This research was supported by the Intramural Research Program at the National Institutes of Health, NINDS. The authors would like to thank all the members of the Laboratory of Molecular Medicine and Neuroscience, Rick Dreyfuss for helping with microscopy and Pamela C. Sieving for helping with the editing.

Note: For routine culturing of neural progenitor and oligodendrocytic lineage cells, incubation is done at 37 &deg;C in a humidified 5% CO2 atmosphere. Every 2 days, the medium is replaced using 50 to 100% of fresh medium if culture is 40-70% confluent. At the time of near confluency, the cultures are passaged at 2-2.5e6/T75 flask usually on a weekly schedule.
1. Preparing the Coated Flask
<ol>
	<li>To prepare coated flasks dilute 5 mg of poly-D-lysine (PDL) in 10...

<ol>
	<li>Messam, C. A., Hou, J., Gronostajski, R. M., Major, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+pathway+of+human+brain+progenitor+cells+identified+by+JC+virus+susceptibility.">Lineage pathway of human brain progenitor cells identified by JC virus susceptibility.</a> Ann. Neurol. 53, 636-646 (2003).</li><li>Vescovi, A. L., Reynolds, B. A., Fraser, D. D., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bFGF+regulates+the+proliferative+fate+of+unipotent+(neuronal)+and+bipotent+(neuronal/astroglial)+EGF-generated+CNS+progenitor+cells.">bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells.</a> Neuron. 11, 951-966 (1993).</li><li>Johe, K. K., Hazel, T. G., Muller, T., Dugich-Djordjevic, M. M., McKay, R. 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Cell Biol. 161, 169-186 (2003).</li><li>Almazan, G., McKay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oligodendrocyte+precursor+cell+line+from+rat+optic+nerve.">An oligodendrocyte precursor cell line from rat optic nerve.</a> Brain Res. 579, 234-245 (1992).</li><li>Filipovic, R., Zecevic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+role+of+minocycline+in+co-cultures+of+human+fetal+neurons+and+microglia.">Neuroprotective role of minocycline in co-cultures of human fetal neurons and microglia.</a> Exp. Neurol. 211, 41-51 (2008).</li><li>Goldman, S. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+histone+deacetylase+activity+induces+developmental+plasticity+in+oligodendrocyte+precursor+cells.">Inhibition of histone deacetylase activity induces developmental plasticity in oligodendrocyte precursor cells.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 14982-14987 (2007).</li><li>Sim, F. J., Goldman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White+matter+progenitor+cells+reside+in+an+oligodendrogenic+niche.">White matter progenitor cells reside in an oligodendrogenic niche.</a> Ernst Schering Res. Found Workshop. , 61-81 (2005).</li><li>Raff, M. C., Miller, R. H., Noble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glial+progenitor+cell+that+develops+in+vitro+into+an+astrocyte+or+an+oligodendrocyte+depending+on+culture+medium.">A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium.</a> Nature. 303, 390-396 (1983).</li><li>Windrem, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+cells+derived+from+the+adult+human+subcortical+white+matter+disperse+and+differentiate+as+oligodendrocytes+within+demyelinated+lesions+of+the+rat+brain.">Progenitor cells derived from the adult human subcortical white matter disperse and differentiate as oligodendrocytes within demyelinated lesions of the rat brain.</a> J. Neurosci. Res. 69, 966-975 (2002).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat. Protoc. 2, 1044-1051 (2007).</li><li>Armstrong, R. C. <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+immature+oligodendrocyte+lineage+cells.">Isolation and characterization of immature oligodendrocyte lineage cells.</a> Methods. 16, 282-292 (1998).</li><li>Hu, B. Y., Du, Z. W., Li, X. J., Ayala, M., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+oligodendrocytes+from+embryonic+stem+cells:+conserved+SHH+signaling+networks+and+divergent+FGF+effects.">Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects.</a> Development. 136, 1443-1452 (2009).</li><li>Gard, A. L., Williams, W. C., Burrell, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroblasts+distinguished+from+O-2A+glial+progenitors+by+surface+phenotype+(O4+GalC-)+and+response+to+cytokines+using+signal+transducer+LIFR+beta.">Oligodendroblasts distinguished from O-2A glial progenitors by surface phenotype (O4+GalC-) and response to cytokines using signal transducer LIFR beta.</a> Dev. Biol. 167, 596-608 (1995).</li><li>Hu, B. Y., Du, Z. W., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+oligodendrocytes+from+pluripotent+stem+cells.">Differentiation of human oligodendrocytes from pluripotent stem cells.</a> Nat. Protoc. 4, 1614-1622 (2009).</li><li>Reubinoff, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitors+from+human+embryonic+stem+cells.">Neural progenitors from human embryonic stem cells.</a> Nat. Biotechnol. 19, 1134-1140 (2001).</li><li>Pfeiffer, S. E., Warrington, A. E., Bansal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oligodendrocyte+and+its+many+cellular+processes.">The oligodendrocyte and its many cellular processes.</a> Trends Cell Biol. 3, 191-197 (1993).</li><li>Zhang, S. C., Ge, B., Duncan, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+human+oligodendroglial+development+in+vitro.">Tracing human oligodendroglial development in vitro.</a> J. Neurosci. Res. 59, 421-429 (2000).</li><li>Bradl, M., Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocytes:+biology+and+pathology.">Oligodendrocytes: biology and pathology.</a> Acta Neuropathol. 119, 37-53 (2010).</li><li>Chong, S. Y., Chan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tapping+into+the+glial+reservoir:+cells+committed+to+remaining+uncommitted.">Tapping into the glial reservoir: cells committed to remaining uncommitted.</a> J. Cell Biol. 188, 305-312 (2010).</li><li>Jakovcevski, I., Filipovic, R., Mo, Z., Rakic, S., Zecevic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+development+and+the+onset+of+myelination+in+the+human+fetal+brain.">Oligodendrocyte development and the onset of myelination in the human fetal brain.</a> Front Neuroanat. 3, 5 (2009).</li><li>Rao, R. C., Boyd, J., Padmanabhan, R., Chenoweth, J. G., 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=Efficient+serum-free+derivation+of+oligodendrocyte+precursors+from+neural+stem+cell-enriched+cultures.">Efficient serum-free derivation of oligodendrocyte precursors from neural stem cell-enriched cultures.</a> Stem Cells. 27, 116-125 (2009).</li><li>D'Intino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triiodothyronine+administration+ameliorates+the+demyelination/remyelination+ratio+in+a+non-human+primate+model+of+multiple+sclerosis+by+correcting+tissue+hypothyroidism.">Triiodothyronine administration ameliorates the demyelination/remyelination ratio in a non-human primate model of multiple sclerosis by correcting tissue hypothyroidism.</a> J. Neuroendocrinol. 23, 778-790 (2011).</li><li>Cui, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+oligodendrocyte+progenitor+cells+from+different+gestational+stages+exhibit+substantially+different+potential+to+myelinate.">Human fetal oligodendrocyte progenitor cells from different gestational stages exhibit substantially different potential to myelinate.</a> Stem Cells Dev. , (2012).</li><li>Messam, C. A., Hou, J., Major, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexpression+of+nestin+in+neural+and+glial+cells+in+the+developing+human+CNS+defined+by+a+human-specific+anti-nestin+antibody.">Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody.</a> Exp. Neurol. 161, 585-596 (2000).</li></ol>

This protocol describes how to derive fetal oligodendrocytes from primary human neural progenitor cells and characterize their phenotype using both flow cytometry and immunofluorescence staining. The expansion and growth of neural progenitors from fetal CNS has been very well described1-4. However, obtaining sufficient human oligodendrocytes for in vitro experimentation remains difficult, even though it is possible to identify and isolate glial precursors from adult human white matter5-13</s...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>DME/HAMS F12 1:1</td>
 <td>Omega Scientist</td>
 <td>DM-251</td>
 <td>1X</td>
 </tr>
 <tr>
 <td>Bovine Albumin</td>
 <td>Sigma</td>
 <td>A9418</td>
 <td>1%</td>
 </tr>
 <tr>
 <td>Gentamicin</td>
 <td>Quality Biologicals</td>
 <td>120-098-031</td>
 <td> 50 &mu;g/ml</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Quality Biologicals</td>
 <td>118-084-061</td>
 <td>2 mM</td>
 </tr>
 <tr>
 <td>T3</td>
 <td>Sigma</td>
 <td>T2877</td>
 <td>3 nM</td>
 </tr>
 <tr>
 <td>N2 Components</td>
 <td>Gibco BRL</td>
 <td>17502</td>
 <td>1:100</td>
 </tr>
 <tr>
 <td>NT-3</td>
 <td>PeproTech Inc</td>
 <td>450-03</td>
 <td>2 ng/ml</td>
 </tr>
 <tr>
 <td>Shh</td>
 <td>R&amp;D System</td>
 <td>1314-SH/CF</td>
 <td>2 ng/ml</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>PeproTech Inc</td>
 <td>100-18B</td>
 <td>20 ng/ml</td>
 </tr>
 <tr>
 <td>PDGF-AA</td>
 <td>PeproTech Inc</td>
 <td>100-13A</td>
 <td>10 ng/ml</td>
 </tr>
 <tr>
 <td>PDL</td>
 <td>Sigma</td>
 <td>P6407</td>
 <td>50 &mu;g/m</td>
 </tr>
 <tr>
 <td>PFA</td>
 <td>Electron Microscopy Sciences</td>
 <td>15712</td>
 <td>2%</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Quality Biologicals</td>
 <td> 118-087-721</td>
 <td></td>
 </tr>
 <tr>
 <td>Papain</td>
 <td>Worthington</td>
 <td>LK003178</td>
 <td>20 U/ml</td>
 </tr>
 <tr>
 <td>DNase vials</td>
 <td>Worthington</td>
 <td>LK003172</td>
 <td>0.005%</td>
 </tr>
 <tr>
 <td>EBSS</td>
 <td>Worthington</td>
 <td>LK003188</td>
 <td></td>
 </tr>
 <tr>
 <td>ProLong Gold with DAPI</td>
 <td>Invitrogen</td>
 <td>P36931</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 1. Reagents.</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td>Primary Abs(all at 1 &mu;g/ml)</td>
 <td></td>
 <td>Species Isotype</td>
 <td>Source </td>
 <td>Secondary Abs</td>
 </tr>
 <tr>
 <td colspan="5">Flow Cytometry</td>
 </tr>
 <tr>
 <td>A2B5-Biotin </td>
 <td>Mouse </td>
 <td> IgM </td>
 <td>Gift J. Nielson </td>
 <td> Streptavidin PETR, Invitrogen, CA </td>
 </tr>
 <tr>
 <td>O4FITC </td>
 <td>Mouse </td>
 <td> IgM </td>
 <td> Gift from J. Nielson</td>
 <td></td>
 </tr>
 <tr>
 <td>A2B5 </td>
 <td>Mouse </td>
 <td>IgM </td>
 <td>Millipore, MA </td>
 <td>g&alpha;m IgM-FITC, Invitrogen, CA</td>
 </tr>
 <tr>
 <td>O4 </td>
 <td>Mouse </td>
 <td>IgM</td>
 <td> Millipore, MA </td>
 <td> g&alpha;m IgM-FITC, Invitrogen, CA</td>
 </tr>
 <tr>
 <td>GalC </td>
 <td>Mouse </td>
 <td> IgG3</td>
 <td> Millipore, MA </td>
 <td> g&alpha;m IgG3-PE, Southern Biotech, AL</td>
 </tr>
 <tr>
 <td>MBP</td>
 <td>Chicken </td>
 <td>IgY </td>
 <td> Millipore, MA </td>
 <td> d&alpha;ck IgY-AMCA, Jackson Immu., PA </td>
 </tr>
 <tr>
 <td>Nestin </td>
 <td>Mouse </td>
 <td>IgG1 </td>
 <td> Messam et al. 200028</td>
 <td>g&alpha;m IgG1-PECy5, Invitrogen, CA</td>
 </tr>
 <tr>
 <td>GFAP </td>
 <td> Rabbit </td>
 <td>IgG </td>
 <td> Millipore, MA </td>
 <td> g&alpha;rb IgG-PE, Jackson Immu, PA </td>
 </tr>
 <tr>
 <td>&beta;III tubulin </td>
 <td> Mouse </td>
 <td>IgG2 </td>
 <td>Covance, CA </td>
 <td> g&alpha;m IgG2a-PETR, Invitrogen, CA</td>
 </tr>
 <tr>
 <td colspan="6">Immunocytochemistry</td>
 </tr>
 <tr>
 <td>&beta;III tubulin (1:1,500) </td>
 <td>Mouse </td>
 <td> IgG2a </td>
 <td> Covance, CA </td>
 <td> g&alpha;m IgG2a-FITC, Invitrogen, CA (1:1,000)</td>
 </tr>
 <tr>
 <td>GFAP 
 (1:1,000) </td>
 <td>Rabbit </td>
 <td> IgG </td>
 <td> Millipore, MA </td>
 <td>g&alpha;rb IgG-FITC, Jackson Immu, PA 
 (1:500)</td>
 </tr>
 <tr>
 <td>MBP 
 (1:50)</td>
 <td>Chicken </td>
 <td> IgY </td>
 <td> Millipore, MA </td>
 <td>d&alpha;ck IgY-FITC, Jackson Immu, PA 
 (1:100) </td>
 </tr>
 <tr>
 <td>O4 
 (1:100) </td>
 <td>Mouse </td>
 <td> IgM </td>
 <td> Millipore, MA </td>
 <td> g&alpha;m IgM-AF546, Invitrogen, CA 
 (1:100)</td>
 </tr>
 <tr>
 <td>GalC 
 (1:10) </td>
 <td>Rabbit </td>
 <td>IgG </td>
 <td>Millipore, MA </td>
 <td>g&alpha;m IgM-AF750, Invitrogen, CA 
 (1:100)</td>
 </tr>
 </tbody>
</table>
Table 2. Antibodies (Abs) used for flow cytometry and immunocytochemistry assays. Antibody conjugates: PE, phycoerythrin; PETR, phycoerythrin Texas Red; AMCA, amino-methyl-coumarin-acetate; Cy, cyanine; FITC, fluorescein isothiocyanate. Ig: immunoglobulin. AF: Alexa Fluor; g&#x03B1;m: goat anti-mouse; g&#x03B1;rb: goat anti-rabbit; d&#x03B1;ck: donkey anti-chicken.</td>
 </tr>
 </tbody>
</table>

progenitor-derived oligodendrocyte culture system from human fetal brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

It is very important to start the differentiation process from a 70%-80% confluent neural progenitor cell culture (Figure 1A). Many cells will die out after changing the culture medium from progenitor to oligo medium since it includes specific growth factors. This indicates that the growth of neural progenitor cells not committed to an oligodendrocytic phenotype will not be supported by the new medium (Figure 1B). Incubation in oligo medium + GF for one week resulted in an interm...

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Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

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14.8K vistas.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. Primario, fetales humanos derivado del cerebro, células progenitoras multipotenciales proliferan In vitro Mientras se mantiene la capacidad de diferenciarse en neuronas y astrocitos. Este trabajo muestra que los progenitores neurales pueden ser inducidas a diferenciarse a través de las etapas del linaje oligodendrocíticos por aire con factores de crecimiento seleccionados.