eoe sub articles

EoE Sub Articles

1. Thawing Neural Progenitor Cells (NPC) cultures
<ol>
	<li>Coat 10 cm plates with polyornithine (PLO) and laminin the day prior to thawing the cells.
	<ol>
		<li>Add 5 mL of PLO diluted to 100 &#181;g/mL in phosphate buffered saline (PBS) or distilled water (dH2O) to plates. Incubate at 37 &#176;C for a minimum of 1 h up to overnight.</li>
		<li>Aspirate the PLO solution and rinse the plates three times with PBS. (PLO is toxic if not properly washed.)</li>
		<li>Add laminin diluted in PBS t...

differentiation of spinal cord neural progenitor cells into motor neurons

Source: Taga, A. et al., <a href="https://app.jove.com/v/62726">Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons.</a> J. Vis. Exp. (2021).
This video showcases a method for differentiating spinal cord neural progenitor cells (NPCs) into motor neurons. It details the culturing process of the NPCs on a coated culture plate using specialized media enriched with specific inhibitors aimed at facilitating NPC differentiation into mature motor neurons.

Differentiation of Spinal Cord Neural Progenitor Cells into Motor Neurons

1. Thawing Neural Progenitor Cells (NPC) cultures

	Coat 10 cm plates with polyornithine (PLO) and laminin the day prior to thawing the cells.
	
		Add 5 ...

Begin with a culture plate coated with a synthetic polymer and an adhesion protein.&#160;
Add spinal cord neural progenitor cells resuspended in a motor neuron differentiation medium, supplemented with a small-molecule inhibitor.
The coated plate surface facilitates cell adhesion.
The small-molecule inhibitor blocks specific protein kinases, preventing cellular apoptosis.
Remove the spent medium and add fresh differentiation medium.
The medium contains a compound that promotes neural progenitor differentiation into motor neuron progenitors.
Additionally, glial committed progenitors emerge.
Next, replace with differentiation medium supplemented with a DNA synthesis inhibitor.
These inhibitors selectively induce DNA damage and death in glial progenitors, while motor neuron progenitors differentiate into mature neurons.
Wash the cells with buffer and add the differentiation medium without inhibitors to maintain the neurons.
During culturing, regularly replace the medium to replenish nutrients.
Additionally, replace the medium with medium supplemented with adhesion protein to enhance neuronal attachment, establishing a homogenous motor neuron culture.

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Research

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Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

JoVE Journal

Developmental Biology

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Differentiation of Spinal Cord Neural Progenitor Cells into Motor Neurons

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