eoe sub articles

EoE Sub Articles

1. Mouse embryonic stem cell culture
<ol>
	<li>Prepare 0.1% gelatin coated cell culture dishes or plates.
	<ol>
		<li>Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in water) to 60 mm cell culture dishes. Rock gently to ensure even coating of the cell culture dishes.</li>
		<li>Put the dishes into a 5% CO2 incubator at 37 &#176;C and allow coating for 1 h.</li>
		<li>Remove the 0.1% gelatin solution before seeding the cells. 
		NOTE: After removing the gelatin, there is ...

a technique for generating neural progenitor cells from mouse embryonic stem cells

Source: Mao, X., et al. <a href="https://app.jove.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

<img alt="Figure 1" src="/files/ftp_upload/22409/22409_Figure1.jpg" /> 
Figure 1: The morphology of the embryoid bodies. (A) Embryoid bodies cultured for 4 days. (B) Embryoid bodies cultured for 2 days.

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

Take mouse embryonic stem cells, or mESCs, suspended in a differentiation medium, and incubate.
In the suspension culture, mESCs aggregate to form a structure termed embryoid bodies or EBs.
EBs are three-dimensional aggregates that mimic a developing embryo and can be differentiated into neural cells.
Transfer the EBs into a tube and allow them to settle by gravity.
Remove the nutrient-depleted medium; resuspend the EBs in a fresh medium, and transfer them to a culture dish.
Incubate the suspension culture to facilitate the growth of EBs.
Transfer the EBs suspended in the medium onto a multiwell plate. The wells are coated with an adhesion protein, allowing the EBs to attach.
Introduce retinoic acid, which enters the cells and triggers the formation of a transcription complex.
The complex induces gene expression, promoting the differentiation of the cells into neural progenitor cells, or NPCs, which exhibit extended cellular processes.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

JoVE Journal

Developmental Biology

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

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