generating cortical pyramidal neurons from human neural stem cells

Generating Cortical Pyramidal Neurons from Human Neural Stem Cells

To prepare neuronal cultures, place glass coverslips into six-well culture dishes, and add polyornithine. Incubate overnight under a flow hood. The following day, use DPBS to wash the coverslips three times and add laminin. Incubate under a flow hood for at least 10 hours.
Prepare culture medium containing the following components. Then, use the medium to plate and dispatch neural stem cells or NSCs at a density of 50,000 cells per square centimeter, pipetting with slow rotating movements in order to reduce cell clustering.

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<ol>
	<li>Neuronal Culture 
	Note: Fibroblast reprogramming in pluripotent stem cells, commitment to the dorsal telencephalon lineage, derivation, amplification, and banking of late cortical progenitors (LCP) were described in Boissart et al. Neuronal differentiation of LCP-like cells was also performed according to Boissart et al with slight modifications. Other procedures have been developed for direct reprogramming of fibroblasts into induced pluripotent stem cells, followed by their differentiation into neurons. This protocol was retained since it allows the selective production of pyramidal glutamatergic neurons.
	<ol>
		<li>Treat 6-well culture plates with glass coverslips with poly-ornithine (diluted to 1/6 in DPBS (Dulbecco s Phosphate Buffered Saline), stock concentration 0.01%) O/N, followed by three washes in DPBS. Then add laminin (stock concentration 1 mg/ml, diluted 500 times in DPBS) for at least 10 hr under the flow hood.</li>
		<li>Plate and dispatch NSC at low density (50,000 cells/cm2) in 6-well culture plates with glass coverslips in 3 ml of culture medium consisting of DMEM (Dulbecco&#8242;s Modified Eagle&#8242;s Medium)/F12 (500 ml), 2 vials (5 ml each) of N2 supplement, 2 vials (10 ml each) of B27 supplement, 10 ml of Pen-Streptomycin (Penicillin = 10,000 units/ml and Streptomycin = 10,000 units/ml), 1 ml of 2-mercaptoethanol (Stock solution: 50 mM) and laminin (1/500), without growth factors. CRITICAL STEP: Carry out this step carefully by adding cells with slow rotating movements to reduce cell clustering.</li>
		<li>Remove the culture medium. Add fresh N2B27 medium containing 2 &#181;g/ml of fresh laminin solution to keep the neuron attached to the glass coverslips and avoid clumping. Change the medium every 3 days. Keep some of the remaining medium (200 &#181;l) before adding fresh medium to prevent the cell from drying. Alternatively, proceed rapidly and change the total volume (3 ml).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PD-PBS (1X), sans Calcium, Magnesium et Phenol Red</td>
			<td>Gibco/ Life Technologies</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine Solution Bioreagent</td>
			<td>Sigma Aldrich</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse laminin</td>
			<td>Dutscher Dominique</td>
			<td>354232</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco/ Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement w/o vit A (50X)</td>
			<td>Gibco/ Life Technologies</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/NUT.MIX F-12 W/GLUT-I</td>
			<td>Gibco/ Life Technologies</td>
			<td>31331028</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Med SFM</td>
			<td>Gibco/ Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco/ Life Technologies</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Steptomycin</td>
			<td>Gibco/ Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverglass 13 mm</td>
			<td>VWR</td>
			<td>631-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent avec DAPI</td>
			<td>Gibco/ Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween(R) 20 Bioextra, Viscous Liquid</td>
			<td>Sigma Aldrich Chimie</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Fibroblasts</td>
			<td>Coriell Cell Line Biorepository</td>
			<td>GM 4603 and GM 1869</td>
			<td>Coriell Institute for Medical Research, Camden, NJ, USA</td>
		</tr>
		<tr>
			<td>CO2 incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6 well culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Piptette&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Gouder, L., et al,. <a href="https://app.jove.com/v/53197">Three-dimensional quantification of dendritic spines from pyramidal neurons derived from human induced pluripotent stem cells.</a> J. Vis. Exp. (2015)
This video describes a method for preparing glass coverslips coated with polyornithine and laminin to enhance the adhesion and growth of neural stem cells. The technique involves sequential incubations and washes, facilitating the formation of a supportive matrix that promotes neuronal cell growth and differentiation, mimicking structures found in the neurons.


	Neuronal Culture
	Note: Fibroblast reprogramming in pluripotent stem cells, commitment to the dorsal telencephalon lineage, derivation, amplification, ...

In a culture plate, coat a coverslip with polyornithine, a positively charged polymer, and incubate.
Polyornithine forms a mesh-like layer that helps negatively charged cells stick to the coverslip.
Remove the solution and wash the coverslips with a buffered salt solution to remove unbound polyornithine.
Add a solution containing an extracellular matrix protein called laminin and incubate.
Laminin forms a matrix over the polyornithine-coated coverslip, enhancing cell attachment.
Discard the solution and take a small number of neural stem cells suspended in a growth factor-free medium.
Add this low-density culture over the prepared coverslip in a slow rotating movement to evenly distribute the cells.
Initially, growth factor deprivation promotes cell division.
Over time, stem cells differentiate into neurons with long projections
Eventually, neurons produce spine-like structures over their projections, like the cortical pyramidal neurons.

12 Views. Generowanie neuronów piramidowych korowych z ludzkich nerwowych komórek macierzystych

Video: Generowanie neuronów piramidowych korowych z ludzkich nerwowych komórek macierzystych - Experiment

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

JoVE Journal

Developmental Biology

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Neuronal dendritic morphology often underlies function. Indeed, many disease processes that affect the development of neurons manifest with a morphological phenotype. This protocol describes a simple and powerful method for analyzing intact dendritic arbors and their associated spines.

Generating Cortical Pyramidal Neurons from Human Neural Stem Cells

Transkrypcja

Generating Cortical Pyramidal Neurons from Human Neural Stem Cells