This work was supported in part by the National Institutes of Health: HD054347 and NS063997-01 to VLS. This work was also supported in part by the Empire State Stem Cell Fund through the New York State Department of Health Contract #C024324 to VLS. The opinions expressed here are solely those of the author and do not necessarily reflect those of the Empire State Stem Cell Board, the New York State Department of Health, or the State of New York. VLS is a Doris Duke Clinical Scientist Developmental Award Recipient. We also thank Professor Timothy Vartanian for his gift of Anti-O1, Anti-O4 antibodies.

1. Preparation of solutions and materials for dissection and maintenance of neural stem cell culture
<ol>
	<li>Prepare 100ml dissection medium (KNOCKOUT DMEM/F12, Invitogen) ahead of time and refrigerate.</li>
<li>Prepare100 ml culture medium (Stem Pro NSC SFM, Invitrogen) and keep at 37 &deg;C in a water bath.</li>
<li>Prepare cell-freezing medium (KNOCKOUT DMEM/F12+10%FBS+ 5%DMSO) for long term cryopreservation of cells.</li>
<li>If desired, prepare 4% paraformaldehyde (PFA) for tissue fixation.</li>
<li>Sterile, autoclaved forceps and scalpel blades with handles are used for dissection.</li>
<li>Set aside a pipette gun, 10 ml transfer pipettes, and 40 &mu;M cell strainer (BD Falcon 352340) for dissociation.</li>
<li>Set aside several 10 cm culture dishes (BD) for dissection, 50 ml centrifuge tubes (BD) for dissociation, 1.5 ml centrifuge tubes for tissue storage and frozen vials (BD) for freezing cells.</li>
</ol>
2. Isolating human neural stem cells from the human fetal brain
<ol>
<li>Harvesting of viable tissue immediately after termination of the fetus is vital to the success of the procedure. For elective procedures, timing to obtain samples can be arranged in advance so as to minimize time after fetal demise. The products of conception are harvested within 2 hours post procedure, but ideally can be achieved on elective procedures within minutes. The fetal tissues often are fragmented. However, in general a significant portion of the brain remains intact for visual identification. Limitations of gestational age (GA) are determined by statutory law but have been performed using this protocol between 18-22 weeks GA.</li> 
<li>Fetal brain is placed in a 10 ml Petri dish containing ice-cold KNOCKOUT DMEM/F12 solution. Identify different parts of the cortex by anatomical landmarks. Boundaries for the frontal and parieto- occipital cortices are oriented by the extrapolated intersection of the central sulcus and sylvian fissure. Dissect tissue from the frontal cortex anterior to the central sulcus and along the border of the sylvian fissure with surgical blades, making sure to keep the ventricle intact and undamaged.</li>
<li>Remove any residual blood and meninges from the separated block of frontal cortex. If the sample is of sufficient quality, it is ideal to dissect the block into several smaller samples for various purposes: sections (fixed in 4% paraformaldehyde, PFA) and protein/mRNA assays (fast frozen in -80&deg;C).</li>
<li>Transfer the selected brain block to a 50 ml centrifuge tube, and add ice-cold KNOCKOUT DMEM/F12 solution at about 3 times of the tissue&#x2019;s volume. Gently dissociate the tissue by mechanical pipetting with a 10 ml transfer pipette until all the tissues becomes fragmented (generally 20-30 times), and then filter the cells through a 40 &mu;M cell strainer (BD Falcon 352340) to obtain single or near single cell suspension.</li>
<li>Centrifuge the cell suspension at 2000 rpm and room temperature for 5 min, resuspend the cell pellet in 10 ml fresh warm culture medium (Stem Pro NSC SFM), and count the cell number with a hemocytometer.</li>
<li>Add 5 ml warm culture medium into each 25 cm2 culture flasks, and transfer 2X106 cells to each flask. Cultures are maintained in a 37&deg;C/5% CO2 incubator for 1 week before the analysis. Change half the medium once a week for further cultures or experiments.</li> 
</ol>
3. Manipulaton of neural stem cells for further characterization or experimentations
<ol>
	<li>Neurospheres usually form in 1 to 2 weeks under the recommended culture conditions with NSC diameter ranging between 200 and 400 &mu;m. Neurospheres at this stage can be dissociated with 0.2 g/L EDTA in calcium and magnesium free Hanks medium (Hanks) at 37&deg;C for 15 min to obtain single cells. Cells suspension are spun at 2000 rpm, rinsed in fresh Hanks, and replated in warm culture medium for subculture.</li> 
<li>To initiate differentiation, dissociated cells are plated on poly-D-lysine/laminin 1 coated coverslips at a density of 1X105 cells per coverslip (24mmX24mm). Oligodendrocyte differentiation is achieved by maintaining the cells in KNOCKOUT DMEM/F12 (Invitrogen, Main, MD)+2% B27(50X, Invitrogen, Main, MD)+10ng/ml bFGF+100ng/ml SHH+ 10ng/ml PDGF-AA for 2days, then switching to the same medium without growth factors for another 5 days. Neuronal differentiation is achieved by maintaining cells in KNOCKOUT DMEM/F12 +2% B27 (50X) for 7 days. Astrocyte differentiation is done by culturing cells in KNOCKOUT DMEM/F12 +1%FBS for a week. </li>
<li>Transfection of neural stem cells with genes can be done with dissociated cells from the preformed neurospheres. Here, we showed the neural stem cells transfected with EGFP-C1 by electroporation. The electroporation of EGFP-C1 construct was done using AMAXA Nucleofector Kits for Mouse Neural Stem Cells (VPG-1004) with AMAXA Nucleofector Device (Lonza AAD-1001), following the company&#x2019;s instruction. In short, 5 &mu;g DNA with 1 X 106 cells were mixed with 100 &mu;l transfection medium, and after pulse electroporation, the cells were resuspended in the neural stem cells maintaining medium for further culture. The differentiation of transfected cells were processed with the dissociation of neurospheres 3-4 days after the electroporation, following the same procedures described in step 3.2. </li>
</ol>
4. Freezing neural stem cells and subcultures
<ol>
<li>Dissociated cell suspensions are centrifuged and resuspended in freezing medium with a concentration of 1X107 cells/vial/ml. Slowly freeze the cells in -20&deg;C, -80&deg;C then transfer to liquid nitrogen for long time storage.</li>
<li>The cells are fast thawed with 37&deg;C water bath and resuspended in warmed DMEM/F12+10% serum for a wash, centrifuged to remove the freezing medium, and resuspended in the warmed culture medium.</li> 
</ol>
5. Representative Results: 
Neural stem cells from a normal fetus at 18 weeks gestational age were cultured following the described methods and neurospheres can be seen after one week with round, smooth borders and fairly homogenous size (Fig.2A). These neurospheres can be transfected with EGFP-C1 or other constructs and followed under fluorescence microscopy (Fig.2B,). Established neurospheres were then dissociated with EDTA and plated as dispersed cells on coated coverslips. Cells differentiated under the respective protocols were fixed with 4% parafamaldehyde, and stained with different cell type specific markers. Multipotentiality is observed with expression of markers indicative of neurons (Fig2C, D, rhodamine) astrocytes (Fig2E, F, rhodamine) and oligodendrocytes (Fig2G, H, rhodamine). The cells not undergone electroporation of EGFP were also differentiated into different cell types and stained with different cell specific markers. Multipotentiality is observed with expression of markers indicative of neurons (Fig3A, B, fluoroscein) astrocytes (Fig3C, rhodamine and Fig3D, fluoroscein) and oligodendrocytes (Fig3E, F, rhodamine).
<img src="/files/ftp_upload/2681/2681fig1.jpg" alt="Figure 1"/> 
Figure 1. Schematic of the experimental procedure to isolate neural stem cells from discarded human fetal brain
<img src="/files/ftp_upload/2681/2681fig2.jpg" alt="Figure 2"/> 
Figure 2. Undifferentiated and differentiated human neural cells propogated in vitro. (A) Neurospheres are shown under phase contrast microscopy demonstrate smooth, round borders and rapid growth after culture for over 1 week. (B) Introduction of various plasmids and constructs can be achieved through transfection. Three days following EGFP-C1 transfection, multiple cells show expression of the green fluorescent protein as seen under fluoroscein immunostaining and fluorescence microscopy. EGFP-C1 transfected neurospheres are dissociated and differentiated under different conditions into neurons (C, D), astrocytes (E, F) and oligodendrocytes (G, H) and seen under rhodamine fluorescence. Concurrently, transfected EGFP positive cells are shown under fluoroscein fluorescence. Transfected cells (white arrow heads) are indistinguishable from untransfected cells (white arrows). The cell nuclei are stained with Hoechst33342. Scale bars are 200 &mu;m for A, 100 &mu;m for B and 25 &mu;m for C-H.
<img src="/files/ftp_upload/2681/2681fig3.jpg" alt="Figure 3"/> 
Figure 3. Neurospheres without transfection are dissociated and differentiated under different conditions into neurons (A, B, fluoroscein), astrocytes (C, rhodamine, D, fluoroscein) and oligodendrocytes (E, F, rhodamine). The cell nuclei are stained with Hoechst33342. Scale bars are 25 &mu;m for A-F.

<ol>
	<li>Gage, F. H., Ray, J., Fisher, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization,+and+use+of+stem+cells+from+the+CNS.">Isolation, characterization, and use of stem cells from the CNS.</a> Annu. Rev. Neurosci. 18, 159-192 (1995).</li><li>Vescovi, A. L., Snyder, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+properties+of+neural+stem+cell+clones:+plasticity+in+vitro+and+in+vivo.">Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo.</a> Brain Pathol. 9, 569-598 (1999).</li><li>Schwartz, P., Bryant, P., Fuja, T., Su, H., O'Dowd, D., 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><li>Martinez-Serrano, A., Rubio, F. J., Navarro, B., Bueno, C., Villa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+and+progenitor+cells:+in+vitro+and+in+vivo+properties,+and+potential+for+gene+therapy+and+cell+replacement+in+the+CNS.">Human neural stem and progenitor cells: in vitro and in vivo properties, and potential for gene therapy and cell replacement in the CNS.</a> Curr Gene Ther. 1, 279-299 (2001).</li><li>Rajan, P., Snyder, E. <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+cells+and+their+manipulation.">Neural stem cells and their manipulation.</a> Methods Enzymol. 419, 23-52 (2006).</li><li>Ruiz-Lozano, P., Rajan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+as+in+vitro+models+of+disease.">Stem cells as in vitro models of disease.</a> Curr Stem Cell Res Ther. 2, 280-292 (2007).</li><li>Sheen, V., Ferland, R., Harney, M., Hill, R., Neal, J., Banham, A., Brown, P., Chenn, A., Corbo, J., Hecht, J., Folkerth, R., Walsh, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+proliferation+and+migration+in+human+Miller-Dieker+neural+precursors.">Impaired proliferation and migration in human Miller-Dieker neural precursors.</a> Ann Neurol. 60, 137-144 (2006).</li><li>Bahn, S., Mimmack, M., Ryan, M., Caldwell, M., Jauniaux, E., Starkey, M., Svendsen, C., Emson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+target+genes+of+the+neuron-restrictive+silencer+factor+in+neurospheres+derived+from+fetuses+with+Down's+syndrome:+a+gene+expression+study.">Neuronal target genes of the neuron-restrictive silencer factor in neurospheres derived from fetuses with Down's syndrome: a gene expression study.</a> Lancet. 359, 310-315 (2002).</li><li>Ferland, R. J., Batiz, L. F., Neal, J., Lian, G., Bundock, E., Lu, J., Hsiao, Y. C., Diamond, R., Mei, D., Banham, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+neural+progenitors+along+the+ventricular+and+subventricular+zones+in+periventricular+heterotopia.">Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia.</a> Hum Mol Genet. 18, 497-516 (2009).</li><li>Esposito, G., Imitola, J., Lu, J., De Filippis, D., Scuderi, C., Ganesh, V. S., Folkerth, R., Hecht, J., Shin, S., Iuvone, T., Chesnut, J., Steardo, L., Sheen, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+functional+profiling+of+human+Down+syndrome+neural+progenitors+implicates+S100B+and+aquaporin+4+in+cell+injury.">Genomic and functional profiling of human Down syndrome neural progenitors implicates S100B and aquaporin 4 in cell injury.</a> Hum Mol Genet. 17, 440-457 (2008).</li><li>Esposito, G., Scuderi, C., Lu, J., Savani, C., De Filippis, D., Iuvone, T., Steardo, L. J. r., Sheen, V., Steardo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S100B+induces+tau+protein+hyperphosphorylation+via+Dickopff-1+up-regulation+and+disrupts+the+Wnt+pathway+in+human+neural+stem+cells.">S100B induces tau protein hyperphosphorylation via Dickopff-1 up-regulation and disrupts the Wnt pathway in human neural stem cells.</a> J Cell Mol Med. 12, 914-927 (2008).</li><li>Flax, J. D., Aurora, S., Yang, C., Simonin, C., Wills, A. M., Billinghurst, L. L., Jendoubi, M., Sidman, R. L., Wolfe, J. H., Kim, S. U., Snyder, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftable+human+neural+stem+cells+respond+to+developmental+cues,+replace+neurons,+and+express+foreign+genes.">Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.</a> Nat Biotechnol. 16, 1033-1039 (1998).</li><li>Fults, D., Pedone, C. A., Morse, H. G., Rose, J. W., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+primitive+neuroectodermal+tumor+cell+line+from+the+cerebral+hemisphere.">Establishment and characterization of a human primitive neuroectodermal tumor cell line from the cerebral hemisphere.</a> J Neuropathol Exp Neurol. 51, 272-280 (1992).</li><li>Conti, L., Cattaneo, E. <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+systems:+physiological+players+or+in+vitro+entities?.">Neural stem cell systems: physiological players or in vitro entities?.</a> Nat Rev Neurosci. 11, 176-187 (2010).</li><li>Svendsen, C. N., ter Borg, M. G., Armstrong, R. J., Rosser, A. E., Chandran, S., Ostenfeld, T., Caldwell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+rapid+and+long+term+growth+of+human+neural+precursor+cells.">A new method for the rapid and long term growth of human neural precursor cells.</a> J Neurosci Methods. 85, 141-152 (1998).</li></ol>

No conflicts of interest declared.

There are various approaches toward culture fresh tissue and producing human cell lines. Historically, fresh tissue has been harvested and cultured immediately to generate various cell types in the central nervous system. This approach however is clearly limited by the number of samples that can be obtained- which in the case for human samples, is usually quite small. Given the minimal degree of manipulation, freshly cultured neural cells provide the most reliable experimental system by limiting potential artifacts from ...

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>KNOCKOUT DMEM/F12</td>
 <td>Invitrogen</td>
 <td>12660-012</td>
 <td>Dissociation medium</td>
 </tr>
 <tr>
 	<td>Stem Pro NSC SFM</td>
 <td>Invitrogen</td>
 <td>A10509-01</td>
 <td>Culture medium</td>
 </tr>
 <tr>
 	<td>Fetal Bovine Serum</td>
 <td>Invitrogen</td>
 <td>10091-148</td>
 <td>Frozen medium</td>
 </tr>
 <tr>
 	<td>Hanks solution (-Ca2+, -Mg2+)</td>
 <td>Invitrogen</td>
 <td>14175-095</td>
 <td>Dissociation medium</td>
 </tr>
 <tr>
 	<td>DMSO</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>Frozen medium</td>
 </tr>
 <tr>
 	<td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>431788</td>
 <td>Dissociation medium</td>
 </tr>
 <tr>
 	<td>Paraformaldehyde</td>
 <td>Sigma-Aldrich</td>
 <td>158127</td>
 <td>Fixation solution</td>
 </tr>
 <tr>
 	<td>bFGF</td>
 <td>R&amp;D</td>
 <td>234-FSE</td>
 <td>Differentiation medium</td>
 </tr>
 <tr>
 	<td>SHH</td>
 <td>R&amp;D</td>
 <td>1845-SH</td>
 <td>Differentiation medium</td>
 </tr>
 <tr>
 	<td>PDGF-AA</td>
 <td>R&amp;D</td>
 <td>221-AA</td>
 <td>Differentiation medium</td>
 </tr>
 <tr>
 	<td>B27</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td>Differentiation medium</td>
 </tr>
 <tr>
 	<td>Mouse Anti-MAP2</td>
 <td>Sigma-Aldrich</td>
 <td>M2320</td>
 <td>1:200</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-DCX</td>
 <td>Cell signaling </td>
 <td>4604s</td>
 <td>1:200</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-GFAP</td>
 <td>DAKO</td>
 <td>Z0334</td>
 <td>1:200</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-S100B</td>
 <td>DAKO</td>
 <td>Z0311</td>
 <td>1:200</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-O1</td>
 <td>gifts of Professor Timothy Vartanian*</td>
 <td>&nbsp;</td>
 <td>1:50</td>
 </tr>
 <tr>
 	<td>Rabbit Anti-O4</td>
 <td>Gifts of Professor Timothy Vartanian*</td>
 <td>&nbsp;</td>
 <td>1:50</td>
 </tr>
 <tr>
 	<td>40&mu;m cell strainer </td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 
 </tbody>
</table>
* Timothy Vartanian, MD, PhD, Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, USA

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.

Watch this Scientific Journal Video about Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue at JoVE.com

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 efficacy.

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

generation-neural-stem-cells-from-discarded-human-fetal-cortical

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JoVE Journal

Neuroscience

15.4K visualizações.  Beth Israel Deaconess Medical Center. Um método simples e confiável sobre o isolamento e cultura de células-tronco neurais de tecido fetal humano descartado cortical é descrito. Derivados de culturas humanas conhecidas doenças neurológicas podem ser usados ​​para a caracterização de processos patológicos celular e molecular, bem como fornecer uma plataforma para avaliar a eficácia farmacológica.

Vídeo: Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

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

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

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

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

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

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

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 presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...