Name: es cell-derived neuroepithelial cell cultures
Uploaded: 2006-11-30T00:00:00.000+00:00
Duration: 7 min 45 s
Description: Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

Step 1
<ol>
<li>Plate mitomycin-C ggrowth-inhibited (10 &micro;g/ml for 2.5 hours) MS5 cells at a density of 70,000/cm2 on gelatin-coated (0.01 % for 30 minutes) 6 well plates in &alpha;-MEM media. </li>
<li>When cells are attached and have formed a monolayer (over night growth), switch to SRM. </li>
<li>Manually isolate ES cell colonies from the ES cell cultures using a syringe with a 27 &frac12; G needle. </li>
<li>Tritrurate the colonies carefully with a 1 ml blue tip and plate at low density on the MS5 cells (usually 2-3 colonies per 6 well plate).</li>
</ol>
Note: The ES cells can also be taken from enzymatic propagation using collagenase or trypsin.
Step 2
<ol>
<li>Grow the ES cells on the MS5 feeders for 14-21 days until rosette-containing colonies form. </li>
</ol>
 Note: Change media often. The rosettes are usually at the outer edges of the colonies and their formation can be greatly enhanced if 300-500 ng/ml Noggin is added to the SRM (3). In some differentiation protocols, SRM can be exchanged with N2-A media and supplemented with growth or other factors to promote cell specification (2-5). 
Step 3
<ol>
<li>Isolate and pick the rosettes with a 27 &frac12; G needle under a microscope. </li>
</ol>
Note: Avoid cutting and picking the MS5 stromal cells. If colonies are packed with rosettes, one can also isolate the entire colony. 
 Step 4
<ol>
<li>Aspirate the clusters of rosettes, tritrurate them carefully with a 1 ml blue tip, and plate them on poly-L-ornithine (0.001%) and fibronectin (1 &micro;g/ml) or laminin (1 &micro;g/ml) -coated 6 wells in N2-A media usually supplemented with bFGF (20 ng/ml). The rosettes will reform and expand.</li>
</ol>
Note: To enhance cell survival, one can plate the small clusters in droplets to increase cell density. This step can also be modified by supplementing the N2-A media with additional growth or other factors to promote cell specification (2-5).

<ol>
	<li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28, 31-40 (2000).</li><li>Perrier, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proc Natl Acad Sci U S A. 101, 12543-12548 (2004).</li><li>Pruszak, J., Isacson, O., Sullivan, S., Cowan, C., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+dopaminergic+neurons.">Directed differentiation of human embryonic stem cells into dopaminergic neurons.</a> Human Embryonic Stem Cells: The Practical Handbook. , (2007).</li><li>Pruszak, J., Sonntag, K. C., Aung, M. H., Sanchez-Pernaute, R., Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+and+methods+for+cell+sorting+of+human+embryonic+stem+cell-derived+neural+cell+populations.">Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.</a> Stem Cells. 25, 2257-2268 (2007).</li><li>Sonntag, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+yield+of+neuroepithelial+precursors+and+midbrain-like+dopaminergic+neurons+from+human+embryonic+stem+cells+using+the+bone+morphogenic+protein+antagonist+noggin.">Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin.</a> Stem Cells. 25, 411-418 (2007).</li><li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dopaminergic+neurons+and+pigmented+epithelia+from+primate+ES+cells+by+stromal+cell-derived+inducing+activity.">Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.</a> Proc Natl Acad Sci U S A. 99, 1580-1585 (2002).</li><li>Ko, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+neural+precursors+as+a+continuous,+stable,+and+on-demand+source+for+human+dopamine+neurons.">Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.</a> J Neurochem. 103, 1417-1429 (2007).</li><li>Hong, S., Kang, U. J., Isacson, O., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+precursors+derived+from+human+embryonic+stem+cells+maintain+long-term+proliferation+without+losing+the+potential+to+differentiate+into+all+three+neural+lineages,+including+dopaminergic+neurons.">Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons.</a> J Neurochem. , (2007).</li><li>Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+subtype+specification+from+embryonic+stem+cells.">Neural subtype specification from embryonic stem cells.</a> Brain Pathol. 16, 132-142 (2006).</li><li>Pankratz, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Lazzari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+derivation+of+neural+rosettes+from+cloned+bovine+blastocysts:+a+model+of+early+neurulation+events+and+neural+crest+specification+in+vitro.">Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.</a> Stem Cells. 24, 2514-2521 (2006).</li><li>Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B., Goldstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+peripheral+sensory+and+sympathetic+neurons+and+neural+crest+cells+from+human+embryonic+stem+cells.">Generation of peripheral sensory and sympathetic neurons and neural crest cells from human embryonic stem cells.</a> Stem Cells. 23, 923-930 (2005).</li></ol>

The authors have nothing to disclose.

This protocol demonstrates the different steps in generating and isolating neuroepithelial cells from human ES cells using SDIA. The application of this method is manifold and has been used in many protocols to produce specified neurons (e.g. 1, 2, 5-9). The rosettes are thought to resemble neural tube cells with an anterior phenotype (2, 5, 10) and also contain neural crest progenitors (11, 12). In addition, they retain a certain level of plasticity, since they can be patterned by specific factors in defined culture con...

<table><tbody><tr><td>L-Glutamine</td><td></td><td>GIBCO, by Life Technologies</td><td>25030</td><td></td></tr><tr><td>alpha-MEM</td><td></td><td>GIBCO, by Life Technologies</td><td>12571</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td></td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>Knockout-DMEM</td><td></td><td>GIBCO, by Life Technologies</td><td>10829</td><td></td></tr><tr><td>Knockout serum replacement</td><td></td><td>GIBCO, by Life Technologies</td><td>10828</td><td></td></tr><tr><td>MEM nonessential amino acid solution</td><td></td><td>GIBCO, by Life Technologies</td><td>12383</td><td></td></tr><tr><td>DMEM/F12</td><td></td><td>GIBCO, by Life Technologies</td><td>11330</td><td></td></tr><tr><td>N2-A supplement</td><td></td><td>Stem Cell Technologies</td><td>07152</td><td></td></tr><tr><td>Mitomycin-C</td><td></td><td>Sigma-Aldrich</td><td>M0503</td><td></td></tr><tr><td>Gelatine type-A</td><td></td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>poly-L-ornithine</td><td></td><td>Sigma-Aldrich</td><td>P4957</td><td>0.01% solution</td></tr><tr><td>Laminin</td><td></td><td>Sigma-Aldrich</td><td>L-2020</td><td></td></tr><tr><td>Fibronectin</td><td></td><td>Sigma-Aldrich</td><td>F2006</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td></td><td>Invitrogen</td><td>13256</td><td></td></tr><tr><td>1 mL Syringe with 27 1/2 G needle</td><td></td><td>BD Biosciences</td><td>309623</td><td></td></tr><tr><td>N2-A media</td><td>medium</td><td></td><td></td><td>DMEM/F12 + 1% N2-A supplement </td></tr><tr><td>Serum replacement media (SRM)</td><td>medium</td><td></td><td></td><td>Knockout-DMEM + 20% Knockout serum replacement +1% MEM nonessential amino acid solution + 2 mM L-glutamine</td></tr><tr><td>&amp;alpha;-MEM media</td><td>medium</td><td></td><td></td><td>&amp;alpha;-MEM + 10% FBS + 2 mM L-glutamine + 1% penicillin/streptomycin</td></tr><tr><td>MS5</td><td>cell line</td><td></td><td></td><td>stromal cells </td></tr><tr><td>Microscope</td><td></td><td></td><td></td><td></td></tr><tr><td>6-well Plates</td><td></td><td></td><td></td><td>for tissue culture</td></tr></tbody></table>

es cell-derived neuroepithelial cell cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

ES Cell-derived Neuroepithelial Cell Cultures

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

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Research

JoVE Journal

Biology

13.5K Views.  Harvard Medical School. Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

Video: ES Cell-derived Neuroepithelial Cell Cultures

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Developmental Biology

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Neuroscience

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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 ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...