RESUMO
Pituitary cells that express the transcription factor SOX2 are stem cells because they can self-renew and differentiate into multiple pituitary hormone-producing cell types as organoids. Wounding and physiological challenges can activate pituitary stem cells, but cell numbers are not fully restored, and the ability to mobilize stem cells decreases with increasing age. The basis of these limitations is still unknown. The regulation of stem cell quiescence and activation involves many different signalling pathways, including those mediated by WNT, Hippo and several cytokines; more research is needed to understand the interactions between these pathways. Pituitary organoids can be formed from human or mouse embryonic stem cells, or from human induced pluripotent stem cells. Human pituitary organoid transplantation is sufficient to induce corticosterone release in hypophysectomized mice, raising the possibility of therapeutic applications. Today, pituitary organoids have the potential to assess the role of individual genes and genetic variants on hormone production ex vivo, providing an important tool for the advancement of exciting frontiers in pituitary stem cell biology and pituitary organogenesis. In this article, we provide an overview of notable discoveries in pituitary stem cell function and highlight important areas for future research.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Hipófise/metabolismo , Fatores de Transcrição/metabolismo , Transdução de Sinais , Diferenciação CelularRESUMO
Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial.W In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G - cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.
Assuntos
Edição de Genes/métodos , Antígenos HLA-G/genética , Antígenos HLA-G/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Antígenos HLA-G/imunologia , Humanos , Imunoterapia/métodos , RNA Guia de Cinetoplastídeos , TransfecçãoRESUMO
The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin.
Assuntos
Fertilização in vitro , Zigoto , Animais , Bovinos , Células Epiteliais , Feminino , Humanos , Masculino , Oócitos , Oviductos , Interações Espermatozoide-Óvulo , Espermatozoides , SuínosRESUMO
The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Integrina alfa5/genética , Miócitos Cardíacos/citologia , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Nicho de Células-TroncoRESUMO
Deep learning is a significant step forward for developing autonomous tasks. One of its branches, computer vision, allows image recognition with high accuracy thanks to the use of convolutional neural networks (CNNs). Our goal was to train a CNN with transmitted light microscopy images to distinguish pluripotent stem cells from early differentiating cells. We induced differentiation of mouse embryonic stem cells to epiblast-like cells and took images at several time points from the initial stimulus. We found that the networks can be trained to recognize undifferentiated cells from differentiating cells with an accuracy higher than 99%. Successful prediction started just 20 min after the onset of differentiation. Furthermore, CNNs displayed great performance in several similar pluripotent stem cell (PSC) settings, including mesoderm differentiation in human induced PSCs. Accurate cellular morphology recognition in a simple microscopic set up may have a significant impact on how cell assays are performed in the near future.
Assuntos
Diferenciação Celular , Aprendizado Profundo , Redes Neurais de Computação , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células Cultivadas , Humanos , Processamento de Imagem Assistida por Computador , Aprendizado de Máquina , MicroscopiaRESUMO
In vivo, the oviduct provides appropriate microenvironment conditions for monospermic fertilization and early embryo development. In addition, glycosaminoglycans such as heparin are present in the oviduct and have been shown to modulate the activity of oviduct-secreted proteins on the regulation of sperms parameters. Thus, the present study was designed to evaluate the effect of porcine oocytes exposure to oviduct fluid (OF) before in vitro fertilization (IVF; incubation of oocytes in OF for 30 minutes before IVF), during IVF (supplementation of IVF medium with 10% OF), and during IVF in combination with heparin (10% OF + 10-µg/mL heparin) on IVF parameters. Regardless of sperm concentration used (0.5, 1.5, or 4.5 × 10(5) cells/mL), exposure of oocytes to OF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control group. This resulted in a general increase (P < 0.05) in the final output of the IVF system in terms of zygotes with two pronuclei in OF-exposed groups: 56 ± 9% (OF before) and 60 ± 7% (10% OF during IVF), compared with control (21 ± 8%), when IVF was performed with 4.5 × 10(5) cells/mL. The combination of 10% OF with heparin during IVF induced a decrease (P < 0.05) of the penetration rate, with no effect (P > 0.05) on the monospermy rate in comparison with 10% OF alone. This resulted in a general reduction (P < 0.05) in the final output of the IVF system (%), which was 33 ± 6% and 52 ± 8%, for 10% OF + heparin and 10% OF, respectively. In conclusion, the OF, used in porcine IVF, exerted a beneficial effect on oocytes by reducing the incidence of polyspermy without decreasing the penetration rate. However, the association of the OF with heparin reduced the efficiency of monospermic zygotes' production.