Name: derivation of leptomeninges explant cultures from postmortem human brain donors
Uploaded: 2017-01-21T13:00:00
Description: Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Development of this protocol was funded by private donations directed to the Parkinson's Institute Brain Donation Program.

The brain donation registration includes documentation by the registrant of their intent to donate. The autopsy permission for tissue retrieval is provided by the next of kin as permitted by law. Research studies using collected autopsy specimens are reviewed by the institutional review board (IRB) to ensure compliance with Health Insurance Portability and Accountability Act (HIPAA) regulations.
NOTE: Leptomeninges samples are collected by the brain dissector or neuropathologist during a brain...

<ol>
	<li>Decimo, I., Fumagalli, G., Berton, V., Krampera, M., Bifari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meninges:+from+protective+membrane+to+stem+cell+niche.">Meninges: from protective membrane to stem cell niche.</a> Am J Stem Cells. 1 (2), 92-105 (2012).</li><li>Bifari, F., 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=Novel+stem/progenitor+cells+with+neuronal+differentiation+potential+reside+in+the+leptomeningeal+niche.">Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche.</a> J Cell Mol Med. 13 (9B), 3195-3208 (2009).</li><li>Bifari, F., 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=Meninges+harbor+cells+expressing+neural+precursor+markers+during+development+and+adulthood.">Meninges harbor cells expressing neural precursor markers during development and adulthood.</a> Front Cell Neurosci. 9, 383 (2015).</li><li>Decimo, I., Bifari, F., Krampera, M., Fumagalli, G. <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+niches+in+health+and+diseases.">Neural stem cell niches in health and diseases.</a> Curr Pharm Des. 18 (13), 1755-1783 (2012).</li><li>Hayashi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meningeal+cells+induce+dopaminergic+neurons+from+embryonic+stem+cells.">Meningeal cells induce dopaminergic neurons from embryonic stem cells.</a> Eur J Neurosci. 27 (2), 261-268 (2008).</li><li>Nakagomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptomeningeal-derived+doublecortin-expressing+cells+in+poststroke+brain.">Leptomeningeal-derived doublecortin-expressing cells in poststroke brain.</a> Stem Cells Dev. 21 (13), 2350-2354 (2012).</li><li>Hjelm, 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=Induction+of+pluripotent+stem+cells+from+autopsy+donor-derived+somatic+cells.">Induction of pluripotent stem cells from autopsy donor-derived somatic cells.</a> Neurosci Lett. 502 (3), 219-224 (2011).</li><li>Hjelm, 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=In+vitro-differentiated+neural+cell+cultures+progress+towards+donor-identical+brain+tissue.">In vitro-differentiated neural cell cultures progress towards donor-identical brain tissue.</a> Hum Mol Genet. 22 (17), 3534-3546 (2013).</li><li>Meske, V., Albert, F., Wehser, R., Ohm, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+autopsy-derived+fibroblasts+as+a+tool+to+study+systemic+alterations+in+human+neurodegenerative+disorders+such+as+Alzheimer's+disease--methodological+investigations.">Culture of autopsy-derived fibroblasts as a tool to study systemic alterations in human neurodegenerative disorders such as Alzheimer's disease--methodological investigations.</a> J Neural Transm (Vienna). 106 (5-6), 537-548 (1999).</li><li>Bliss, L. 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=Use+of+postmortem+human+dura+mater+and+scalp+for+deriving+human+fibroblast+cultures.">Use of postmortem human dura mater and scalp for deriving human fibroblast cultures.</a> PLoS One. 7 (9), e45282 (2012).</li><li>Sproul, A. 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=Generation+of+iPSC+lines+from+archived+non-cryoprotected+biobanked+dura+mater.">Generation of iPSC lines from archived non-cryoprotected biobanked dura mater.</a> Acta Neuropathol Commun. 2, 4 (2014).</li><li>Adler, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+clinical+diagnostic+accuracy+of+early+vs+advanced+Parkinson+disease:+clinicopathologic+study.">Low clinical diagnostic accuracy of early vs advanced Parkinson disease: clinicopathologic study.</a> Neurology. 83 (5), 406-412 (2014).</li><li>Hughes, A. J., Daniel, S. E., Kilford, L., Lees, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+clinical+diagnosis+of+idiopathic+Parkinson's+disease:+a+clinico-pathological+study+of+100+cases.">Accuracy of clinical diagnosis of idiopathic Parkinson's disease: a clinico-pathological study of 100 cases.</a> J Neurol Neurosurg Psychiatry. 55 (3), 181-184 (1992).</li><li>Hughes, A. J., Daniel, S. E., Lees, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+accuracy+of+clinical+diagnosis+of+Lewy+body+Parkinson's+disease.">Improved accuracy of clinical diagnosis of Lewy body Parkinson's disease.</a> Neurology. 57 (8), 1497-1499 (2001).</li><li>Langston, J. W., Sch&#252;le, B., Rees, L., Nichols, R. J., Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisystem+Lewy+body+disease+and+the+other+parkinsonian+disorders.">Multisystem Lewy body disease and the other parkinsonian disorders.</a> Nature Genetics. 47 (12), 1378-1384 (2015).</li><li>Vangipuram, M., Ting, D., Kim, S., Diaz, R., Sch&#252;le, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+punch+biopsy+explant+culture+for+derivation+of+primary+human+fibroblasts.">Skin punch biopsy explant culture for derivation of primary human fibroblasts.</a> J Vis Exp. (77), e3779 (2013).</li><li>Byers, B., 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=SNCA+triplication+Parkinson's+patient's+iPSC-derived+DA+neurons+accumulate+alpha-synuclein+and+are+susceptible+to+oxidative+stress.">SNCA triplication Parkinson's patient's iPSC-derived DA neurons accumulate alpha-synuclein and are susceptible to oxidative stress.</a> PLoS One. 6 (11), e26159 (2011).</li><li>Sanders, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+mutations+cause+mitochondrial+DNA+damage+in+iPSC-derived+neural+cells+from+Parkinson's+disease+patients:+reversal+by+gene+correction.">LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.</a> Neurobiol Dis. 62, 381-386 (2014).</li><li>Beevers, J. E., Caffrey, T. M., Wade-Martins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+(iPSC)-derived+dopaminergic+models+of+Parkinson's+disease.">Induced pluripotent stem cell (iPSC)-derived dopaminergic models of Parkinson's disease.</a> Biochem Soc Trans. 41 (6), 1503-1508 (2013).</li></ol>

This protocol describes a simple and robust protocol to derive a meningeal fibroblast culture from human postmortem leptomeninges collected in conjunction with a brain donation. There are very few descriptions of protocols to derive cell cultures from postmortem human material. Two studies describe skin-derived fibroblast cultures7,8,9, one study describes dura samples10, and another describes non-cryopre...

Meninges consist of three membranes that protect the brain: dura mater, arachnoid and pia mater. More recently, it has been recognized that meninges also play an important role in brain development and brain homeostasis1. Meninges are derived from mesenchymal and neural crest-derived cells and interestingly, it has been shown that progenitor cells residing in meninges can give rise to neurons in vitro and after transplantation in vivo2,3,4. Meninges cultures have been also successfully used as feeder layers, as they possess stromal cell-derived inducing activity for differentiation of embryonic stem cells into dopaminergic neurons5. Moreover, leptomeninges have the potential to directly differentiate into neurons, astrocytes, and oligodendrocytes under ischemic conditions6.

For this protocol, human postmortem meninges samples are collected from the arachnoid and pia mater, collectively called the leptomeninges, and are procured as part of a human brain donation for research purposes. Dissection of the brain is performed within 24 h of death and the leptomeninges sample is placed in cold growth media for further processing within the next 6-8 h as illustrated here in this protocol.
This protocol describes the dissection and preparation of human meninges samples for the development of patient primary leptomeninges cell culture. The tissue is cut into 25-30 pieces of approximately 3 mm x 3 mm squares. Three pieces are placed in each 6-well gelatin-coated well and held down with round glass coverslips. The meninges dissection takes about 25-35 min. The main challenge with this culture is sterility as the brain procurement, transport, and dissection are generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B and use multi-well dishes to separately culture tissue pieces.
Outgrowth of meningeal fibroblasts occurs usually within the first week. Media is changed every two to three days until cells are confluent and the cells are enzymatically passaged. The meningeal fibroblasts are cryopreserved at 1 million cells per mL/vial in cryopreservation media. With this protocol, 20-30 million meningeal fibroblasts can be derived in 6-8 weeks for cryopreservation. Downstream applications of these meningeal fibroblasts are primary cultures for disease research, direct neuronal differentiation or derivation of induced pluripotent stem cells from the leptomeninges for understanding of disease mechanisms and for drug development.

<table border="0" cellpadding="0" cellspacing="0" width="1256">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:459px;">Corning Petri dishes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:127px;">351029</td>
			<td style="width:485px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc 6-well plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-mm cover slips</td>
			<td>Fisher Scientific</td>
			<td colspan="2">12-545-83 15CIR-1D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels, sterile blade, No. 15</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved precision tip forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>22-230490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution</td>
			<td>Fisher Scientific</td>
			<td>SH30264.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 500 mL filter unit</td>
			<td>Fisher Scientific</td>
			<td>430770</td>
			<td>Combine media components and filter.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Nunc Cell Culture Treated Flasks with Filter Caps, T175 cm2&#160;</td>
			<td>Thermo Scientific</td>
			<td>178883</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Growth Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hyclone DMEM</td>
			<td>Fisher Scientific</td>
			<td>SH30081.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone FBS</td>
			<td>Fisher Scientific</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100x)</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B (Yellow Solution/250 &#181;g/mL)</td>
			<td>Fisher Scientific</td>
			<td>BP264520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bambanker Freeze 120 mL</td>
			<td>Fisher Scientific</td>
			<td>NC9582225</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fibronectin Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">8 well chamber slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>1256518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Glycerol&#160;</td>
			<td>BioRad</td>
			<td>9455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% normal goat serum&#160;</td>
			<td>Fisher Scientific</td>
			<td>101098-382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Fibronectin antibody [F1]</td>
			<td>Abcam</td>
			<td>ab32419</td>
			<td>1:300 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SERPINH1</td>
			<td>Sigma</td>
			<td>S5950-200ul</td>
			<td>1:250 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SOX2</td>
			<td>Millipore</td>
			<td>MAB4343</td>
			<td>1:100 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td>1:200 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TUJ1</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>1:1000 dilution in blocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 anti-rabbit</td>
			<td>Thermo Fisher</td>
			<td>A11029</td>
			<td>1:400 dilution in blocking solution; (green channel; Ex/Em2 495/519 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 555 anti-mouse</td>
			<td>Thermo Fisher</td>
			<td>A21424</td>
			<td>1:400 dilution in blocking solution; (red channel; Ex/Em2 590/617 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td>dilute to a final concentration of 1.0 &#956;g/ml; (blue channel; Ex/Em2 358/461 nm)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Suppliers are suggestions, similar products from alternative vendors can be used as well.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation of leptomeninges explant cultures from postmortem human brain donors

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of Parkinson&#39;s disease is not pathologically confirmed in up to 25% of clinically diagnosed Parkinson&#39;s disease. Therefore, tissue collected from clinically diagnosed patients with idiopathic Parkinson&#39;s disease can have a high rate of misdiagnosis; hence in vitro studies from such tissues to study Parkinson&#39;s disease as a preclinical model can become futile.
By collecting postmortem human leptomeninges with a confirmed neuropathological diagnosis of Parkinson&#39;s disease and characterized by nigrostriatal cell loss and intracellular protein inclusions called Lewy bodies, one can be certain that clinically observed parkinsonism is not caused by another underlying disease process (e.g. tumor, arteriosclerosis).
This protocol presents the dissection and preparation of postmortem human leptomeninges for derivation of a meningeal fibroblast culture. This procedure is robust and has a high success rate. The challenge of the culture is sterility as the brain procurement is generally not performed under sterile conditions. Therefore, it is important to supplement the culture media with a cocktail of penicillin, streptomycin, and amphotericin B.
The derivation of meningeal fibroblasts from autopsy-confirmed cases with Parkinson&#39;s disease provides the foundation for in vitro modeling of Parkinson&#39;s disease. Meningeal fibroblasts appear 3-9 days after sample preparation and about 20-30 million cells can be cryopreserved in 6-8 weeks. The meningeal fibroblast culture is homogenous and the cells express fibronectin, a commonly used marker to identify meninges.

When the leptomeninges processing protocol has been successful, outgrowth of meningeal fibroblasts is first observed three to nine days after dissection, although this can depend on the length of post-mortem interval for the brain. Figure 1 demonstrates meningeal fibroblast cultures of four different donors. Figure 1A shows a leptomeninges piece held down by a glass cover slip (dark diagonal line) and fibroblast outgrowth around the tissue four days after...

Watch this Scientific Journal Video about Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors at JoVE.com

Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

The leptomeninges explant culture protocol from human postmortem brain is a technically robust and simple way to derive fibronectin-positive meningeal fibroblasts within 6-8 weeks and cryopreserve approximately 20-30 million cells.

Even though great progress has been made in the clinical characterization of Parkinson&#39;s disease, several studies report that the diagnosis of ...

I'm Birgitt Schule, the Director of the Brain Donation Program at the Parkinson's Institute and Clinical Center. In this video, we describe a technique for an explant cell culture and duration of primary fibroblast from leptomeninges of postmortem human brain from patients with Parkinson's disease. These fibroblasts can be used for variety of applications, including cell-based assays.This is important, because we can study cell lines from autopsy-proven cases of sporadic Parkinson's disease with Lewy body pathology. Begin by placing all required materials into the tissue culture hood. All objects entering the hood must be sprayed with 70%ethanol and wiped down.The first step is to transfer the meninges from the tube to the cutting dish. Start by pouring approximately 10 milliliters of phosphate-buffered saline into a sterile 10-centimeter cutting dish. Here you can see the meninges.Using sterile forceps, carefully transfer the meninges into the cutting dish. Perform this step slowly to prevent ripping. The next step is to wash the meninges in PBS to remove as much blood as possible.If your sample is too bloody, you may consider transferring it to another cutting dish. Add another 10 milliliters of PBS into a fresh 10-centimeter dish. Repeat this wash to continue removing blood from the meninges.Finally, transfer the meninges into a fresh 10-centimeter dish containing PBS. When finished, cover the dish and transfer it to the dissecting microscope in the laminar flow Hood. This next step is to continue sectioning the meninges into smaller pieces.Using two sterile scalpels, gently separate the blood vessels from the meninges with a shallow scraping motion. Prepare a region large enough to cut roughly 18 3-millimeter by 3-millimeter pieces, as shown in this image. Then, take the scalpel and, using a rocking motion, section the prepared region into 3-millimeter by 3-millimeter pieces.It is easier to do this in a stepwise process by first cutting the meninges into larger pieces and then cutting those pieces into smaller sections. Cover the cutting dish, and transfer it back into the tissue culture hood. Next, prepare to transfer the meninges into a six-well tissue culture plate that has been pretty coated with 1 milliliter of 0.1 percent gelatin for 30 to 60 minutes.Add one milliliter of meninges media into each well. Then, with sterile forceps, transfer three to four meninges pieces into the center of each well. The cells grown on this plate will be a passage zero.The next step uses coverslips to ensure that the meninges pieces are stationary and remain in contact with the culture dish. Place a sterile 15-millimeter circular coverslip over the meninges pieces. If necessary, reposition the meninges pieces with a coverslip.Then, using the forceps, firmly press down on the coverslip to ensure that the pieces are touching the plate. This image shows the meninges pieces held in place with a coverslip. Place the cover over the six-well plate and carefully transfer the plate into an incubator set to 37 degrees Celsius and 5%carbon dioxide.After two days in culture, add an additional one milliliter of fresh meninges media to each well. At seven days, aspirate the media. Then, refill each well with 2 milliliters of fresh media.After day seven, continue to replenish the wells with 2 milliliters of fresh media every other day. This image shows the initial day of the meninges pieces and culture under an inverted microscope. After roughly six to nine days, cellular attachment in the first outgrowth can be observed.Between 15 to 19 days, the cell number increases and growth becomes denser. Meningeal fibroblasts are characterized by the expression of fibronectin. These images show cultured meningeal cells that have been stained and are positive for fibronectin.After watching this video, you should have a good understanding of how to derive leptomeninges explant cultures from postmortem human brain donors. This technique provides a simple and robust way to derive meningeal fibroblast within three to six weeks, and allows to bank 15 to 20 million cells at a low passage number.

derivation-leptomeninges-explant-cultures-from-postmortem-human-brain

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

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.

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 ...

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 ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...