without epithelium. METHODS: Limbal epithelial cell cultures were established from cadaveric cor neo-scleral rim explants derived from 6 different donors. The explants from each donor were placed under 3 different groups: on human preserved AM with epithelium (Group 1), AM deepithelialized with trypsin (Group 2) and control (Group 3). The epithelial cell migration was evaluated under phase contrast microscopy. After 15 days, the amniotic membrane with cells cultures were removed and submitted to scanning and transmission electron microscopy to check for epithelial migration and adhesion. RESULTS: All epithelial cell cultures from the controls grew over the botton of the culture plate wells until reaching confluence. Epithelial cultures grew over all but one denuded amniotic membrane. In the group amniotic membrane with epithelium, epithelial cell growing was observed only in 1 well. CONCLUSIONS: Using this model, denuded amniotic membrane appeared to be the best substrate for epithelial cell migration and adhesion comparing to amniotic membrane with epithelium. Removal of amniotic membrane epithelial seems to be an important step for establishing limbal epithelial cell culture on amniotic membrane.
- ItemAcesso aberto (Open Access)Cytological features of live limbal tissue donor eyes for autograft or allograft limbal stem cell transplantation(Conselho Brasileiro de Oftalmologia, 2011-08-01) Barros, Jeison de Nadai [UNIFESP]; Santos, Myrna Serapião dos [UNIFESP]; Barreiro, Telma Regina Maria Pereira [UNIFESP]; Belfort, Rubens Junior [UNIFESP]; Gomes, José Álvaro Pereira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)PURPOSE: To evaluate by impression cytology (IC) the corneal surface of live limbal tissue donor eyes for autograft or allograft limbal stem cell transplantation (LSCT). METHODS: Twenty limbal donors were enrolled (17 for autograft LSCT and 3 for allograft). Impression cytology was performed before transplantation of superior and inferior limbal grafts and after the third postoperative month. RESULTS: Impression cytology analysis showed sheets of corneal epithelial cells and goblet cell absence beyond the edge of the keratectomy sites in all patients, suggesting that conjunctival invasion towards the center did not occur in any eye. Partial conjunctivalization within 2 to 3 clock hours, confirmed by the presence of goblet cells, was limited to the keratectomy site in 10% of the cases. CONCLUSION: A clear central corneal surface was demonstrated in all eyes following surgery leading to the conclusion that limbal donation was a safe procedure in this group of patients. A small percentage of eyes can have donor sites re-epithelized with conjunctival cells at the periphery of the cornea.
- ItemAcesso aberto (Open Access)Development of a rabbit's urethral sphincter deficiency animal model for anatomical-functional evaluation(Sociedade Brasileira de Urologia, 2012-02-01) Skaff, M. [UNIFESP]; Pinto, E.r.s. [UNIFESP]; Leite, Kátia Ramos Moreira [UNIFESP]; Almeida, F.g. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)OBJECTIVE: The aim of the study was to develop a new durable animal model (using rabbits) for anatomical-functional evaluation of urethral sphincter deficiency. MATERIALS AND METHODS: A total of 40 New Zealand male rabbits, weighting 2.500 kg to 3.100 kg, were evaluated to develop an incontinent animal model. Thirty-two animals underwent urethrolysis and 8 animals received sham operation. Before and at 2, 4, 8 and 12 weeks after urethrolysis or sham operation, it was performed cystometry and leak point pressure (LPP) evaluation with different bladder distension volumes (10, 20, 30 mL). In each time point, 10 animals (8 from the study group and 2 from the sham group) were sacrificed to harvest the bladder and urethra. The samples were evaluated by H&E and Masson's Trichrome to determine urethral morphology and collagen/smooth muscle density. RESULTS: Twelve weeks after urethrolysis, it was observed a significant decrease in LPP regardless the bladder volume (from 33.7 ± 6.6 to 12.8 ± 2.2 cmH2O). The histological analysis evidenced a decrease of 22% in smooth muscle density with a proportional increase in the collagen, vessels and elastin density (p < 0.01). CONCLUSIONS: Transabdominal urethrolysis develops urethral sphincter insufficiency in rabbits, with significant decrease in LPP associated with decrease of smooth muscle fibers and increase of collagen density. This animal model can be used to test autologous cell therapy for stress urinary incontinence treatment.
- ItemSomente MetadadadosEarly modulation of inflammation by mesenchymal stem cell after acute kidney injury(Elsevier B.V., 2009-06-01) Semedo, Patricia [UNIFESP]; Palasio, Carolina G. [UNIFESP]; Oliveira, Cassiano D. [UNIFESP]; Feitoza, Carla Q. [UNIFESP]; Goncalves, Giselle M. [UNIFESP]; Cenedeze, Marcos Antonio [UNIFESP]; Wang, Pamella M. H. [UNIFESP]; Teixeira, Vicente P. A.; Reis, Marlene A.; Pacheco-Silva, Alvaro [UNIFESP]; Saraiva Camara, Niels Olsen [UNIFESP]; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Triangulo Mineiro Med SchTherapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3-5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. in addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 x 105 cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 +/- 0.21 vs. 3.23 +/- 0.89 mg/dl, p<0.05). the improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-alpha and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. the decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. and also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. (C) 2009 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Efeito dos processos do cultivo celular de células-tronco endometriais humanas e sua influência na taxa gestacional de murinos submetidos à transferência embrionária(Universidade Federal de São Paulo (UNIFESP), 2017-12-05) Rochetti, Raquel Cellin [UNIFESP]; Lo Turco, Edson Guimarães [UNIFESP]; Barretto, Letícia Siqueira de Sá [UNIFESP]; http://lattes.cnpq.br/8335307682492956; http://lattes.cnpq.br/0251394799200508; http://lattes.cnpq.br/5659059615745740; Universidade Federal de São Paulo (UNIFESP)As células-tronco vêm sendo fortemente estudadas devido ao seu papel terapêutico. No ambiente uterino, as células-tronco endometriais (EnMSC) são participantes ativas da implantação embrionária atuando como imunomoduladoras dos processos inflamatórios decorrentes desse evento, porém o bom funcionamento e a quantidade dessas células podem influenciar no sucesso de adesão do embrião ao útero. Em terapias celulares, é comum a manipulação in vitro das células-tronco a fim de expandir e/ou armazenar as células para futuras aplicações, porém, processos in vitro podem interferir na qualidade das células influenciando o resultado final do tratamento. Desse modo, o objetivo desse estudo foi avaliar os efeitos dos processos do cultivo celular das EnMSC e observar a influência do transplante dessas células na taxa gestacional de murinos submetidos à transferência embrionária. Para isso, foram coletados tecidos endometriais de 5 voluntárias, isolados e cultivados in vitro para expansão das EnMSC até a terceira passagem. As EnMSC foram caracterizadas e avaliadas quanto ao crescimento celular e à peroxidação lipídica. Além disso, foi avaliado o perfil metabolômico presente no meio de cultivo condicionado. Em seguida, embriões murinos em fase de blastocisto foram transferidos para camundongas, com e sem o acréscimo de 1x105 células em cada lado do útero. Após o período gestacional foi avaliado a taxa de prenhez, a quantidade de camundongos nascidos e peso ao nascer. Como resultado, pudemos observar que em passagens baixas as EnMSC mantêm um padrão de crescimento in vitro similar entre as passagens, sem alteração no estresse oxidativo. Através da análise metabolômica foi observado um perfil metabólico diferenciado entre as passagens pré e pós-congelamento, de forma que a passagem pós-congelada teve maior abundancia de esfingosina e esfingosina-1-fosfato. Já na transferência embrionária, foi observado estresse das camundongas receptoras acompanhado de ato canibal, além disso, não foi observada diferença significante quanto à taxa de prenhez, número de nascidos e peso ao nascer. Assim, de acordo com os resultados foi possível concluir que o padrão de cultivo utilizado nesse estudo é viável para expansão in vitro das EnMSC e que o congelamento dessas células altera seu perfil metabólico, embora esses fatores não tenham influenciado na taxa de implantação de embriões transplantados às camundongas.
- ItemAcesso aberto (Open Access)Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector(Conselho Brasileiro de Oftalmologia, 2010-10-01) Oliveira, Lauro Augusto De [UNIFESP]; Kim, Charles; Sousa, Luciene Barbosa De [UNIFESP]; Schwab, Ivan R.; Rosenblatt, Mark I.; Universidade Federal de São Paulo (UNIFESP); University of California Department of Ophthalmology and Visual Science; Weill Cornell Medical College Department of OphthalmologyPURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP) reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80% confluency) prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS). We evaluated the transduction efficiency (TE) over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells). RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1%) and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7%. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.
- ItemAcesso aberto (Open Access)Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano(Conselho Brasileiro de Oftalmologia, 2008-10-01) Cristovam, Priscila Cardoso [UNIFESP]; Glória, Maria Aparecida da [UNIFESP]; Melo, Gustavo Barreto de [UNIFESP]; Gomes, José Álvaro Pereira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)PURPOSE: To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR). METHODS: Corneo-scleral rims from different donors (n=6) had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A). The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B) or without (Group C) irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM), the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining. RESULTS: Epithelial cell growth was observed in 4/6 rims (Group A). All epithelial cell suspensions that were cultured with 3T3 cells (Group B) formed clones. No adhesion or true clone formation (holo- or meroclones) was observed in the cell suspensions that were cultivated without 3T3 (Group C) (p=0.009). CONCLUSIONS: Epithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.
- ItemAcesso aberto (Open Access)Impression cytology and in vivo confocal microscopy in corneas with total limbal stem cell deficiency(Conselho Brasileiro de Oftalmologia, 2013-10-01) Araújo, Aline Lütz de [UNIFESP]; Ricardo, José Reinaldo da Silva [UNIFESP]; Sakai, Vivian Naomi [UNIFESP]; Barros, Jeison de Nadai [UNIFESP]; Gomes, José Álvaro Pereira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)PURPOSES: To describe corneal changes seen on in vivo confocal microscopy in patients with total limbal stem cell deficiency and to correlate them with cytological findings. METHODS: A prospective case series including 13 eyes (8 patients) with total limbal deficiency was carried out. Stem cell deficiency was diagnosed clinically and by corneal impression cytology. Confocal images of the central cornea were taken with the Heidelberg Retina Tomograph II, Rostock Corneal Module (Heidelberg Engineering, Heidelberg, Germany). RESULTS: Impression cytology of the cornea revealed conjunctival epithelial cells and goblet cells in all cases. In vivo confocal microscopy showed disruption of normal layers of the corneal epithelium in all eyes. Confocal images showed cells with characteristics of conjunctival epithelium at the cornea in 76.9% of the total. These findings on confocal microscopy were compatible to limbal stem cell deficiency. Additionally, goblet cells, squamous metaplasia, inflammatory cells and dendritic cells were observed. The sub-basal nerve plexus was not identified in any of the corneas. Corneal neovessels were observed at the epithelium and stroma. All cases showed diffuse hyper-reflective images of the stroma corresponding to opacity of the tissue. CONCLUSIONS: Limbal stem cell deficiency had been confirmed by impression cytology in all cases, and 76.9% of the cases could also be diagnosed by in vivo confocal microscopy through the conjunctival epithelial cell visualization on the corneal surface. Frequent confocal microscopy findings were abnormal cells at the cornea (conjunctival epithelial, goblet and inflammatory cells), corneal neovessels and diffuse hyper-reflection of the stroma.
- ItemSomente MetadadadosA Novel Approach for Subretinal Implantation of Ultrathin Substrates Containing Stem Cell-Derived Retinal Pigment Epithelium Monolayer(Karger, 2012-01-01) Hu, Yuntao; Liu, Laura; Lu, Bo; Zhu, Danhong; Ribeiro, Ramiro; Diniz, Bruno [UNIFESP]; Thomas, Padmaja B.; Ahuja, Ashish K.; Hinton, David R.; Tai, Yu-Chong; Hikita, Sherry T.; Johnson, Lincoln V.; Clegg, Dennis O.; Thomas, Biju B.; Humayun, Mark S.; Univ So Calif; CALTECH; Univ Calif Santa Barbara; Peking Univ; Chang Gung Mem Hosp; Hosp Evangel Curitiba; Universidade Federal de São Paulo (UNIFESP)Objective: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats. Methods: A platform device was used for the implantation of 4-mu m-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study. Spectral-domain optical coherence tomography (SD-OCT) scanning and histological examinations were performed to confirm placement location of the implant. hESC- RPE cells attached to the substrate before and after implantation were evaluated using standard cell counting techniques. Results: SD-OCT scanning and histological examination revealed that the substrates were precisely placed in the rat's subretinal space. the hESC-RPE cell monolayer that covered the surface of the substrate was found to be intact after implantation. Cell counting data showed that less than 2% of cells were lost from the substrate due to the implantation procedure (preimplantation count 2,792 +/- 74.09 cells versus postimplantation count 2,741 +/- 62.08 cells). Detailed microscopic examination suggested that the cell loss occurred mostly along the edges of the implant. Conclusion: With the help of this platform device, it is possible to implant ultrathin substrates containing an RPE monolayer into the rat's subretinal space. This technique can be a useful approach for stem cell-based tissue bioengineering techniques in retinal transplantation research. Copyright (c) 2012 S. Karger AG, Basel
- ItemSomente MetadadadosP27(Kip1) overexpression regulates IL-1 beta in the microenvironment of stem cells and eutopic endometriosis co-cultures(Academic Press Ltd- Elsevier Science Ltd, 2017) Gonçalves, Giovana Aparecida [UNIFESP]; Invitti, Adriana Luckow [UNIFESP]; Parreira, Rafael Martins [UNIFESP]; Kopelman, Alexander [UNIFESP]; Schor, Eduardo [UNIFESP]; Girão, Manoel João Batista Castello [UNIFESP]Endometriosis is a gynecological benign chronic disease defined as the growth of endometrial glands and stroma in extra-uterine sites, most commonly implanted over visceral and peritoneal surfaces within the female pelvis causing inflammatory lesions. It affects around 10% of the female population and is often accompanied by chronic pelvic pain, adhesion formation and infertility. Therefore, endometriosis could be considered a "social disease", since it affects the quality of life, reproductivity and also has a socioeconomic impact. The expression of cell cycle and inflammatory proteins is modified in the endometriotic tissues. Immunostaining of glandular and stromal cells in endometrial biopsies obtained from patients with endometriosis compared with those of healthy control demonstrated that endometriotic tissues have lower levels of p27(kaP1) protein. Endometriosis endometrial cells cultures have also lower levels of p27(kip1) compared to health endometrial cells cultures and restore the cell cycle balance when transduced with an adenoviral vector carring the p27(kiP1) coding gene (Adp27EGFP). The low levels of p27(kip1) are related to the S phase in the cell cycle, whereas higher levels lead to a G1 cell cycle arrest. The inflammatory cytokine IL-1 beta was recently identified as another key protein in the endometriosis proliferation. This cytokine has elevated levels during the proliferative and secretory phases of the menstrual cycle. In endometriosis endometrial cells cultures the IL-beta stimulates the production of IL-6 and IL-8, increasing the cell proliferation and reducing the apoptosis and Bax expression in these cells. According to these remarks, this work aims to evaluate the inflammatory effects in vitro, but more next to what happens in a woman's body, associating endometrial cells with stem cells, thus mimicking the endometrial microenvironment, with gene therapy using Adp27, notoriously known as controller cell cycle, apoptosis and potent modulator of VEGF expression. (C) 2015 Elsevier Ltd. All rights reserved.
- ItemSomente MetadadadosPhysicochemical characterization of ferumoxytol, heparin and protamine nanocomplexes for improved magnetic labeling of stem cells(Elsevier Science Bv, 2017) Bryant, L. Henry, Jr.; Kim, Saejeong J.; Hobson, Matthew; Milo, Blerta; Kovacs, Zsofia I.; Jikaria, Neekita; Lewis, Bobbi K.; Aronova, Maria A.; Sousa, Alioscka A. {UNIFESP}; Zhang, Guofeng; Leapman, Richard D.; Frank, Joseph A.Stem cell-based therapies have become a major focus in regenerative medicine and to treat diseases. A straightforward approach combining three drugs, heparin (H), protamine (P) with ferumoxytol (F) in the form of nanocomplexes (NCs) effectively labeled stem cells for cellular MRI. We report on the physicochemical characteristics for optimizing the H, P, and F components in different ratios, and mixing sequences, producing NCs that varied in hydrodynamic size. NC size depended on the order in which drugs were mixed in media. Electron microscopy of HPF or FHP showed that F was located on the surface of spheroidal shaped HP complexes. Human stem cells incubated with FHP NCs resulted in a significantly greater iron concentration per cell compared to that found in HPF NCs with the same concentration of F. These results indicate that FHP could be useful for labeling stem cells in translational studies in the clinic. Published by Elsevier Inc.
- ItemSomente MetadadadosPluripotent stem cell transcription factors during human odontogenesis(Springer, 2013-09-01) Cunha, Juliana Malta da [UNIFESP]; Costa-Neves, Adriana da; Kerkis, Irina; Pereira da Silva, Marcelo Cavenaghi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Inst ButantanStem cells are capable of generating various cell lines and can be obtained from adult or embryonic tissues for clinical therapies. Stem cells from deciduous dental pulp are among those that are easily obtainable from adult tissues and have been widely studied because of their ability to differentiate into a variety of cell lines in the presence of various chemical mediators. We have analyze the expression of several proteins related to the differentiation and proliferative potential of cell populations that compose the tooth germ of human fetuses. We evaluate 20 human fetuses of both genders. After being paraffin-embedded, cap and bell stages of tooth germ development were subjected to immunohistochemistry for the following markers: Oct-4, Nanog, Stat-3 and Sox-2. the studied antibodies showed nuclear or cytoplasmic immunnostaining within various anatomical structures and with various degrees of expression, indicating the action of these proteins during tooth development. We conclude that the interrelationship between these transcription factors is complex and associated with self-renewal and cell differentiation. Our results suggest that the expression of Oct-4, Nanog, Sox-2 and Stat-3 are related to differentiation in ameloblasts and odontoblasts.
- ItemSomente MetadadadosRobot-assisted ureteral reconstruction using a tubularized peritoneal flap: a novel technique in a chronic porcine model(Springer, 2017) Brandao, Luis Felipe [UNIFESP]; Laydner, Humberto; Akca, Oktay; Autorino, Riccardo; Zargar, Homayoun; De, Shubha; Krishnam, Jayram; Pallavi, Patil; Monga, Manoj; Stein, Robert J.; Magi-Galluzzi, Cristina; Andreoni, Cassio [UNIFESP]; Kaouk, Jihad H.To evaluate the feasibility and functional outcomes in porcine models of a novel robotic surgical technique for the treatment of complex ureteral injuries and strictures. Six pigs underwent robotic ureteral reconstruction using a long tabularized peritoneal flap and followed for 6-9 weeks after the surgery. Ureteral flap vascularity, intra-renal pressure, patency of the conduct, endoscopic aspect of the flap, renal function and histopathology were evaluated. All animals successfully underwent ureteral reconstruction using a tubularized peritoneal flap. Median operative time was 223 min (162-360). Flap tubularization suture took 31 min (19-47), and proximal anastomosis took 20 min (15-38). Bladder mobilization with psoas hitch and distal anastomosis took 9 min (7-12) and 23 min (13-46), respectively. On follow-up, significant shrinkage of the ureteral flap in both length and width was observed. Antegrade pyelograms confirmed dilation and tortuosity of the proximal ureter, dilation of the renal pelvis, and major and minor calyxes without any definitive strictures. Microscopically, focal urothelial lining was seen in the neoureter. Creatinine level was significantly higher at the end of the follow-up period (p = 0.003). Robot-assisted ureteral reconstruction using a tubularized peritoneum flap is technically feasible and reproducible. The flap sustained abundant vascular supply after different intervals of follow-up and the peritoneal mesenchymal cells differentiated into urothelium and myofibroblasts. Further studies are needed to address the issue of functional obstruction to improve long-term renal function outcomes.
- ItemAcesso aberto (Open Access)Terapia intravenosa com células-tronco derivadas de tecido adiposo (ADSC) de ratas com trauma vaginal : uma avaliação da musculatura uretral quanto aos aspectos histopatológicos e à expressão imunohistoquímica de MYH11, MYH2 e desmina(Universidade Federal de São Paulo (UNIFESP), 2017) Sé, Alexandre Brandão [UNIFESP]; Castro, Rodrigo de Aquino [UNIFESP]; Bortolini, Maria Augusta Tezelli; http://lattes.cnpq.br/1150368284144393; http://lattes.cnpq.br/6590913930590292; http://lattes.cnpq.br/4355231135859354; Universidade Federal de São Paulo (UNIFESP)Objetivo: Analisar, no trigésimo dia pós-trauma, o efeito da injeção intravenosa de ADSC na uretra de ratas 72 horas após a realização de trauma por distensão vaginal. A análise foi realizada por meio de avaliação histopatológica e da expressão imunohistoquímica das proteínas MYH11, MYH2 e Desmina. Métodos: As ratas foram divididas em grupos Controle, distensão vaginal (VD) sem tratamento (trauma) e distensão vaginal seguido por uma injeção de ADSCs na veia da cauda da rata (trauma + ADSC). Os grupos com VD foram sacrificados 30 dias após o trauma. As uretras foram incubadas com anticorpos primários dirigidos contra desmina, miosina de cadeia pesada de músculo estriado (MYH2) e miosina de cadeia pesada de músculo liso (MYH11). Foram realizadas duas análises nos três diferentes grupos (Controle, Trauma e Trauma + ADSC). A primeira análise foi histopatológica qualitativa. A segunda, foi uma avaliação imunohistoquímica, que denominamos de quantitativa. Resultados: A avaliação histopatológica no trigésimo dia após o trauma mostrou que as uretras das ratas do grupo Trauma apresentaram maior edema, dissociação das fibras musculares e alargamento do lúmen em relação às uretras das ratas controle e das tratadas com células-tronco. As uretras de ratas tratadas com células-tronco apresentaram maior espessura das camadas musculares (miosina de cadeia pesada do músculo liso e estriado e desmina) em relação aos grupos Controle e Trauma. Nesse mesmo período, a expressão imunohistoquímica de MYH11, MYH2 e Desmina não mostrou diferenças estatísticas significativas entre os grupos Controle, Trauma e Trauma + ADSC (p=0,43; p=0,48 e p=0,54) respectivamente. Conclusão: Na avaliação qualitativa após trinta dias do trauma simulado e a injeção na veia da cauda da rata de ADSC observamos uma recuperação do tecido uretral. Já na avaliação quantitativa das proteínas musculares MYH11, MYH2 e Desmina não observamos alterações estatisticamente significativas.