IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals

AbstractThe study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON–NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca2+]i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

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New Endeavors of (Micro)Tissue Engineering: Cells Tissues Organs on-Chip and Communication Thereof

Cells Tissues Organs ◽

10.1159/000516356 ◽

2021 ◽

pp. 1-15

Author(s):

Haysam M.M.A.M. Ahmed ◽

Liliana S. Moreira Teixeira

Keyword(s):

Tissue Engineering ◽

Food Additives ◽

Market Potential ◽

Model Systems ◽

Human Model ◽

Human Stem Cells ◽

Animal Testing ◽

Preclinical Research ◽

On Chip

The development of new therapies is tremendously hampered by the insufficient availability of human model systems suitable for preclinical research on disease target identification, drug efficacy, and toxicity. Thus, drug failures in clinical trials are too common and too costly. Animal models or standard 2D in vitro tissue cultures, regardless of whether they are human based, are regularly not representative of specific human responses. Approaching near human tissues and organs test systems is the key goal of organs-on-chips (OoC) technology. This technology is currently showing its potential to reduce both drug development costs and time-to-market, while critically lessening animal testing. OoC are based on human (stem) cells, potentially derived from healthy or disease-affected patients, thereby amenable to personalized therapy development. It is noteworthy that the OoC market potential goes beyond pharma, with the possibility to test cosmetics, food additives, or environmental contaminants. This (micro)tissue engineering-based technology is highly multidisciplinary, combining fields such as (developmental) biology, (bio)materials, microfluidics, sensors, and imaging. The enormous potential of OoC is currently facing an exciting new challenge: emulating cross-communication between tissues and organs, to simulate more complex systemic responses, such as in cancer, or restricted to confined environments, as occurs in osteoarthritis. This review describes key examples of multiorgan/tissue-on-chip approaches, or linked organs/tissues-on-chip, focusing on challenges and promising new avenues of this advanced model system. Additionally, major emphasis is given to the translation of established tissue engineering approaches, bottom up and top down, towards the development of more complex, robust, and representative (multi)organ/tissue-on-chip approaches.

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α-Synuclein oligomers induce early axonal dysfunction in human iPSC-based models of synucleinopathies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1713129115 ◽

2018 ◽

Vol 115 (30) ◽

pp. 7813-7818 ◽

Cited By ~ 60

Author(s):

Iryna Prots ◽

Janina Grosch ◽

Razvan-Marius Brazdis ◽

Katrin Simmnacher ◽

Vanesa Veber ◽

...

Keyword(s):

Axonal Transport ◽

Disease Patient ◽

In Vivo Models ◽

Human Neurons ◽

Anterograde Axonal Transport ◽

Axonal Dysfunction ◽

Underlying Mechanisms ◽

Human Ipsc

α-Synuclein (α-Syn) aggregation, proceeding from oligomers to fibrils, is one central hallmark of neurodegeneration in synucleinopathies. α-Syn oligomers are toxic by triggering neurodegenerative processes in in vitro and in vivo models. However, the precise contribution of α-Syn oligomers to neurite pathology in human neurons and the underlying mechanisms remain unclear. Here, we demonstrate the formation of oligomeric α-Syn intermediates and reduced axonal mitochondrial transport in human neurons derived from induced pluripotent stem cells (iPSC) from a Parkinson’s disease patient carrying an α-Syn gene duplication. We further show that increased levels of α-Syn oligomers disrupt axonal integrity in human neurons. We apply an α-Syn oligomerization model by expressing α-Syn oligomer-forming mutants (E46K and E57K) and wild-type α-Syn in human iPSC-derived neurons. Pronounced α-Syn oligomerization led to impaired anterograde axonal transport of mitochondria, which can be restored by the inhibition of α-Syn oligomer formation. Furthermore, α-Syn oligomers were associated with a subcellular relocation of transport-regulating proteins Miro1, KLC1, and Tau as well as reduced ATP levels, underlying axonal transport deficits. Consequently, reduced axonal density and structural synaptic degeneration were observed in human neurons in the presence of high levels of α-Syn oligomers. Together, increased dosage of α-Syn resulting in α-Syn oligomerization causes axonal transport disruption and energy deficits, leading to synapse loss in human neurons. This study identifies α-Syn oligomers as the critical species triggering early axonal dysfunction in synucleinopathies.

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In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Nanomedicine Nanotechnology Biology and Medicine ◽

10.1016/j.nano.2008.05.002 ◽

2008 ◽

Vol 4 (4) ◽

pp. 330-339 ◽

Cited By ~ 11

Author(s):

L.F. Gamarra ◽

L.F. Pavon ◽

L.C. Marti ◽

W.M. Pontuschka ◽

J.B. Mamani ◽

...

Keyword(s):

Stem Cells ◽

Iron Oxide ◽

Iron Oxide Nanoparticles ◽

Superparamagnetic Iron Oxide ◽

In Vitro Study ◽

Superparamagnetic Iron Oxide Nanoparticles ◽

Vitro Study ◽

Oxide Nanoparticles ◽

Human Stem Cells

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Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504393112 ◽

2015 ◽

Vol 112 (20) ◽

pp. E2725-E2734 ◽

Cited By ~ 147

Author(s):

Cedric Bardy ◽

Mark van den Hurk ◽

Tameji Eames ◽

Cynthia Marchand ◽

Ruben V. Hernandez ◽

...

Keyword(s):

Neuronal Activity ◽

Basal Medium ◽

Physiological Conditions ◽

Synaptic Functions ◽

Human Neurons ◽

Nervous System Function ◽

Neurophysiological Activity

Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.

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

10.1101/2021.12.10.472063 ◽

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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Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein

International Journal of Molecular Sciences ◽

10.3390/ijms21051644 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1644 ◽

Cited By ~ 7

Author(s):

Oksana M. Subach ◽

Vladimir P. Sotskov ◽

Viktor V. Plusnin ◽

Anna M. Gruzdeva ◽

Natalia V. Barykina ◽

...

Keyword(s):

Calcium Ions ◽

Fluorescent Protein ◽

Fast Response ◽

Two Photon ◽

Calcium Indicator ◽

Peptide Pair ◽

Calcium Indicators ◽

Green Fluorescent

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.

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Inhibitory Effect of Multimodal Nanoassemblies against Glycative and Oxidative Stress in Cancer and Glycation Animal Models

BioMed Research International ◽

10.1155/2021/8892156 ◽

2021 ◽

Vol 2021 ◽

pp. 1-17

Author(s):

Hamda Khan ◽

Mohd Waseem ◽

Mohammad Faisal ◽

Abdulrahman A. Alatar ◽

Ahmed A. Qahtan ◽

...

Keyword(s):

Oxidative Stress ◽

Lung Cancer ◽

Mrna Expression ◽

Strong Association ◽

In Vitro Study ◽

Model Systems ◽

Fluorescence Studies ◽

And Oxidative Stress

In recent years, there has been a progress in the study of glycation reaction which is one the possible reason for multiple metabolic disorders. Glycation is a nonenzymatic reaction between nucleic acids, lipids, and proteins resulting into the formation of early glycation products that may further lead to the accumulation of advanced glycation end products (AGEs). The precipitation of AGEs in various cells, tissues, and organs is one of the factors for the initiation and progression of various metabolic derangements including the cancer. The AGE interaction with its receptor “RAGE” activates the inflammatory pathway; yet, the downregulation of RAGE and its role in these pathways are not clear. We explore the effect of anticancer novel nanoassemblies on AGEs to determine its role in the regulation of the expression of RAGE, NFƙB, TNF-α, and IFN-γ. This paper is based on the in vivo and in vitro study in glycation and lung cancer model systems. Upon the treatment of nanoassemblies in both the model systems, we observed a protective effect of nanoassemblies over the inhibition of glycative and oxidative stress via mRNA expression analysis. The mRNA expression results corroborated with the reactive oxygen species (ROS), carboxy-methyl-lysine (CML), and fluorescence studies. In this study, we found that the presence of common factors for glycation and lung cancer is oxidative and glycative stress. This oxidation and glycation might be responsible for the initiation of inflammation which may further lead to uncontrolled growth of cells leading to cancer. This can be a strong association between lung cancer and glycation reaction. The intervention of the anticancer and antiglycation effects of multimodal nanoassemblies throughout the study promises a new pathway for cancer research.

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Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons

10.1101/2020.04.21.052498 ◽

2020 ◽

Author(s):

Feline W. Lindhout ◽

Robbelien Kooistra ◽

Sybren Portegies ◽

Lotte J. Herstel ◽

Riccardo Stucchi ◽

...

Keyword(s):

Neuronal Development ◽

Developmental Stages ◽

Expression Profiles ◽

Model Systems ◽

Human Model ◽

Neuronal Stem Cells ◽

Axon Development ◽

Electrophysiological Responses ◽

Human Neurons ◽

Human Ipsc

ABSTRACTEarly neuronal development is a well-coordinated process in which neuronal stem cells differentiate into polarized neurons. This process has been well studied in classical non-human model systems, but to what extent this is recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured human iPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. We found extensive remodeling of the neuron transcriptome and proteome, with altered mRNA expression of ~1,100 genes and different expression profiles of ~1,500 proteins during neuronal differentiation and polarization. We also identified a distinct stage in axon development marked by an increase in microtubule remodeling and apparent relocation of the axon initial segment from the distal to proximal axon. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.

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Human relevance of blocking the Rac1-TRPC5 pathway as a podocyte-protective strategy for progressive kidney diseases

10.1101/2020.08.28.272344 ◽

2020 ◽

Author(s):

Yiming Zhou ◽

Choah Kim ◽

Juan Lorenzo B. Pablo ◽

Fan Zhang ◽

Ji Yong Jung ◽

...

Keyword(s):

Kidney Diseases ◽

Kidney Tissue ◽

3D Culture ◽

Cytoskeletal Proteins ◽

Model Systems ◽

Sprague Dawley ◽

Puromycin Aminonucleoside ◽

Podocyte Injury ◽

Human Ipsc

AbstractPodocyte injury and the appearance of proteinuria are key features of progressive kidney diseases including focal segmental glomerulosclerosis (FSGS). Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of disease; however, less is known about the human relevance and translatability of TRPC5 inhibition into effective drug development programs. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated human iPSC-derived podocytes and kidney organoids.A single i.p. injection of PAN (50mg/kg) was given to wild-type Sprague-Dawley rats (Male, 4-5 weeks, Charles River). AC1903 was administered twice a day for 7 days after PAN injection. 24-hour urine albumin levels were measured on day 7. Human iPS cells were used to generate podocyte and kidney organoid. PAN treatment was used to induce human podocyte injury in these in vitro model systems. PAN treatment triggered the Rac1-TRPC5 injury pathway in human iPSC-derived podocyte cultures and kidney organoids. The TRPC5 inhibitor AC1903 reversed the effects of PAN-induced injury providing the first evidence for therapeutic applicability of TRPC5 inhibition to human podocytes in both 2D and 3D culture systems. Cross-validation in rats with PAN-induced nephrosis, an established model of podocyte injury and progressive kidney disease, confirmed that inhibition of TRPC5 by AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria. Taken together, our results confirmed the relevance of the TRPC5-Rac1 pathway in human kidney tissue thus highlighting the potential of this therapeutic strategy for patients.

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