scholarly journals CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons

2019 ◽  
Author(s):  
Ruilin Tian ◽  
Mariam A. Gachechiladze ◽  
Connor H. Ludwig ◽  
Matthew T. Laurie ◽  
Jason Y. Hong ◽  
...  
Keyword(s):  
Cell Biology ◽  
Cell Types ◽  
Neuronal Morphology ◽  
Genetic Screens ◽  
Crispr Interference ◽  
Disease States ◽  
Differentiated Cells ◽  
Human Neurons ◽  
Longitudinal Imaging ◽  
Induced Pluripotent

SUMMARYCRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.

scholarly journals Present and future challenges of induced pluripotent stem cells

2015 ◽  
Vol 370 (1680) ◽  
pp. 20140367 ◽  
Author(s):  
Mari Ohnuki ◽  
Kazutoshi Takahashi
Keyword(s):  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Disease Modelling ◽  
Future Perspectives ◽  
Differentiated Cells ◽  
Human Ipscs ◽  
Future Challenges ◽  
Induced Pluripotent

Growing old is our destiny. However, the mature differentiated cells making up our body can be rejuvenated to an embryo-like fate called pluripotency which is an ability to differentiate into all cell types by enforced expression of defined transcription factors. The discovery of this induced pluripotent stem cell (iPSC) technology has opened up unprecedented opportunities in regenerative medicine, disease modelling and drug discovery. In this review, we introduce the applications and future perspectives of human iPSCs and we also show how iPSC technology has evolved along the way.

scholarly journals ∆9-tetrahydrocannabinol negatively regulates neurite outgrowth and Akt signaling in hiPSC-derived cortical neurons

2018 ◽  
Author(s):  
Carole Shum ◽  
Lucia Dutan ◽  
Emily Annuario ◽  
Katherine Warre-Cornish ◽  
Samuel E. Taylor ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Cortical Neurons ◽  
Endocannabinoid System ◽  
Neuronal Morphology ◽  
Cannabinoid Type 1 Receptor ◽  
Cellular Processes ◽  
Extracellular Signal ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Arachidonoyl Glycerol

AbstractEndocannabinoids regulate different aspects of neurodevelopment. In utero exposure to the exogenous psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC), has been linked with abnormal cortical development in animal models. However, much less is known about the actions of endocannabinoids in human neurons. Here we investigated the effect of the endogenous endocannabinoid 2-arachidonoyl glycerol (2AG) and Δ9-THC on the development of neuronal morphology and activation of signaling kinases, in cortical glutamatergic neurons derived from human induced pluripotent stem cells (hiPSCs). Our data indicate that the cannabinoid type 1 receptor (CB1R), but not the cannabinoid 2 receptor (CB2R), GPR55 or TRPV1 receptors, is expressed in young, immature hiPSC-derived cortical neurons. Consistent with previous reports, 2AG and Δ9-THC negatively regulated neurite outgrowth. Interestingly, acute exposure to both 2AG and Δ9-THC inhibited phosphorylation of serine/threonine kinase extracellular signal-regulated protein kinases (ERK1/2), whereas Δ9-THC also reduced phosphorylation of Akt (aka PKB). Moreover, the CB1R inverse agonist SR 141716A attenuated the negative regulation of neurite outgrowth and ERK1/2 phosphorylation induced by 2AG and Δ9-THC. Taken together, our data suggest that hiPSC-derived cortical neurons express CB1Rs and are responsive to both endogenous and exogenous cannabinoids. Thus, hiPSC-neurons may represent a good cellular model for investigating the role of the endocannabinoid system in regulating cellular processes in human neurons.

scholarly journals Human ES and iPS Cells Display Less Drug Resistance Than Differentiated Cells, and Naïve-State Induction Further Decreases Drug Resistance

2020 ◽  
Author(s):  
Yulia Panina ◽  
Junko Yamane ◽  
Kenta Kobayashi ◽  
Hideko Sone ◽  
Wataru Fujibuchi
Keyword(s):  
Drug Resistance ◽  
Drug Exposure ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Ips Cells ◽  
Toxicity Assessment ◽  
Research Knowledge ◽  
Differentiated Cells ◽  
Induced Pluripotent

AbstractPluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.

Stem Cells: In Sickness and in Health

2020 ◽  
Vol 15 ◽  
Author(s):  
Hisham F. Bahmada ◽  
Mohamad K. Elajami ◽  
Reem Daouk ◽  
Hiba Jalloul ◽  
Batoul Darwish ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Therapy Resistance ◽  
The Body ◽  
Differentiated Cells ◽  
Undifferentiated Cells ◽  
Potential Applications ◽  
Induced Pluripotent

: Stem cells are undifferentiated cells with the ability to proliferate and convert to different types of differentiated cells that make up the various tissues and organs in the body. They exist both in embryos as pluripotent stem cells that can differentiate into the three germ layers and as multipotent or unipotent stem cells in adult tissues to aid in repair and homeostasis. Perturbations in these cells’ normal functions can give rise to a wide variety of diseases. In this review, we discuss the origin of different stem cell types, their properties and characteristics, their role in tissue homeostasis, current research, and their potential applications in various life-threatening diseases. We focus on neural stem cells, their role in neurogenesis and how they can be exploited to treat diseases of the brain including neurodegenerative diseases and cancer. Next, we explore current research in induced pluripotent stem cell (iPSC) techniques and their clinical applications in regenerative and personalized medicine. Lastly, we tackle a special type of stem cells called cancer stem cells (CSCs) and how they can be responsible for therapy resistance and tumor recurrence and explore ways to target them.

Dendritic trafficking for neuronal growth and plasticity

2013 ◽  
Vol 41 (6) ◽  
pp. 1365-1382 ◽  
Author(s):  
Michael D. Ehlers
Keyword(s):  
Cell Biology ◽  
Neural Circuit ◽  
Cell Communication ◽  
Neuronal Cell ◽  
Cell Types ◽  
The Body ◽  
Plastic Properties ◽  
Electrophysiological Properties ◽  
Differentiated Cells ◽  
Unique Cell

Among the largest cells in the body, neurons possess an immense surface area and intricate geometry that poses many unique cell biological challenges. This morphological complexity is critical for neural circuit formation and enables neurons to compartmentalize cell–cell communication and local intracellular signalling to a degree that surpasses other cell types. The adaptive plastic properties of neurons, synapses and circuits have been classically studied by measurement of electrophysiological properties, ionic conductances and excitability. Over the last 15 years, the field of synaptic and neural electrophysiology has collided with neuronal cell biology to produce a more integrated understanding of how these remarkable highly differentiated cells utilize common eukaryotic cellular machinery to decode, integrate and propagate signals in the nervous system. The present article gives a very brief and personal overview of the organelles and trafficking machinery of neuronal dendrites and their role in dendritic and synaptic plasticity.

scholarly journals Neuronal differentiation strategies: insights from single-cell sequencing and machine learning

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev193631
Author(s):  
Nikolaos Konstantinides ◽  
Claude Desplan
Keyword(s):  
Machine Learning ◽  
Single Cell ◽  
Neuronal Differentiation ◽  
Cell Types ◽  
Specific Cell ◽  
Single Cell Sequencing ◽  
Differentiated Cells ◽  
History Of ◽  
Induced Pluripotent

ABSTRACTNeuronal replacement therapies rely on the in vitro differentiation of specific cell types from embryonic or induced pluripotent stem cells, or on the direct reprogramming of differentiated adult cells via the expression of transcription factors or signaling molecules. The factors used to induce differentiation or reprogramming are often identified by informed guesses based on differential gene expression or known roles for these factors during development. Moreover, differentiation protocols usually result in partly differentiated cells or the production of a mix of cell types. In this Hypothesis article, we suggest that, to overcome these inefficiencies and improve neuronal differentiation protocols, we need to take into account the developmental history of the desired cell types. Specifically, we present a strategy that uses single-cell sequencing techniques combined with machine learning as a principled method to select a sequence of programming factors that are important not only in adult neurons but also during differentiation.

scholarly journals Deficiency in MT5-MMP Supports Branching of Human iPSCs-Derived Neurons and Reduces Expression of GLAST/S100 in iPSCs-Derived Astrocytes

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1705
Author(s):  
Nikita Arnst ◽  
Pedro Belio-Mairal ◽  
Laura García-González ◽  
Laurie Arnaud ◽  
Louise Greetham ◽  
...  
Keyword(s):  
Physiological Role ◽  
Neuronal Morphology ◽  
App Processing ◽  
Amyloidogenic Processing ◽  
Model Of Alzheimer’S Disease ◽  
Human Neurons ◽  
Human Ipscs ◽  
Induced Pluripotent ◽  
The Impact

For some time, it has been accepted that the β-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the β-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer’s disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100β expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology.

scholarly journals Applying CRISPR Screen in Diabetes Research

2021 ◽  
Author(s):  
Peng Yi ◽  
Noelle Morrow
Keyword(s):  
Cell Biology ◽  
Cancer Biology ◽  
Cell Types ◽  
Diabetes Research ◽  
Genetic Screens ◽  
Multiple Organs ◽  
Genome Wide ◽  
Forward Genetic Screens ◽  
Crispr Screen ◽  
Single Cell Type

The CRISPR/Cas9 genome editing system has been one of the greatest scientific discoveries in the last decade. The highly efficient and precise editing ability of this technology is of great therapeutic value and benefits the basic sciences as an advantageous research tool. In recent years, forward genetic screens utilizing CRISPR technology have been widely adopted, with genome-wide or pathway-focused screens leading to important and novel discoveries. CRISPR screens have been used primarily in cancer biology, virology and basic cell biology; but they have rarely been applied to diabetes research. A potential reason for this is that diabetes related research can be more complicated, often involving cross-talk between multiple organs or cell types. Nevertheless, many questions can still be reduced to the study of a single cell type if assays are carefully designed. Here we review the application of CRISPR screen technology and provide perspective on how it can be used in diabetes research.

scholarly journals Cell type determination for cardiac differentiation occurs soon after seeding of human induced pluripotent stem cells

2021 ◽  
Author(s):  
Connie L Jiang ◽  
Yogesh Goyal ◽  
Naveen Jain ◽  
Qiaohong Wang ◽  
Rachel E Truitt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cell Types ◽  
Cardiac Differentiation ◽  
Marker Genes ◽  
Directed Differentiation ◽  
Cell Type ◽  
Differentiated Cells ◽  
Induced Pluripotent

Cardiac directed differentiation of human induced pluripotent stem cells consistently produces a mixed population of cardiomyocytes and non-cardiac cell types even when using very well-characterized protocols. We wondered whether differentiated cell types might result from intrinsic differences in hiPS cells prior to the onset of differentiation. By associating individual differentiated cells that share a common hiPS cell precursor, we were able to test whether expression variability in differentiated cells was pre-determined from the hiPS cell state. Although within a single experiment, differentiated cells that share an hiPS cell progenitor were more transcriptionally similar to each other than to other cells in the differentiated population, when the same hiPS cells were differentiated in parallel, we did not observe high transcriptional similarity across differentiations. Additionally, we found that substantial cell death occurred during differentiation in a manner that suggested that all cells were equally likely to survive or die, suggesting that there was no intrinsic selection bias for cells descended from particular hiPS cell progenitors. These results led us to wonder about how cells grow out spatially during the directed differentiation process. Labeling cells by their expression of a few canonical cell type marker genes, we showed that cells expressing the same marker tended to occur in patches observable by visual inspection, suggesting that cell type determination across multiple cell types, once initiated, is maintained in a cell-autonomous manner for multiple divisions. Altogether, our results show that while there is substantial heterogeneity in the initial hiPS cell population, that heterogeneity is not responsible for heterogeneous outcomes, and that the window during which cell type specification occurs is likely to begin shortly after the seeding of hiPS cells for differentiation.

scholarly journals Interferon-γ signaling synergizes with LRRK2 in human neurons and microglia

2020 ◽  
Author(s):  
Silvia De Cicco ◽  
Dina Ivanyuk ◽  
Wadood Haq ◽  
Vasiliki Panagiotakopoulou ◽  
Aleksandra Arsić ◽  
...  
Keyword(s):  
Neuronal Loss ◽  
Cell Types ◽  
Interferon Γ ◽  
Akt Phosphorylation ◽  
Human Neurons ◽  
Lrrk2 G2019s ◽  
Nfat Activation ◽  
Activation Profile ◽  
Ifn Γ ◽  
Induced Pluripotent

AbstractIncreasing evidence suggests a role for interferons (IFNs) in neurodegeneration. Parkinson’s disease (PD) associated kinase LRRK2 has been implicated in IFN type II (IFN) response in infections and nigral neuronal loss. However, whether and how LRRK2 synergizes with IFN-γ still remains unclear. Here, we employed dopaminergic (DA) neurons and microglia differentiated from patient induced pluripotent stem cells to unravel the role of IFN-γ in LRRK2-PD. We show that IFN-γ induces LRRK2 expression in both DA neurons and microglial cells. LRRK2-G2019S, the most common PD-associated mutation, sensitizes DA neurons to IFN-γ by decreasing AKT phosphorylation. IFN-γ suppresses NFAT activity in both neurons and microglia and synergistically enhances LRRK2-induced defects of NFAT activation. Furthermore, LRRK2-G2019S negatively regulates NFAT via calcium and microtubule dynamics. Importantly, we uncover functional consequences of the reduction of NFAT activity in both cell types, namely defects of neurite elongation and alteration of microglial activation profile and motility. We propose that synergistic IFN-γ/LRRK2 activation serves as a direct link between inflammation and neurodegeneration in PD.

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