22 research outputs found
Might synthetic cannabinoids influence neural differentiation?
Abstract in proceedings of the Fourth International Congress of CiiEM: Health, Well-Being and Ageing in the 21st Century, held at Egas Moniz’ University Campus in Monte de Caparica, Almada, from 3–5 June 2019.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.info:eu-repo/semantics/publishedVersio
hiPSC-based model of prenatal exposure to cannabinoids: effect on neuronal differentiation
Copyright © 2020 Miranda, Barata, Vaz, Ferreira, Quintas and Bekman. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Phytocannabinoids are psychotropic substances ofcannabis with the ability to bind endocannabinoid (eCB) receptors that regulate synaptic activity in the central nervous system (CNS). Synthetic cannabinoids (SCs) are synthetic analogs of Δ9-tetrahydrocannabinol (Δ9-THC), the psychotropic compound of cannabis, acting as agonists of eCB receptor CB1. SC is an easily available and popular alternative to cannabis, and their molecular structure is always changing, increasing the hazard for the general population. The popularity of cannabis and its derivatives may lead, and often does, to a child's exposure to cannabis both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with an altered rate of mental development and significant changes in nervous system functioning. However, the understanding of mechanisms of its action on developing the human CNS is still lacking. We investigated the effect of continuous exposure to cannabinoids on developing human neurons, mimicking the prenatal exposure by drug-consuming mother. Two human induced pluripotent stem cells (hiPSC) lines were induced to differentiate into neuronal cells and exposed for 37 days to cannabidiol (CBD), Δ9-THC, and two SCs, THJ-018 and EG-018. Both Δ9-THC and SC, at 10 μM, promote precocious neuronal and glial differentiation, while CBD at the same concentration is neurotoxic. Neurons exposed to Δ9-THC and SC show abnormal functioning of voltage-gated calcium channels when stimulated by extracellular potassium. In sum, all studied substances have a profound impact on the developing neurons, highlighting the importance of thorough research on the impact of prenatal exposure to natural and SC.This work was supported by the Fundação para a Ciência e a Tecnologia (FCT), Portugal (SFRH/BPD/81627/2011 to SV), by iBB — Institute for Bioengineering and Biosciences — project UIDB/04565/2020, and by Egas Moniz Higher Institute of Health Science (Egas Moniz, CRL). Funding was also received from the European Union’s Horizon 2020 Research and Innovation programme, under the Grant Agreement number 739572—The Discoveries Centre for Regenerative and Precision Medicine H2020-WIDESPREAD-01-2016-2017 to EB.info:eu-repo/semantics/publishedVersio
Neural Differentiation of Embryonic Stem Cells In Vitro: A Road Map to Neurogenesis in the Embryo
Background: The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. Methodology/Principal Findings: In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expresse
Generation and characterization of induced pluripotent stem cell line (IBBISTi004-A) from an Angelman syndrome patient carrying a class II deletion of the maternal chromosome 15q11.2-q13
© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).Angelman Syndrome is a rare neurodevelopmental disorder caused by several (epi)genetic alterations. The patients present strong neurological impairment due to the absence of a functional maternal UBE3A gene in neurons. Here, we generated and characterized a new induced pluripotent stem cell (iPSC) line from a female child with Angelman syndrome harbouring a class II deletion. iPSCs were reprogrammed from fibroblasts using Sendai viruses. The new iPSCs express pluripotency markers, are capable of trilineage in vitro differentiation and have the expected imprinting status of Angelman syndrome. These iPSCs are a valuable tool to elucidate the pathophysiological mechanisms associated with this disease.This work is funded by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the projects UIDB/04565/2020 and UIDP/04565/2020 of Institute for Bioengineering and Biosciences – iBB, the project PTDC/BIA-MOL/29320/2017 of the Instituto de Medicina Molecular João Lobo Antunes and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB, as well by the Pedro Maria José de Mello Costa Duarte grant by Fundação Amélia de Mello. C. Maranga is supported by a PhD fellowship (PD/BD/135505/2018) and S.T. da Rocha is supported by an assistant research contract (CEECIND/01234/2017) from FCT.info:eu-repo/semantics/publishedVersio
Scalable generation of cerebellar neurons from pluripotent stem cells
Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, our ability to differentiate cerebellar neurons from pluripotent stem cells is still limited. Recently, we described the long-term culture of cerebellar neuroepithelium formed from human iPSCs, recapitulating the early developmental events of the cerebellum. Additionally, an efficient maturation of replated cerebellar progenitors into distinct types of functional cerebellar neurons was also achieved under defined and feeder-free conditions. However, developing a scalable protocol that allows to produce large numbers of organoids and high yields of mature neurons in a 3D bioreactor culture systems is still a difficult challenge. In this work, we present a new approach for the reproducible and scalable generation of mid-hindbrain organoids under chemically defined conditions by using the novel PBS 0.1 (100 mL) Vertical-Wheel single-use bioreactor. In this system, an efficient cell aggregation with shape and size-controlled aggregates can be obtained, which is important for homogeneous and efficient differentiation. Moreover, a larger amount of iPSC-derived aggregates can be generated without being excessively labour-intensive, achieving 431 ± 53.6 aggregates/mL at 24 hours after seeding. After differentiation, distinct types of cerebellar neurons were generated, including Purkinje cells (Calbindin+), Granule cells (BARHL1+ and Pax6+), Golgi cells (Neurogranin+ and GAD65+), Deep cerebellar nuclei projection neurons (TBR1+) and Non-Golgi-type interneurons (Parvalbumin+ and Calbindin-). These cells show signs of efficient maturation, staining positive for MAP2, and are able to change intracellular Ca2+ concentration following KCl stimulation. In this system, human iPSC-derived organoids are able to mature into different mature cerebellar neurons and to survive for up to 3 months, without replating and co-culture with feeder layers
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Modeling Rett syndrome with human patient-specific forebrain organoids
Copyright © 2020 Gomes, Fernandes, Vaz, Silva, Bekman, Xapelli, Duarte, Ghazvini, Gribnau, Muotri, Trujillo, Sebastião, Cabral and Diogo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Engineering brain organoids from human induced pluripotent stem cells (hiPSCs) is a powerful tool for modeling brain development and neurological disorders. Rett syndrome (RTT), a rare neurodevelopmental disorder, can greatly benefit from this technology, since it affects multiple neuronal subtypes in forebrain sub-regions. We have established dorsal and ventral forebrain organoids from control and RTT patient-specific hiPSCs recapitulating 3D organization and functional network complexity. Our data revealed a premature development of the deep-cortical layer, associated to the formation of TBR1 and CTIP2 neurons, and a lower expression of neural progenitor/proliferative cells in female RTT dorsal organoids. Moreover, calcium imaging and electrophysiology analysis demonstrated functional defects of RTT neurons. Additionally, assembly of RTT dorsal and ventral organoids revealed impairments of interneuron's migration. Overall, our models provide a better understanding of RTT during early stages of neural development, demonstrating a great potential for personalized diagnosis and drug screening.We also acknowledge financial support from Fundação para a Ciência e a Tecnologia (FCT), Portugal, through iBB, Institute for Bioengineering and Biosciences (UIDB/04565/2020) and from Programa Operacional Regional de Lisboa 2020 (Project No. 007317). AG was supported by FCT (PD/BD/128373/2017).info:eu-repo/semantics/publishedVersio
Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis
Copyright: Pereira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.We propose a novel single-deoxynucleoside-based assay that is easy to perform and provides accurate values for the absolute length (in units of time) of each of the cell cycle stages (G1, S and G2/M). This flow-cytometric assay takes advantage of the excellent stoichiometric properties of azide-fluorochrome detection of DNA substituted with 5-ethynyl-2'-deoxyuridine (EdU). We show that by pulsing cells with EdU for incremental periods of time maximal EdU-coupled fluorescence is reached when pulsing times match the length of S phase. These pulsing times, allowing labelling for a full S phase of a fraction of cells in asynchronous populations, provide accurate values for the absolute length of S phase. We characterized additional, lower intensity signals that allowed quantification of the absolute durations of G1 and G2 phases.Importantly, using this novel assay data on the lengths of G1, S and G2/M phases are obtained in parallel. Therefore, these parameters can be estimated within a time frame that is shorter than a full cell cycle. This method, which we designate as EdU-Coupled Fluorescence Intensity (E-CFI) analysis, was successfully applied to cell types with distinctive cell cycle features and shows excellent agreement with established methodologies for analysis of cell cycle kinetics.João A. Ferreira received support from a Calouste Gulbenkian Foundation grant (96526) and Pedro Pereira is an FCT fellow (SFRH/BD/45502/2008). Evguenia Bekman is the recipient of an IMM-Lisbon fellowship (iMM/BPD/60-2016; project PTDC/BEXBCM/5899/2014).info:eu-repo/semantics/publishedVersio
Generation and characterization of a novel mouse embryonic stem cell line with a dynamic reporter of Nanog expression.
The pluripotent state in embryonic stem (ES) cells is controlled by a core network of transcription factors that includes Nanog, Oct4 and Sox2. Nanog is required to reach pluripotency during somatic reprogramming and is the only core factor whose overexpression is able to oppose differentiation-promoting signals. Additionally, Nanog expression is known to fluctuate in ES cells, and different levels of Nanog seem to correlate with ES cells' ability to respond to differentiation promoting signals. Elucidating how dynamic Nanog levels are regulated in pluripotent cells and modulate their potential is therefore critical to develop a better understanding of the pluripotent state.We describe the generation and validation of a mouse ES cell line with a novel Nanog reporter (Nd, from Nanog dynamics), containing a BAC transgene where the short-lived fluorescent protein VNP is placed under Nanog regulation. We show that Nanog and VNP have similar half-lives, and that Nd cells provide an accurate and measurable read-out for the dynamic levels of Nanog. Using this reporter, we could show that ES cells with low Nanog levels indeed have higher degree of priming to differentiation, when compared with high-Nanog cells. However, low-Nanog ES cells maintain high levels of Oct4 and Sox2 and can revert to a state of high-Nanog expression, indicating that they are still within the window of pluripotency. We further show that the observed changes in Nanog levels correlate with ES cell morphology and that Nanog dynamic expression is modulated by the cellular environment.The novel reporter ES cell line here described allows an accurate monitoring of Nanog's dynamic expression in the pluripotent state. This reporter will thus be a valuable tool to obtain quantitative measurements of global gene expression in pluripotent ES cells in different states, allowing a detailed molecular mapping of the pluripotency landscape
Morphology and Nanog:VNP levels correlation.
<p>(A) Bright field images of Nd cells grown in different culture media: serum; serum/LIF; 2i; and 2i with reduced inhibitors (FGF/ERK and GSK3ß inhibitors) concentration (1∶10, 1∶5 and 1∶2). The addition of increasing amounts of inhibitors results in more tightly packed morphology of ES cell colonies and in the reduction in flattened differentiated cells and a more tightly packed morphology of ES cell colonies. When ES cells are grown in serum alone, the inverse is observed. (B) Representative FC histograms of Nanog:VNP expression for Nd cells grown in different culture media.The addition of increasing amounts of inhibitors results in the increasing expression of Nanog:VNP (to up 95%), while cell grown in serum alone decreases significantly Nanog:VNP expression (to around 15%). The negative control cells (E14tg2a) are represented in gray. (C) Nanog:VNP expression quantifications for Nd cells grown in different culture media (n = 3). Statistically significant differences (p-value <0.002) observed between “serum/LIF” and other conditions are denoted with (*).</p
<i>Nanog</i> expression in FACS-sorted Nd ES cells.
<p>(A) Representative histogram of FACS-sorted Nd sub-populations, grown in serum/LIF. VNP-low (VNP<sub>L</sub>) and VNP-high (VNP<sub>H</sub>) populations were collected for posterior analysis. (B) Nanog:VNP expression after re-plating sorted populations of Nd ES cells in serum/LIF. After 2–4 days, normal levels of heterogeneity are re-established, and expression of Nanog:VNP is similar between the three populations, either derived from the sorted VNP<sub>L</sub> and VNP<sub>H</sub> subsets, or from the whole population (“All”). Representative dot blots for FC analysis of VNP in non-fixed cells are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059928#pone.0059928.s004" target="_blank">Figure S4</a>. Statistically significant differences (p-value <0.05) observed between “All” and VNP sub-populations are denoted with (*), while statistically significant differences between VNP<sub>L</sub> and VNP<sub>H</sub> are denoted with (**). (C) mRNA expression analysis by RT-PCR of cells collected immediately after sorting (day 0) and four days after re-plating (day 4). The purified VNP<sub>L</sub> subpopulation (immediately after sorting) shows lower Nanog mRNA levels and higher expression of lineage-affiliated genes (Fgf5, Gata6 and T-brachyury). After culture for 4 days, both VNP<sub>L</sub> and VNP<sub>H</sub> subsets show similar Nanog mRNA expression. Expression of lineage markers in the VNP<sub>L</sub> subpopulation is still more elevated after 4 days of culture, most likely reflecting the slower reversion to heterogeneity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059928#pone.0059928.s004" target="_blank">Figure S4</a>). Similar levels of Oct4 and Sox2 expression are observed for all analysed samples.</p