scholarly journals Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived neurons and astrocytes

2021 ◽  
Author(s):  
Jaideep Kesavan ◽  
Orla Watters ◽  
Klaus Dinkel ◽  
Michael Hamacher ◽  
Jochen H.M. Prehn ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
P2x7 Receptor ◽  
Functional Expression ◽  
Extracellular Adenosine ◽  
Functional Studies ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractThe P2X7 receptor (P2X7R) is a cation membrane channel activated by extracellular adenosine 5′-triphosphate. Activation of this receptor results in numerous downstream events including the modulation of neurotransmission, release of pro-inflammatory mediators, cell proliferation or cell death. While the expression of P2X7Rs is well documented on microglia and oligodendrocytes, the presence of functional P2X7Rs on neurons and astrocytes remains debated. Furthermore, to date, functional studies on the P2X7R are mostly limited to studies in cells from rodents and immortalised cell lines expressing human P2X7Rs. To assess the functional expression of P2X7Rs in human neurons and astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into forebrain cortical neurons that co-express FOXG1 and βIII-tubulin as well as S100 β-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining on the soma and processes of hiPSC-derived neurons and astrocytes. In addition, our data show that stimulation with the potent nonselective P2X7R agonist BzATP induces robust calcium rises in hiPSC-derived neurons and astrocytes, which were blocked by the selective P2X7R antagonist AFC-5128. Together, our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived forebrain cortical neurons and astrocytes demonstrating that these cells offer the potential for investigating P2X7R-mediated pathophysiology and drug screening in vitro.

scholarly journals Differentiation of human induced pluripotent stem cells into erythroid cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohsen Ebrahimi ◽  
Mehdi Forouzesh ◽  
Setareh Raoufi ◽  
Mohammad Ramazii ◽  
Farhoodeh Ghaedrahmati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Promising Alternative ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractDuring the last years, several strategies have been made to obtain mature erythrocytes or red blood cells (RBC) from the bone marrow or umbilical cord blood (UCB). However, UCB-derived hematopoietic stem cells (HSC) are a limited source and in vitro large-scale expansion of RBC from HSC remains problematic. One promising alternative can be human pluripotent stem cells (PSCs) that provide an unlimited source of cells. Human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are self-renewing progenitors that can be differentiated to lineages of ectoderm, mesoderm, and endoderm. Several previous studies have revealed that human ESCs can differentiate into functional oxygen-carrying erythrocytes; however, the ex vivo expansion of human ESC-derived RBC is subjected to ethical concerns. Human iPSCs can be a suitable therapeutic choice for the in vitro/ex vivo manufacture of RBCs. Reprogramming of human somatic cells through the ectopic expression of the transcription factors (OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG) has provided a new avenue for disease modeling and regenerative medicine. Various techniques have been developed to generate enucleated RBCs from human iPSCs. The in vitro production of human iPSC-derived RBCs can be an alternative treatment option for patients with blood disorders. In this review, we focused on the generation of human iPSC-derived erythrocytes to present an overview of the current status and applications of this field.

191 ROBUST GENERATION OF NEURAL STEM CELLS FROM PIG INDUCED PLURIPOTENT STEM CELLS FOR TRANSLATIONAL NEURAL REGENERATIVE MEDICINE

2014 ◽  
Vol 26 (1) ◽  
pp. 210
Author(s):  
A. Gallegos-Cardenas ◽  
K. Wang ◽  
E. T. Jordan ◽  
R. West ◽  
F. D. West ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Neural Differentiation ◽  
Neural Induction ◽  
Differentiation Potential ◽  
Induced Pluripotent ◽  
Human Ipsc

The generation of pig induced pluripotent stem cells (iPSC) opened the possibility to evaluate autologous neural cell therapy as a viable option for human patients. However, it is necessary to demonstrate whether pig iPSC are capable of in vitro neural differentiation similar to human iPSC in order to perform in vitro and in vivo comparative studies. Multiple laboratories have generated pig iPSC that have been characterised using pluripotent markers such as SSEA4 and POU5F1. However, correlations of pluripotent marker expression profiles among iPSC lines and their neural differentiation potential has not been fully explored. Because neural rosettes (NR) are composed of neural stem cells, our goal was to demonstrate that NR from pig iPSC can be generated, isolated, and expanded in vitro from multiple porcine iPSC lines similar to human iPSC and that the level of pluripotency in the starting porcine iPSC population (POUF51 and SSEA4 expression) could influence NRs development. Three lines of pig iPSC L1, L2, and L3 were cultured on matrigel-coated plates in mTeSR1 medium (Stemcell Technologies Inc., Vancouver, BC, Canada) and passaged every 3 to 4 days. For neural induction (NI), pig iPSC were disaggregated using dispase and plated. After 24 h, cells were maintained in N2 media [77% DMEM/F12, 10 ng mL–1 bovine fibroblast growth factor (bFGF), and 1X N2] for 15 days. To evaluate the differentiation potential to neuron and glial cells, NR were isolated, expanded in vitro and cultured for three weeks in AB2 medium (AB2, 1X ANS, and 2 mM L-Glutamine). Immunostaining assays were performed to determine pluripotent (POU5F1 and SSEA4), tight junction (ZO1), neural epithelial (Pax6 and Sox1), neuron (Tuj1), astrocyte (GFAP), and oligodendrocyte (O4) marker expression. Line L2 (POU5F1high and SSEA4low) showed a high potential to form NR (6.3.5%, P < 0.05) in comparison to the other 2 lines L1 (POU5F1low and SSEA4low) and L3 (POU5F1low and SSEA4high) upon NI. The NR immunocytochemistry results from Line L2 showed the presence of Pax6+ and Sox1– NRs cells at day 9 post-neural induction and that ZO1 started to localise at the apical border of NRs. At day 13, NRs cells were Pax6+ and Sox1+, and ZO1 was localised to the lumen of NR. After isolation and culture in vitro, NR cells expressed transcription factors PLAGL1, DACH1, and OTX2 through 2 passages, but were not detected in later passages. However, rosette cytoarchitecture was present up until passage 7 and were still Pax6+/Sox1+. NRs at passage 2 were cryopreserved and upon thaw showed normal NR morphology and were Pax6+/Sox1+. To characterise the plasticity of NRs, cells were differentiated. Tuj1 expression was predominant after differentiation indicating a bias towards a neuron phenotype. These results demonstrate that L2 pig iPSC (POUF51high and SSEA4low) have a high potential to form NR and neural differentiation parallels human iPSC neurulation events. Porcine iPSC should be considered as a large animal model for determining the safety and efficacy of human iPSC neural cell therapies.

Chinese Medicine PaBing-II Protects Human iPSC Derived Dopaminergic Neurons from Oxygen Stress

2021 ◽  
Author(s):  
Shouhai Wu ◽  
Tongxiang Lin ◽  
Yang Xu
Keyword(s):  
Oxidative Stress ◽  
Chinese Medicine ◽  
Pluripotent Stem Cells ◽  
Dopaminergic Neurons ◽  
Protective Effects ◽  
Midbrain Dopaminergic Neurons ◽  
Induced Pluripotent ◽  
6 Hydroxydopamine ◽  
Human Ipsc

Abstract BackgroundPaBing-II Formula (PB-II) is a traditional Chinese medicine developed to treat Parkinson's disease (PD). However, due to the complexity of PB-II and the difficulty of culturing human dopaminergic neurons (DAn) in vitro, the mechanism of PB-II to treat PD remains unclear. MethodsWe established the human induced pluripotent stem cells (iPSCs) and derived DAn from hiPSCs to study the protective effects of PB-II on DAn after oxidative stress, which plays an important role in PD pathogenesis. ResultsWe found that serum derived from rats that had ingested PB-II significantly protect hiPSC-derived DAn from reactive oxygen species (ROS). In addition, PB-II dependent serum can activate nuclear erythroid-derived factor 2 (Nrf2) responses, which are required for the neutralization of ROS. In addition, PB-II can activate the Nrf2/ARE signal pathway of midbrain dopaminergic neurons of PD rats induced with 6-hydroxydopamine (6-OHDA) injury, rescue DAn cells, and improve the symptoms of PD rats. ConclusionsPB-II significantly protects the DA neurons from oxidative stress by activating the Nrf2 pathway.

scholarly journals Opportunities and challenges for the use of induced pluripotent stem cells in modelling neurodegenerative disease

Open Biology ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 180177 ◽  
Author(s):  
Yi-Ying Wu ◽  
Feng-Lan Chiu ◽  
Chan-Shien Yeh ◽  
Hung-Chih Kuo
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Three Dimensional ◽  
Disease Patient ◽  
Neuronal Cultures ◽  
Sporadic Disease ◽  
Induced Pluripotent ◽  
Human Ipsc

Adult-onset neurodegenerative diseases are among the most difficult human health conditions to model for drug development. Most genetic or toxin-induced cell and animal models cannot faithfully recapitulate pathology in disease-relevant cells, making it excessively challenging to explore the potential mechanisms underlying sporadic disease. Patient-derived induced pluripotent stem cells (iPSCs) can be differentiated into disease-relevant neurons, providing an unparalleled platform for in vitro modelling and development of therapeutic strategies. Here, we review recent progress in generating Alzheimer's, Parkinson's and Huntington's disease models from patient-derived iPSCs. We also describe novel discoveries of pathological mechanisms and drug evaluations that have used these patient iPSC-derived neuronal models. Additionally, current human iPSC technology allows researchers to model diseases with 3D brain organoids, which are more representative of tissue architecture than traditional neuronal cultures. We discuss remaining challenges and emerging opportunities for the use of three-dimensional brain organoids in modelling brain development and neurodegeneration.

scholarly journals 16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maria Sundberg ◽  
Hannah Pinson ◽  
Richard S. Smith ◽  
Kellen D. Winden ◽  
Pooja Venugopal ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Wide Spectrum ◽  
Copy Number Variations ◽  
Network Activity ◽  
Neuron Networks ◽  
Soma Size ◽  
Marker Expression ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractReciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition.

scholarly journals Electrophysiological measures from human iPSC-derived neurons are associated with schizophrenia clinical status and predict individual cognitive performance

2021 ◽  
Author(s):  
Stephanie Cerceo Page ◽  
Srinidhi Rao Sripathy ◽  
Federica Farinelli ◽  
Zengyou Ye ◽  
Yanhong Wang ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
Clinical Characteristics ◽  
Clinical Status ◽  
Risk Scores ◽  
Na Channel ◽  
Physiological Properties ◽  
Induced Pluripotent ◽  
Human Ipsc ◽  
The Relationship

Neurons derived from human induced pluripotent stem cells (hiPSCs) have been used to model basic cellular aspects of neuropsychiatric disorders, but the relationship between the emergent phenotypes and the clinical characteristics of donor individuals has been unclear. We analyzed RNA expression and indices of cellular function in hiPSC-derived neural progenitors and cortical neurons generated from 13 individuals with high polygenic risk scores (PRS) for schizophrenia and a clinical diagnosis of schizophrenia, along with 15 neurotypical individuals with low PRS. We identified electrophysiological measures associated with diagnosis that implicated altered Na+ channel function and GABA-ergic neurotransmission. Importantly, electrophysiological measures predicted cardinal clinical and cognitive features found in these schizophrenia patients. The identification of basic neuronal physiological properties related to core clinical characteristics of illness may prove useful in generating leads that enable development of novel therapeutics.

scholarly journals Topologically controlled circuits of human iPSC-derived neurons for electrophysiology recordings

2021 ◽  
Author(s):  
Sophie Girardin ◽  
Blandine Clément ◽  
Stephan J. Ihle ◽  
Sean Weaver ◽  
Jana B. Petr ◽  
...  
Keyword(s):  
Information Processing ◽  
Neurological Disorders ◽  
Induced Pluripotent Stem Cell ◽  
Animal Origin ◽  
Great Promise ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Human Ipsc ◽  
The Brain

Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with mostly unidirectional connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs). Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain.

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