ISCIII - Contribuciones a congresoshttp://hdl.handle.net/20.500.12105/21112024-03-29T14:38:05Z2024-03-29T14:38:05ZHighly efficient generation of human Cerebral Organoids bypassing embryoid body stageGonzález-Sastre, RosaCoronel Lopez, RaquelBernabeu-Zornoza, AdelaRosca, AndreeaMateos-Martínez, PatriciaMaeso, LauraMartín Benito, SabelaLópez-Alonso, VictoriaListe, Isabelhttp://hdl.handle.net/20.500.12105/172312024-01-19T02:00:54Z2023-01-01T00:00:00ZHuman cerebral organoids (hCOs) are a promising in vitro model that may overcome some of the limitations that currently exist when studying human brain development and disease. Since Lancaster et al. first generated hCOs, efforts have been made to better recapitulate the physiology of the human brain and improve the efficiency and reproducibility of protocols. Different groups employed dual-SMAD inhibition (double inhibition of the transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) pathways) to achieve rapid neural induction. The method developed here, outlines the generation of homogeneous organoids by rapid neuroepithelial induction, avoiding the Embryoid Body (EB) stage. The efficiency of this protocol to form neuroepithelial structures and subsequently organoids is almost 100% due to the use of dual-SMAD inhibition in combination with CHIR99021 (a GSK3β inhibitor/Wnt activator) at the neural induction stage. This is a simple and reproducible protocol as we do not need to use Matrigel or bioreactors which standardizes the methodology. It is also a robust protocol as we have successfully performed it on human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). We performed IHC and Q-RT-PCR assays for cell cycle, neural precursors, neuronal and synaptic vesicle markers. The generated hCOs are highly homogeneous and show ventricular zones (VZs) with radial glia in the center that differentiate to give rise to neurons located around these VZs. These neurons acquire a mature state and are able to form synapses. In addition, we also performed IHC and Q-RT-PCR assays for different markers of oligodendrocytes, astrocytes, microglial cells and vasculature obtaining promising results. We believe that this protocol will be a breakthrough in the generation of organoids for use as a model for the study of neurodevelopmental and neurological diseases, as well as for drug testing.
2023-01-01T00:00:00ZEffects of Polystyrene Nanoplastics on the biology of human Neural Stem Cells and human Cerebral OrganoidsZapata Ortiz, VeronicaGarcía Lopez, CarlaGallego Rodríguez, MaríaHernández Suárez, AnaMateos-Martínez, PatriciaCoronel, RaquelGonzález-Sastre, RosaGonzález-Caballero, MCarmenListe-Noya, Isabelhttp://hdl.handle.net/20.500.12105/172302024-01-19T02:00:51Z2023-01-01T00:00:00ZGlobal plastic production has increased exponentially in recent decades and a significant proportion persists in the environment, where it is degraded by mechanical and physical processes giving rise to micro (< 5mm) and nanoplastics (< 1000 nm; NP) and can reach humans through ingestion, inhalation and the dermal route. There is growing concern about the effects that NPs may cause on human health, in particular there are few studies that assess the effect of NPs on the developing brain, although they have been shown to be able to cross the blood-brain barrier and the placenta. In this study, we evaluate the effects of 30 nm polysteren NPs (PSNPs) on human neural stem cells (hNSCs) and human cerebral organoids (hCOs). In these models we perform studies on cell death, cell proliferation and phenotypic differentiation. As initial results, it can be said that the NPs penetrate cells and organoids, observing an increase of apoptotic markers at higher concentrations, indicative of cell death and alterations in cell proliferation and phenotypic differentiation. Overall, our study suggests the vulnerability of human stem cells to PSNPs exposure, resulting in functional disturbance that might lead to neurodevelopmental disorders.
2023-01-01T00:00:00ZNeurodevelopmental effects of Cypermethrin in human Neural Stem CellsRosca, AndreeaCoronel Lopez, RaquelGonzález-Sastre, RosaMateos-Martínez, PatriciaLópez-Alonso, VictoriaGonzález-Caballero, MCarmenListe-Noya, Isabelhttp://hdl.handle.net/20.500.12105/172292024-01-19T02:00:49Z2023-01-01T00:00:00ZThe lack of adequate models for the study of developmental neurotoxicity has made difficult to correctly assess the risk and effects of toxic exposure. Despite the large body of results on animals, these studies are costly, time consuming and the results are not always reliable to assess the impact of chemical compounds on the developing human brain because animal models do not perfectly reflect human physiology. In vitro systems are becoming a promising tool to assess the toxicological effects of chemical compounds on developmental neurotoxicity with promising results. Currently, stem cells are becoming a useful model to study this type of toxicity. Stem cells are undifferentiated cells with the potential to differentiate into more specialized cell types. They are present during brain development and into adult life, making them a more appropriate model for mimicking key events that take place during embryonic brain development. Cypermethrin (CYP) is one of the most widely used and highly effective synthetic pyrethroids. CYP can enter the body mainly through skin contact but also through inhalation or ingestion of food or water. The main mechanism of action of pyrethroids is the interaction with Na channels and the induction of prolonged depolarization in neurons. In this study, the human neural stem cell line hNS1 was used to evaluate the effects of CYP on early developmental stages. hNS1 cells were exposed to different concentrations of the pesticide and cell death, proliferation and cell fate specification were analyzed under differentiation conditions by immunocytochemistry and RT-qPCR. The results showed that this compound induces apoptotic cell death at the highest doses tested and a decrease in cells in the cell cycle. Besides, CYP causes a decrease in both neurogenesis and gliogenesis in hNS1 cells. In conclusion, CYP has toxic effects on hNS1 cells and should be further studied.
2023-01-01T00:00:00ZRNA-SEQ analysis reveals the implication of Amyloid Precursor Protein (APP) in cell fate specification of human neural Stem Cells by several Signaling Pathways. IBRO Neuroscience ReportsCoronel Lopez, RaquelRosca, AndreeaGonzález-Sastre, RosaMateos-Martínez, PatriciaGallego, Marta InesListe-Noya, IsabelLópez-Alonso, Victoriahttp://hdl.handle.net/20.500.12105/172282024-01-19T02:00:48Z2023-01-01T00:00:00ZNumerous studies have focused on the pathophysiological role of amyloid precursor protein (APP) in Alzheimer's disease (AD), being the aggregation of β-amyloid (Aβ) peptide a central event. However, many authors consider that alterations in physiological functions of APP are likely to play a key role in AD pathogenesis. APP is a glycoprotein expressed ubiquitously in a wide variety of tissues, being especially abundant in the brain. The APP expression is detected at early stages of nervous system development, as well as in adult brain, suggesting that this protein plays a key and important role at various stages of life. Previous studies in our laboratory revealed that APP playing an important role on differentiation of human neural stem cells (hNSCs), favoring glial differentiation (gliogenesis), and preventing the differentiation of them towards neuronal phenotype (neurogenesis). In that case, we proposed to the APP/AICD/GSK-3β system as a possible molecular mechanism involved in the observed effects. However, given the multifunctionality of APP, we think that other molecular mechanisms could also be implicated. In the present study, we have evaluated the effects of APP overexpression in hNSCs at a global level by a transcriptomic analysis using the massive RNA sequencing (RNA-seq) technology. Specifically, we have focused on differentially expressed genes (DEGs) that are related to neuronal and glial differentiation processes, as well as on groups of DEGs associated with different signaling pathways, to find a possible interaction between them and APP. Our data indicate a differential expression in genes related to gliogenesis and neurogenesis processes, as well as in the pathways of Notch signaling, Wnt signaling, PI3K-AKT signaling, and JAK-STAT signaling, among other. The knowledge of physiological functions of APP, as well as the possible signaling pathways that could be implicated, are essential to advance the understanding of the pathogenesis of AD.
2023-01-01T00:00:00ZSINGLE-CELL sequencing workflow to study cellular composition and cell type specific expression profiles of human Cerebral OrganoidsGonzález-Sastre, RosaCoronel Lopez, RaquelMateos-Martínez, PatriciaJimenez Sancho, Maria PilarRosca, AndreeaMaeso, LauraMartín Benito, SabelaZaballos, ÁngelListe-Noya, IsabelLópez-Alonso, Victoriahttp://hdl.handle.net/20.500.12105/172272024-01-24T22:04:29Z2023-01-01T00:00:00ZHuman cerebral organoid culture is a technology with immense potential in the areas of developmental neurobiology and neurodegeneration for example to study cell types, mechanisms involved, to discover of new biomarkers, to propose specific therapeutic strategies or to study the effects of compound-induced toxicity. Single-cell RNA sequencing (scRNA-seq) is a promising technology that will help to define the identity of the cerebral organoids and to understand cellular composition and cell type specific expression profiles. Standardization of workflows to do the scRNA-seq analysis is an important means to improve the use of this technology. We present the workflow and results of the scRNA-seq performed for cerebral organoids generated from the AND-2 cell line of human embryonic stem cells (hESCs). Dissociated cerebral organoid samples were loaded on the 10X Chromium and single cell libraries were prepared according to 10X Genomics standard procedures and sequenced on the Novaseq sequencer (Illumina).The data were checked and aligned to the GRCh38 human reference genome with CellRanger v6.0.2 and analyzed with Seurat v4.0. After quality filtering and data normalization with the SCTransform function, we performed Principal component analysis (PCA) using the highly variable genes, built a Shared Nearest Neighbor (SNN) graph using the Louvain method. To visualize data, Uniform Manifold Approximation and Projection (UMAP) dimensional reduction was performed. The identities of the cell clusters were assigned using the expression of genes specific of each cell type. We annotate in the AND2 cerebral organoids clusters for intermediate progenitor cells, astrocytes, oligodendrocyte precursor cells, excitatory neurons, inhibitory neurons, and mesodermal cells. We find also some cells in these organoids with expression of endothelial and microglial gene markers. Enrichment analysis of the highly variable differentially expressed genes (DEGs) was utilized to characterize the assigned cell types with Gene Ontology (GO), PanglaoDB and Cellmarker databases.
2023-01-01T00:00:00ZStudying the cellular and molecular evolution of familiar Alzheimer'sdisease using human Cerebral OrganoidsMateos-Martínez, PatriciaGonzález-Sastre, RosaCoronel Lopez, RaquelRosca, AndreeaMaeso, LauraMartín Benito, SabelaLuque, DanielTerrón, Maria CarmenAlonso, Victoria LópezListe, Isabelhttp://hdl.handle.net/20.500.12105/172262024-01-19T02:00:44Z2023-01-01T00:00:00ZThe leading cause of dementia in the elderly is Alzheimer's disease (AD), and its increase is expected in the coming years. The histopathological hallmarks of AD are associated with the presence in brain of neurofibrillary tangles, due to the increase in hyperphosphorylated Tau protein, as well as amyloid plaques, due to the increase in amyloid peptide. There is no cure for AD and the treatments effective in slowing neurodegeneration. This lack of cure/treatments may be due to the lack of good study models. Until now, in vivo or in vitro monolayer cellular models have been used that do not allow recapitulating the complexity of the human brain, as well as the histopathology of AD in early stages of its development. For all these reasons, in this work we consider using the technology of three-dimensional cultures: human cerebral organoids (hCOs). In this work, using the protocol developed in our laboratory, we present the generation and characterisation of hCOs with mutations associated with familial AD (fAD) as compared with control hCOs. For this purpose, we used human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) as controls. As fAD models, we used hiPSCs with mutations in PSEN1 and APP duplication. Using immunohistochemistry and RT-qPCR we have analysed these hCOs for markers of neural precursors, brain cell types and synaptic markers, as well as the progression of AD phenotype (amyloid plaques). We have also performed electron microscopy studies to observe differences in the ultrastructure of the hCOs. We present the differences found that show the hCOs generated from hiPSCs with AD variants are experimental in vitro models that will allow further study of pathology.
2023-01-01T00:00:00ZPonencia: Estrategias para Lograr una Cura Funcional del VIHde la Torre-Tarazona, Humberto Erickhttp://hdl.handle.net/20.500.12105/128342022-11-29T14:59:20Z2019-01-01T00:00:00Z2019-01-01T00:00:00Z