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Inflammation is observed in many tumors, which affects metastasis, infiltration, and immune escape and causes poor differentiation of the cancer cells. However, the molecular basis underlying the relationship between inflammation and poor differentiation in tumors has not been identified. In this study, we demonstrate that angiopoietin-like protein-8 (ANGPTL8), which is induced by stress stimuli such as inflammation, is involved in the maintenance of the undifferentiated state of clear cell renal cell carcinoma (ccRCC) cells. ANGPTL8 is also involved in the production of chemokines that attract immune suppressor cells to the tumor microenvironment. ANGPTL8 sustains the continuous production of chemokines by activating the NF-κB signaling pathway and maintains the undifferentiated state of ccRCC cells. Finally, ANGPTL8 is induced by STAT3 signaling, which is activated by immune cells in the tumor microenvironment. These results support a role for ANGPTL8 in determining the properties of ccRCC by hampering tumor cell differentiation and establishing the tumor microenvironment.
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Proteína 8 Semelhante a Angiopoietina , Carcinoma de Células Renais , Neoplasias Renais , Hormônios Peptídicos , Humanos , Proteína 8 Semelhante a Angiopoietina/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Diferenciação Celular , Inflamação , Neoplasias Renais/genética , Hormônios Peptídicos/metabolismo , Microambiente TumoralRESUMO
In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.
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Embryonic stem cells (ES cells) express a transient and heterogeneous pattern of molecules, which suggests a notable mechanism to control self-renewal avoid the differentiation into germ layers. We show that 9-O-acetyl GD3 (9OacGD3), a highly expressed b-series ganglioside in neural stem (NS) cells, is expressed in undifferentiated mouse ES cells in a heterogeneous fashion. After sorting, undifferentiated 9OacGD3(+) ES cell population had higher levels of nestin and Sox2 mRNA than the 9OacGD3(-) cells. Even with elevated expression of these neural transcription factors, 9OacGD3(+) cells did not give rise to more neural progenitors than 9OacGD3(-) cells. Expression of 9OacGD3 was recovered from 9OacGD3(-) cell population, demonstrating that expression of this ganglioside in mouse embryonic stem cells is transient, and does not reflect cell fate. Our findings show that the ganglioside 9OacGD3 is expressed heterogeneously and transiently in ES cells, and this expression corresponds to higher levels of Sox2 and Nestin transcripts.
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Células-Tronco Embrionárias/metabolismo , Gangliosídeos/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Gangliosídeos/metabolismo , Camundongos , Nestina/genética , Nestina/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
The presence of residual undifferentiated pluripotent stem cells (PSCs) in PSC-derived cell therapy products (CTPs) is a major safety issue for their clinical application, due to the potential risk of PSC-derived tumor formation. An international multidisciplinary multisite study to evaluate a droplet digital PCR (ddPCR) approach to detect residual undifferentiated PSCs in PSC-derived CTPs was conducted as part of the Health and Environmental Sciences Institute Cell Therapy-TRAcking, Circulation & Safety Technical Committee. To evaluate the use of ddPCR in quantifying residual iPSCs in a cell sample, different quantities of induced pluripotent stem cells (iPSCs) were spiked into a background of iPSC-derived cardiomyocytes (CMs) to mimic different concentrations of residual iPSCs. A one step reverse transcription ddPCR (RT-ddPCR) was performed to measure mRNA levels of several iPSC-specific markers and to evaluate the assay performance (precision, sensitivity, and specificity) between and within laboratories. The RT-ddPCR assay variability was initially assessed by measuring the same RNA samples across all participating facilities. Subsequently, each facility independently conducted the entire process, incorporating the spiking step, to discern the parameters influencing potential variability. Our results show that a RT-ddPCR assay targeting ESRG, LINC00678, and LIN28A genes offers a highly sensitive and robust detection of impurities of iPSC-derived CMs and that the main contribution to variability between laboratories is the iPSC-spiking procedure, and not the RT-ddPCR. The RT-ddPCR assay would be generally applicable for tumorigenicity evaluation of PSC-derived CTPs with appropriate marker genes suitable for each CTP.
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BACKGROUND AND OBJECTIVES: Outbred mice are widely used in toxicology, pharmacology, and fundamental biomedical research. However, there have been no reports of in vitro culture systems for spermatogonial stem cells (SSCs) derived from these mice. METHODS: As a step towards constructing a non-cellular niche supporting the in vitro maintenance of outbred mouse SSC self-renewal, we systematically investigated the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in SSCs derived from Imprinting Control Region (ICR) mice. RESULTS: Among the genes encoding 25 integrin subunits, integrin α1, α5, α6, α9, αV, and αE, and integrin ß1 and ß5 had significantly higher transcriptional levels than the other subunits. Furthermore, at the translational level, integrin α5, α6, α9, αV, αE, and ß1 were localized on the surface of SSCs, but integrin α1 and ß5 not. Moreover, significantly stronger translational expression than integrin α9 and αE was observed in integrin α5, α6, αV, and ß1. SSCs showed significantly increased adhesion to fibronectin, laminin, tenascin C and vitronectin, and functional blocking of integrin α5ß1, α6ß1, α9ß1 or αVß1 significantly inhibited adhesion to these molecules. CONCLUSIONS: We confirmed that integrin α5ß1, α6ß1, α9ß1 and αVß1 actively function on the surface of undifferentiated SSCs derived from outbred ICR mice.
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Originally presented at the Journal's one day conference entitled 'Displacement: Contemporary Traumatic Experience' held in London in November 2019, this paper expands on the author's theory of the implicit psychological organizing gestalt, an associated pattern of psychic functions which operate in an integrated way to simultaneously structure and organize our experience of self-cohesion and self-continuity. The gestalt, which implicitly links the formation of psychic skin, body image, cultural skin and both personal and cultural identity with place, functions as an emergent non-conscious permanent presence or background 'constant'. It develops over time and emerges out of embodied emotional experiencing with the total environment - both human and non-human. The author argues that it is the rupture of this gestalt and the disorganizing consequences of its loss which underlies the experience of displacement trauma. If disruptions in the formation of the gestalt and/or its later rupture remain unrecognized and unrepresented then the absence creates a void which can be intergenerationally transmitted. Case material is presented which describes this and which highlights the ways in which the gestalt can contribute to our understanding of collective displacement anxiety, cultural trauma and cultural complexes.
Cet article a été présenté initialement à la conférence du Journal intitulée « Le Déplacement: Une expérience traumatique contemporaine ¼ qui s'est tenue à Londres en novembre 2019. Il développe la théorie de l'auteur d'une gestalt implicite d'organisation psychologique, un schéma associé de fonctions psychiques qui opère de manière intégrée pour simultanément structurer et organiser notre expérience de cohésion de soi et de continuité de soi. Cette gestalt, qui relie implicitement la formation de la peau psychique, de l'image du corps, de la peau culturelle et de l'identité personnelle et culturelle avec le lieu, fonctionne comme une présence permanente, non-consciente et émergente, une « constante ¼ de fond: elle se développe au fil du temps et émerge à partir de l'expérience émotionnelle incarnée avec l'environnement total - humain et non-humain. L'auteur soutient que c'est la rupture de cette gestalt et les conséquences perturbantes de sa perte qui sous-tendent l'expérience du traumatisme de déplacement. Si des perturbations dans sa formation et/ou sa rupture ultérieure restent non-reconnues et non-représentées, alors l'absence crée un vide qui peut être transmis d'une génération à l'autre. L'article présente du matériel clinique décrivant ceci. Ce matériel souligne les façons dont cette gestalt peut contribuer à notre compréhension de l'angoisse collective de déplacement et du traumatisme culturel.
Presentado inicialmente en la Conferencia del Journal, titulada 'Desplazamiento: Experiencia Traumática Contemporánea' llevada a cabo en Londres, en Noviembre 2019, el presente trabajo amplía - sobre la base de la teoría de la autora sobre la Gestalt de organización psicológica implícita - un patrón asociado de funciones psíquicas, las cuales operan en un modo integrado para estructurar simultáneamente nuestra experiencia de auto-continuidad y auto-cohesión. La Gestalt, la cual implícitamente vincula la formación de la piel psíquica, la imagen corporal, la piel cultural y la identidad personal y cultural, con el lugar, funciona como una presencia permanente, emergente, no-consciente o un 'constante' contexto: se desarrolla a través del tiempo y emerge a partir de la experiencia emocional corporizada con la totalidad del medio ambiente - humano y no-humano. La autora argumenta que es la ruptura de esta Gestalt y las consecuencias desorganizadoras de su pérdida, la cual subyace a la experiencia de trauma por desplazamiento. Si las interrupciones en su formación y/o ruptura permanecen sin ser reconocidas y sin representación, entonces la ausencia crea un vacío que puede transmitirse intergeneracionalmente. Se presenta material de un caso que describe y subraya los modos en los cuales la Gestalt puede contribuir a nuestra comprensión sobre la ansiedad por desplazamiento colectivo y trauma cultural.
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Trauma Psicológico/psicologia , Refugiados/psicologia , Feminino , Trauma Histórico/psicologia , Humanos , Pessoa de Meia-IdadeRESUMO
To date, in vitro culture systems able to sufficiently expand the small population of spermatogonial stem cells (SSCs), a tool for the development of sperm-mediated gene transfer techniques in transgenic pigs, in the porcine seminiferous tubule have not been reported. Therefore, as a step toward engineering a noncellular niche to support the in vitro maintenance of porcine SSC self-renewal, we investigated the types of integrin heterodimers that are expressed and functional on their membrane. The α and ß integrin subunit protein expressions were analyzed using immunocytochemistry and fluorescence immunoassay, and the function of integrin heterodimers was confirmed by attachment and antibody inhibition assays. The integrin subunits, α3, α4, α5, α6, α8, α9, αV, and ß1, were identified on the surface of them. Moreover, they showed significantly increased adhesion to fibronectin, laminin, and vitronectin, and functional blocking of integrin α4ß1, α6ß1, or αVß1 significantly inhibited adhesion to these molecules. They showed significantly decreased adhesion to tenascin C and functional blocking of integrin α5ß1 did not significantly inhibit adhesion to fibronectin. Accordingly, we confirmed that the integrin heterodimers α4ß1, α6ß1, and αVß1 actively function on the surface of undifferentiated porcine SSCs, whereas α3, α5, α8, and α9 are present in inactive forms.
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Células-Tronco Germinativas Adultas/metabolismo , Integrinas/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Masculino , Espermatogônias/metabolismo , Sus scrofaRESUMO
Cell sheets technology is being available for fracture healing. This study was performed to clarify bone healing mechanism of undifferentiated (UCS) and osteogenic (OCS) differentiated mesenchymal stromal cell (MSC) sheets in the fracture model of dogs. UCS and OCS were harvested at 10 days of culture. Transverse fractures at the radius of six beagle dogs were assigned into three groups (n = 4 in each group) i.e. UCS, OCS and control. The fractures were fixed with a 2.7 mm locking plate and six screws. Cell sheets were wrapped around the fracture site. Bones were harvested 8 weeks after operation, then scanned by micro-computed tomography (micro-CT) and analyzed histopathologically. The micro-CT revealed different aspects of bone regeneration among the groups. The percentages of external callus volume out of total bone volume in control, UCS, and OCS groups were 42.1, 13.0 and 4.9% (p < 0.05) respectively. However, the percentages of limbs having connectivity of gaps were 25, 12.5 and 75% respectively. In histopathological assessments, OCS group showed well organized and mature woven bone with peripheral cartilage at the fracture site, whereas control group showed cartilage formation without bone maturation or ossification at the fracture site. Meanwhile, fracture site was only filled with fibrous connective tissue without endochondral ossification and bone formation in UCS group. It was suggested that the MSC sheets reduced the quantity of external callus, and OCS induced the primary bone healing.
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Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells.
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Anticorpos Monoclonais/farmacologia , Carboidratos/química , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polpa Dentária/citologia , Células-Tronco/citologia , Adolescente , Adulto , Anticorpos Monoclonais/isolamento & purificação , Antígenos de Superfície/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Dentina/metabolismo , Epitopos/metabolismo , Humanos , Ligação Proteica/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Tunicamicina/farmacologia , Adulto JovemRESUMO
Human pluripotent stem cells (hPSCs), represented by embryonic stem (hESCs) and induced pluripotent stem cells (hiPSCs), are attracting increasing attention in various research fields. However, their application in a clinical scenario must overcome an important hurdle given that these cells are potentially tumorigenic. This inherent problem becomes more significant as the number of transplanted cells becomes larger. In this Progress Report, recent findings concerning a novel glycan marker for hPSCs are described, as well as attempts made in relation to its practical application to regenerative medicine. In line with current thinking in the glycoscience field, it is assumed that cellular glycomes are closely related to cell functions. Based on this premise, hESCs and hiPSCs are analyzed by an advanced glycan profiling technology--the high-density lectin microarray. It is found that all human iPSCs derived from different tissular origins show essentially the same glycan profiles, which are typified by several characteristic structural features. In addition, a recombinant lectin probe, rBC2LCN, which shows rigorous specificity to H type 1 and 3 glycan structures, is found to serve as an excellent probe for hPSCs.
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Biomarcadores/metabolismo , Pesquisa Biomédica/métodos , Sondas Moleculares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Polissacarídeos/metabolismo , Medicina Regenerativa/métodos , Animais , Membrana Celular , Humanos , Polissacarídeos/químicaRESUMO
BACKGROUND CONTEXT: The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. PURPOSE: We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. STUDY DESIGN/SETTING: A prospective study using a rodent model of posterolateral spinal fusion was carried out. PATIENT SAMPLE: Thirty-six syngeneic Fischer rats comprised the patient sample. METHODS: Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. RESULTS: Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and ß-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). CONCLUSION: In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct.
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Proteína Morfogenética Óssea 2/farmacologia , Células-Tronco Mesenquimais/citologia , Fusão Vertebral/métodos , Fator de Crescimento Transformador beta/farmacologia , Animais , Células Cultivadas , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/farmacologia , Engenharia Tecidual/métodosRESUMO
Understanding of the fundamental mechanisms that govern adhesive properties of human induced pluripotent stem cells (hiPSCs) to culture environments provides surface design strategies for maintaining their undifferentiated state during cell expansion. Polyamidoamine dendrimer surface with first-generation (G1) with dendron structure was used for co-cultures of hiPSCs and SNL feeder cells that formed tightly packed compact hiPSC colonies, similar to those on a conventional gelatin-coated surface. hiPSCs passaged up to 10 times on the G1 surface maintained their undifferentiated state. Immunostaining and reverse transcriptase PCR analysis of fibronectin showed that the secreted fibronectin matrix from feeder cells on the G1 surface contributed to hiPSC attachment. Compared with cells on the gelatin-coated surface, F-actin and paxillin immunostaining revealed a well-organized network of actin stress fibers and focal adhesion formation at cell-substrate sites in hiPSC colonies on the G1 surface. E-cadherin expression levels on these surfaces were almost same, but paxillin and Rac1 expression levels on the G1 surface were significantly higher than those on the gelatin-coated surface. Zyxin showed prominent expression on the G1 surface at sites of focal adhesion and cell-cell contact in colonies, whereas zyxin expression on the gelatin-coated surface was not observed in regions of cell-cell contact. These findings indicate that transduction of mechanical stimuli through actin polymerization at sites of focal adhesion and cell-cell contact results in maintenance of undifferentiated hiPSC colonies on G1 surface. The G1 surface enables a substrate design based on the mechanical cues in the microenvironment from feeder cells to expand undifferentiated hiPSCs in long-term culture.
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Técnicas de Cocultura , Citoesqueleto/metabolismo , Dendrímeros/metabolismo , Fibronectinas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Actinas/metabolismo , Caderinas/metabolismo , Adesão Celular , Diferenciação Celular , Proliferação de Células , Dendrímeros/química , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Gelatina/metabolismo , Humanos , Paxilina/metabolismo , Poliaminas/metabolismo , Zixina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
O presente trabalho tem como objetivo investigar a demanda de tratamento em psicoterapia de casal, buscando compreender as repercussões das contradições entre a queixa inicial e as questões latentes na condução do encaminhamento, bem como o manejo clínico necessário diante da ambiguidade do pedido de ajuda. Desenvolvemos uma investigação clínico-qualitativa, a partir de um estudo de caso. O atendimento ocorreu em uma clínica-escola de uma universidade privada da cidade do Rio de Janeiro. A psicoterapia de casal transcorreu em coterapia, durante três meses, com frequência de uma vez por semana, estando em supervisão semanalmente. Verificamos que, muitas vezes, a demanda do casal por tratamento é fortemente ambígua, podendo desencadear impasses para o psicoterapeuta, como intensas inquietações na condução das intervenções terapêuticas e dúvidas na indicação adequada de tratamento.
This study aims to investigate the demand for treatment in couples psychotherapy. It seeks to understand the repercussions of contradicting initial complaints and latent issues in the conduct requiring referral, as well as the necessary clinical management due to the ambiguity in the request for help. We developed a clinical-qualitative investigation, based on a case study. The treatment took place at a private university school-clinic in the city of Rio de Janeiro. The couples psychotherapy occurred in co-therapy, once a week for three months with weekly supervisions. We verified that the couples' demand for treatment is often very ambiguous, which can lead to deadlocks for the psychotherapist, such as intense concerns about the conduct of therapeutic interventions and doubts regarding the recommendations for the adequate treatment.
El presente trabajo tiene como objetivo investigar la demanda de tratamiento en psicoterapia de parejas. Busca comprender las repercusiones de las contradicciones entre la queja inicial y las cuestiones latentes en la orientación del tratamiento, como el manejo clínico necesario ante la ambigüedad de la solicitud de ayuda. Desarrollamos una investigación clínico-cualitativa, basada en un estudio de caso. El caso tuvo lugar en una clínica-escuela de una universidad privada en la ciudad de Rio de Janeiro. La psicoterapia de pareja transcurrió en coterapia, una vez por semana durante tres meses con supervisión semanal. Verificamos que, a menudo, la demanda de la pareja por el tratamiento es fuertemente ambigua, pudiendo desencadenar dificultad al psicoterapeuta, como las preocupaciones intensas sobre la realización de intervenciones terapéuticas y las dudas con respecto a las recomendaciones de tratamiento adecuadas.
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Humanos , Masculino , Feminino , Adulto , Psicoterapia , Terapia de CasalRESUMO
Human pluripotent embryonic stem cells (hESCs) have great promise for research into human developmental biology, development of cell therapies for the treatment of diseases, toxicology, and drug discovery. Traditionally, undifferentiated hESCs are maintained on mouse embryonic fibroblasts (MEFs), which impede the clinical applications of hESCs. Here we have examined the long-term stability of the Japanese hESC line (KhES-1) in feeder-free culture. KhES-1 cells were cultured with MEF conditioned medium (CM) and different doses of basic fibroblast growth factor (bFGF) in six-well-plates of which the surface was coated with Matrigel. KhES-1 cells were maintained for at least 40 passages. In this culture system, the cells maintained stable proliferation rates and steadily expressed Oct-4, Nanog, and alkaline phosphatase. In addition, KhES-1 cells maintained without direct feeder contact formed embryonic bodies with expression of markers from the three germ layers. Here we demonstrated that Japanese human embryonic stem cells KhES-1 were cultured long term in a feeder-free method, while retaining pluripotency in vitro.