Los autores agradecen el Dr. Philip H. Schwartz de la Comisión Nacional de Recursos Humanos de las células madre neurales en el Hospital de Niños del Condado de Orange el Instituto de Investigación para proporcionar hNSPCs y la instrucción inicial en su cultivo.

Nota: Para el cultivo de la rutina de nuestra hNSPCs, podemos cambiar el 50-100% de los medios de comunicación cada dos días y por lo general les paso 01:02 una vez por semana. La cultura de los medios de comunicación contiene un 20% BIT-9500, 1X factores de antibiótico / antimicótico, y el crecimiento (EGF, FGF y PDGF cada uno a 40 ng / ml) en DMEM: F12 medios de base. Preparación del frasco recubiertas y disociar las células <ol><li> Para preparar nuevos frascos para las células pases, abrigo T25 frascos con 10 mg / ml de fibronectina humana en el EMEM durante 4 horas, o durante la noche, en un 37 ° C incubadora de cultivo de tejidos. Justo antes de los pases de las células, eliminar la solución de fibronectina y enjuague los frascos con PBS. </li><li> La transferencia de la antigua &quot;condicionada&quot; medios de comunicación de las células para ser pasados ​​al nuevo recubiertos de fibronectina frascos (por ejemplo, que la mitad del volumen final de los medios de comunicación para cada frasco será la &quot;condición&quot; los medios de comunicación). Estas condiciones de cultivo para mantener estas células en su estado indiferenciado. </li><li> Una vez que todos los medios de comunicación se elimina de las células para ser pasados, lavar las células una vez con PBS. Tenga cuidado de no secar las células. </li><li> Añadir disociación celular Buffer (CDB) directamente sobre las células (1,0 a 1,5 ml de un frasco T25). Se incuban las células en el búfer durante unos 5 minutos a temperatura ambiente. Golpeando suavemente el frasco ayudará a acelerar el desprendimiento de células. </li></ol> Centrifugación, resuspensión, y las Pilas de Revestimiento <ol><li> Añadir el suero que contienen los medios de comunicación (DMEM: F12 con 10% inactivado por calor suero fetal bovino) (uso ~ 3 veces el volumen de la CDB) en el matraz con células desprendidas. Retire el papel y las células a un tubo cónico de 15 ml. Use una pequeña cantidad de suero fresco contiene los medios de comunicación para enjuagar el frasco y se recuperan las células residuales. </li><li> Centrifugar los medios de comunicación y las células a 1000 rpm (~ 200xg) durante 5 minutos. </li><li> Aspiración de los medios de comunicación que contiene suero y resuspender las células en medio de cultivo fresco, pipeteando arriba y abajo para hacer una suspensión celular homogénea. </li><li> La transferencia de la mitad de las células se resuspendieron en cada nuevo frasco de una división de 1:2. Anote el número de paso de nuevo en el frasco. </li></ol>

<ol>
	<li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> J. Neurosci. Res. 83, 845-856 (2006).</li><li>Nethercott, H. N., Maxwell, H., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+culture.">Neural stem cell culture.</a> Human Stem Cell Manual: A Laboratory Guide. , (2007).</li><li>Palmer, T. D., Schwartz, P. H., Taupin, P., Kaspar, B., Stein, S. A., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture.+Progenitor+cells+from+human+brain+after+death.">Cell culture. Progenitor cells from human brain after death.</a> Nature. 411, 42-43 (2001).</li><li>Schwartz, P. H., Bryant, P. J., Fuja, T. J., Su, H., O'Dowd, D. K., Klassen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+neural+progenitor+cells+from+post-mortem+human+cortex.">Isolation and characterization of neural progenitor cells from post-mortem human cortex.</a> J Neurosci Res. 74, 838-851 (2003).</li></ol>

Hemos encontrado que este protocolo ofrece culturas fiable de hNSPCs. Un factor crítico para nuestras células es que tienen que ser lo bastante denso y las culturas no pueden ser pasados ​​al punto de que las células son escasas. En nuestras manos, culturas escasa crecen muy lentamente o desaparecen completamente división. Por esta razón, se suelen dividir nuestras culturas 1:2 o 1:3 cuando la cultura es extremadamente denso. Revestimiento de la superficie de una placa de cultivo con fibronectina es importante, ya que promueve la u...

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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>BIT-9500 (BSA, Insulin, Transferrin)</td>
<td>Reagent</td>
<td>Stem Cell Technologies</td>
<td>09500</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic-Antimycotic</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>DMEM:F12 (1X)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10437-028</td>
<td>&nbsp;</td>
<tr>
<td>FGF, human basic recombinant</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>100-18B</td>
<td>&nbsp;</td>
<tr>
<td>PDGF-AB</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>100-00AB</td>
<td>&nbsp;</td>
<tr>
<td>EGF, human recombinant</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>CB40052</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Cell Culture Flask, 25 cm2</td>
<td>Tool</td>
<td>Corning</td>
<td>10-126-28</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Fibronectin, human, natural</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>CB40008A</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Human Neural Stem/Precursor Cells</td>
<td>Cells</td>
<td>National Human Neural Stem Cell Resource http://www.nhnscr.org/default.htm </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell Dissociation Buffer</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>13150-016</td>
<td>&nbsp;</td>		</tbody>
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passaging human neural stem cells

La capacidad de manipular humana madre neurales / células precursoras (hNSPCs) in vitro constituye un medio para investigar su utilidad como los trasplantes de células con fines terapéuticos, así como para explorar muchos de los procesos fundamentales del desarrollo humano y la patología neuronal. Este protocolo presenta un método simple de cultivo y hNSPCs pases con la esperanza de la normalización de esta técnica y el aumento de la reproducibilidad de la investigación con células madre humanas. El hNSPCs usamos fueron aislados de cadáveres corteza cerebral postnatal por el Instituto Nacional de Recursos Humanos de las células madre neurales y crecido como adherente culturas en frascos recubiertas con fibronectina (Palmer et al, 2001;.. Schwartz et al, 2003). Nosotros la cultura de nuestro hNSPCs en DMEM: F12 medio libre de suero suplementado con EGF, FGF y PDGF y el paso de las 1:02 aproximadamente cada siete días. Usando estas condiciones, la mayoría de las células en el cultivo mantienen una morfología bipolar y expresan marcadores de células madre indiferenciadas neural (como la nestina y Sox2).

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

Gene Expression | Biology Video Collection

Passaging Human Neural Stem Cells

La capacidad de manipular humana madre neurales / células precursoras (hNSPCs) in vitro permite investigar su utilidad como los trasplantes de células con fines terapéuticos y para explorar el desarrollo neuronal humano. Este protocolo presenta un método de cultivo y hNSPCs pases con la esperanza de la reproducibilidad de cada vez mayor de la investigación con células madre humanas.

Pases células madre neurales humanas

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

Research

JoVE Journal

Biology

14.5K vistas.  University of California, Irvine (UCI). La capacidad de manipular humana madre neurales / células precursoras (hNSPCs) in vitro permite investigar su utilidad como los trasplantes de células con fines terapéuticos y para explorar el desarrollo neuronal humano. Este protocolo presenta un método de cultivo y hNSPCs pases con la esperanza de la reproducibilidad de cada vez mayor de la investigación con células madre humanas.

Video: Passaging Human Neural Stem Cells

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

HUES pases madre de embriones humanos líneas celulares con tripsina

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Investigación

Biología

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagación de la madre embrionarias humanas (ES) Células

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

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.

Transfección de células madre neurales humanas con el Nucleofector Amaxa

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

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.

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.

Conteo de células madre neurales humanas

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 ...

Immunocytochemistry: Human Neural Stem Cells

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.

Inmunocitoquímica: células madre neurales humanas

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.

Derivación de células estaminales embrionarias humanas por 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 ...

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.

Ratón epidérmico de la cresta neural de células madre (EPI-NCSC) Culturas

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.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

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

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.

Congelación y descongelación células estaminales embrionarias humanas

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 ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

La diferenciación de células madre embrionarias en los precursores de oligodendrocitos

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...