The authors would like to acknowledge Dr. Philip H. Schwartz of the National Human Neural Stem Cell Resource at the Children s Hospital, Orange County Research Institute for providing hNSPC cultures.

Passaging Human Neural Stem Cells
Note: Please refer to the <a href="https://www.jove.com/index/details.stp?ID=263">Passaging Human Neural Stem Cells</a> article on how to detach and resuspend the cells. https://www.jove.com/index/Details.stp?ID=263
Preparing the Cell Suspension for Counting
<ol>
<li>Place 25 &micro;l of 0.4% Trypan Blue solution and 20 &micro;l of cell media into an eppe...

<ol>
	<li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Caprette, D. a. v. i. d. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemacytometer.">Using a Hemacytometer.</a> BioEd Online. , (2008).</li><li>Caprette, D. a. v. i. d. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Chamber+(Hemacytometer).">Counting Chamber (Hemacytometer).</a> BioEd Online. , (2008).</li></ol>

This protocol describes a straightforward and rapid way to determine the cell concentration for a variety of experiments involving cells. Using the shortcut described in 3d, one can quickly and accurately determine the cell concentration and total cell number in a given volume.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Phase Contrast Hemacytometer</td>
<td>Tool</td>
<td>Hausser </td>
<td>02-671-54</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Trypan Blue Stain 0.4%</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15250-061</td>
<td>Distributed by Invitrogen under the indicated catalog #</td>
</tr>		</tbody>
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counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Watch this Scientific Journal Video about Counting Human Neural Stem Cells at JoVE.com

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Research

JoVE Journal

Biology

15.5K ビュー.  University of California, Irvine (UCI). 生存細胞の正確な数の知識は、多くの組織培養の操作が必要です。このプロトコルは、生細胞と死細胞を区別し、血球計算盤を用いて細胞を定量化する方法について説明します。それはヒト神経幹/前駆細胞（hNSPCsを）数えて説明していますが、それは他の細胞型に使用することができます。

ビデオ: Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells is required for many tissue culture manipulations.  This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer.  Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell...

transfecting human neural stem cells with the amaxa nucleofector

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo.  This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by...

immunocytochemistry: human neural stem cells

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells.  This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's...

flow cytometry protocols for surface and intracellular antigen analyses of neural cell types

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful...

nutrient regulation by continuous feeding for large-scale expansion of mammalian cells in spheroids

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors.  This study demonstrates the methods required for establishing simple adjusted rate fed...

passaging human neural stem cells

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell...

differentiating human neural stem cells into neural and astrocyte progenitors

This video demonstrates the method to differentiate human neural stem cells from neurospheres into neural and astrocyte progenitors using enzymatic treatment and culturing in a growth factor-rich medium, which eventually leads to maturation into specialized progenitor...

Differentiating Human Neural Stem Cells into Neural and Astrocyte Progenitors

generating neural retina from human pluripotent stem cells

Author Spotlight: Simple and Efficient Neural Retina Organoid Production for Disease Modeling

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and...

Generating Neural Retina from Human Pluripotent Stem Cells

differentiating human embryonic stem cells into neural progenitors

This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Transfer 10 milliliters of medium into two 5-milliliter portions into a clean 15-milliliter tube labeled &#34;CM&#34; for a conditioned medium. Take care not to aspirate any of the cells settled in the flask. Transfer 3 milliliters of medium from the CM tube to a 15-milliliter tube labeled &#34;trypsin inhibitor solution.&#34; Then, set the CM tube...

generating cortical pyramidal neurons from human neural stem cells

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...

Generating Cortical Pyramidal Neurons from Human Neural Stem Cells

Plate the cells on a basement membrane matrix coated 6-well plate at a density of 2 x 105 cells per well in 2 milliliters of mTeSR1 with 10 micromolar ROCK inhibitor. After 24 hours, replace the culture medium with neural induction medium supplemented with 1 micromolar dorsomorphin and 5 micromolar SB431542. Change the medium every other day during the first four days of neural induction, then every day, until confluence is reached at day...

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...

direct induction of human neural stem cells from peripheral blood hematopoietic progenitor cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood...

generation of neural stem cells from discarded human fetal cortical tissue

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

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...

feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is...

differentiating non-transgenic and transgenic human pluripotent stem cells into neural progenitor cells

This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription...

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

culturing h9-embryonic stem cells for generation of human neural retina

Culturing H9-Embryonic Stem Cells for Generation of Human Neural Retina

isolation and culture of neural crest stem cells from human hair follicles

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair...

differentiation of enteric nervous system lineages from human pluripotent stem cells

Author Spotlight: Robust Culture of Human Enteric Neurons from Human Pluripotent Stem Cells

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is...

Derivation of Enteric Neurons from Human Pluripotent Stem Cells

https://cloudfront.jove.com/CDNSource/teasers/66133_b.jpg

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

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 ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...