We wish to thank Dr. Devin Gary for his insight and feedback on the use of GRP cells.

Introduction
In this protocol we show how to extract, select and plate glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic and astrocytic cell lineages and are defined by their expression of A2B5. In the GRP medium supplemented with FGF-2, the A2B5+ GRPs will begin expressing the early oligodendrocytic lineage markers PDGF&alpha;R and NG21,2. These oligodendrocyte lineage cells pass through a series of distinct phenotypic stages, each of them characterized by morphological changes, as well as expression of markers to specific developmental stages.
These precursor cells are currently being studied as a potential source for cell-based therapeutic approaches in disorders of the central nervous system white matter, including multiple sclerosis, leukodystrophies and periventricular leukomalacia (PVL)3,4,5,6. Studies using human brain derived oligodendrocyte lineage precursors have reported successful remyelination in brains of the shiverer mouse dysmyelination model as well as in demyelinated spinal cord rodent models6,7,8,9. These glial precursor cells have also been used in studies of other white matter disease models such as spinal cord injury and amyotrophic lateral sclerosis10,11,12,13.
Our laboratory has developed an early postnatal hypoxia-ischemia mouse model with which we are evaluating the efficacy of these spinal cord derived GRP cells as a restorative approach in PVL, the leading cause of cerebral palsy14,15,16. There is also a rodent brain derived OPC model that has been widely used for studying white matter injury since the GRP cells have a tendency to differentiate into the astrocytic pathway17. The OPC phenotype has already entered the oligodendrocyte lineage pathway, a phenomenon that seems irreversible. However, we are interested in assessing the response of a wider spectrum of oligodendrocyte progenitors including GRPs and possibly even the NEPs derived at E10.5. For this reason we have adopted the spinal cord derived GRP model for our work.
In addition to the use of GRPs for cell replacement, the derivation of these cells from wild type and transgenic rodent models of white matter diseases allows further study into glial differentiation under normal and disease conditions in an in vitro setting. Previous publications show evidence of selective vulnerability of oligodendrocytic lineage precursor cells to various external stressors like maturation dependent glutamate excitotoxicity, oxidative stress, and select factors implicated in neuroinflammation18,19. Our studies have focused on understanding the cellular and molecular mechanisms behind the development of these white matter injuries, evaluating the susceptibility of cells in the oligodendrocyte lineage spectrum to insult as well as analyzing putative therapeutic approaches.
Materials
All procedures involving animals conform to PHS policy and the JHU IACUC. All procedures should be performed in a laminar flow hood in order to maintain aseptic conditions. Commonly dissections are performed in a horizontal flow hood and in vitro work in a vertical flow hood. All media used cold during dissections. The mice used in our study are a wild type CD-1 strain and a transgenic GFP in a C57/BL6 background. One CD-1 mouse dam typically yields 10+ fetuses which are enough to seed 1-2 T25 flasks. Typically, two dams are sacrificed at a time and fetal spinal cords pooled and plated into 1 T25 flask per dam. Once cells have been derived they can be expanded and characterized in vitro for subsequent studies.
1. Preparation of Media and Flasks
<ul type="disc">
 <li>GRP stock medium is used as the Dissection medium: DMEM / F12 1:1 (Invitrogen) + B27 (50x; Invitrogen) N2 (100x; Invitrogen) Supplements and Bovine Serum Albumin (BSA; 0.5% w/v).</li>
 <li>Dissociation medium: same as dissection medium.</li>
 <li>Culture medium : GRP stock medium + FGF-2 (10-20 ng/ml; Invitrogen) and heparin (1 &mu;g/ml; Sigma).</li>
 <li>PLL/laminin coated 75 cm2 or 25 cm2 flasks (T75 or T25).</li>
 <li>Tissue culture flasks (75 cm2, Falcon) were coated with 15-20&mu;g/ml Poly-L-lysine (PLL; Sigma) in distilled H2O, incubated at 37 &deg;C for 1 hr then aspirated. Flasks are washed 1X with PBS then coated with 15-20 &mu;g/ml Laminin (Sigma) in PBS at 37 &deg;C for 1 hr, then aspirated and fresh PBS or media added until cells are ready to be plated. Following coating, flasks should never be allowed to dry but can be kept in PBS or media at 4 &deg;C for brief periods before use.</li>
</ul>
2. Instruments and Other Material
<ul type="disc">
 <li>Petri dishes: 4 per dam: 3 Petri dishes for dissection (75 mm), a 20 mm dish for collecting the harvested spinal cords.</li>
 <li>Large scissors (43 mm Operating scissors, Roboz) and tissue Forceps for opening the dam s abdomen.</li>
 <li>Small scissor for dissecting the uterus and removing the fetuses (15 mm Micro Dissecting Scissors, Roboz).</li>
 <li>Micro Dissecting spring scissors (6 mm) to dissect the spinal cord of the fetuses.</li>
 <li>Micro Dissecting angled Forceps for anchoring the dam and fetal body during surgery and later removing the spinal cord once exposed.</li>
 <li>40 or 70 micrometer cell strainer for removing cell debris before plating.</li>
 <li>0.05% Trypsin prewarmed to 37 &deg;C.</li>
 <li>10 mg/ml DNAse 1 (Sigma), prepared as a 100x stock (1 g/ml).</li>
 <li>Bio-hazard bag for animal corpses.</li>
 <li>15 and 50 ml centrifuge tubes for processing extracted spinal cords.</li>
</ul>
3. Determining Timed Pregnancy
Pregnant mouse dams are either ordered from commercial sources specifying delivery on embryonic day E12 or E13 of fetal development or pregnancy determined in-house. Briefly, two females are placed together with one male in the late afternoon and left overnight together. The next day the females are observed for the presence of the vaginally located mucus plug. The mucus plug is only an indication that mating occurred so animals are subsequently weighed daily to monitor rate of weight increase. The day the mucus plug is observed is defined as embryonic day one (E1).
4. Tissue Derivation
<ol>
 <li>Work under a horizontal flow hood to maintain aseptic conditions.</li>
 <li>Spray down the hood and working area with 70% ethanol.</li>
 <li>Autoclave instruments or spray liberally with 70% ethanol.</li>
 <li>Prepare instruments, media, and microscope for use.</li>
 <li>Pour 20 ml of cold dissection medium into sterile Petri dish.</li>
 <li>Anesthetize animal with chloral hydrate (500 mg/kg) administered intraperitoneally (IP) followed by cervical dislocation or decapitation.</li>
 <li>Spray the abdominal area of dam with 70-90% ethanol (disinfection)</li>
 <li>Open abdomen with large scissor and forceps.
 <ol type="a">
 <li>In CD1 and C57/BL6 strains: Usually 8 to 14 fetuses will be inside the uterus. The number of fetuses is strain dependent.</li>
 </ol>
 </li>
 <li>Extract the mouse uterus including the pups and place in fresh cold dissection medium in a clean Petri dish to wash blood and tissue debris from fetus.</li>
 <li>Extract each fetus from the uterine horn and separate them from the embryonic sac and placenta using the small scissors. Transfer the extracted fetuses into fresh dissection medium in a new Petri dish.
 <ol type="a">
 <li>Dissection medium should be at low temperature to lower metabolic activity and preserve viability of derived cells.</li>
 <li>Work from now on with two forceps and micro dissecting spring-scissors.</li>
 </ol>
 </li>
 <li>Working under dissection microscope, cut the skin along the spinal cord with the micro surgical scissors and peel skin back to expose the spinal cord.</li>
 <li>Transect the spinal cord with the microsurgical scissors at about C1 and at the beginning of the tail.</li>
 <li>Remove the spinal cord with blunt forceps, ensure all bone and cartilage are removed and transfer to 3rd Petri dish of dissecting medium.</li>
 <li>To prevent overgrowth of meningeal tissue in subsequent cell cultures try to remove as much of the meninges as possible from the spinal cord. Due to the nascent protruding peripheral nerves this is more difficult than removing meningeal tissue from extracted cerebrum.</li>
 <li>Once meninges have been removed, transfer spinal cords to 20 mm Petri dish</li>
 <li>Clean up by thoroughly spraying the area with 70-90% ethanol.</li>
 <li>The following steps for dissociation should be done quickly to maintain a high viability of the derived spinal cord tissue.</li>
</ol>
5. Culture Establishment
<ol>
 <li>Trypsin should be pre-warmed for at least 30 min in a 37 &deg;C H2O-bath. An appropriate aliquot should be removed from the stock to reduce the exposure of stock to temperature fluctuations.</li>
 <li>Transfer the harvested spinal cords to a 50 ml centrifuge tube containing 10ml pre-warmed trypsin (0.05%; Quality Biologicals) and 100 &mu;l DNAse (10 mg/ml) and triturate briefly.</li>
 <li>Incubate in 37 &deg;C H2O-bath for 10 minutes.</li>
 <li>Triturate and incubate for another 10 min.</li>
 <li>Add 5 ml GRP medium (defined above) and centrifuge for 5 min at 1000 RPM.</li>
 <li>Aspirate supernatant and resuspend pellet with 10 ml GRP medium and add DNAse-1 (10 mg/ ml; Sigma).</li>
 <li>Incubate in 37 &deg;C H2O-bath for 10 minutes.</li>
 <li>Centrifuge 5 min at 1000 RPM and aspirate supernatant.</li>
 <li>Resuspend pellet with fresh GRP medium then filter debris with a 40 - 70 &mu;m cell strainer.</li>
 <li>Plate on PLL/laminin coated 25 cm2 flasks (T25) and incubate at 37 &deg;C, 95% humidity and 5% CO2.</li>
 <li>100% media change on following day.</li>
 <li>At this point, GRPs have attached and started extending processes.</li>
</ol>
6. Immunopanning the GRP Population
<ol>
 <li>Plate spinal cord tissue in PLL/laminin coated flasks until flask is 85-90% confluent or about a week.</li>
 <li>Harvest cells by scraping flask with a rubber policeman or with 0.05% trypsin, triturate thoroughly but gently, centrifuge at 1000 RPM for 5 min and thoroughly resuspend pellet with 3 ml GRP medium.</li>
 <li>Transfer 1 ml each to 3 T25 flasks or 3 ml to 1 T75 flask add appropriate volumes of medium to completely cover cells and allow flasks to reach 80-90% confluence.</li>
 <li>Change media every other day, sooner if medium depletes quickly.</li>
 <li>Coat Bacteriological Petri dishes (75 mm) overnight at 4 &deg;C with an anti-mouse IgM antibody (Southern Biotech), 10 &mu;g/ml in PBS.</li>
 <li>Aspirate the excess buffer and wash the Petri dishes 3x with PBS.</li>
 <li>Add A2B5 antibody (Millipore) at a concentration of 5 &mu;g/ml diluted in PBS and incubate for 1 hr at room temperature (RT).</li>
 <li>Wash plates again 3x with PBS then add 8 ml of GRP medium to prevent drying of plate.</li>
 <li>Transfer 5 ml of GRP medium from Petri dishes and harvest GRP cells by scraping flasks with a rubber policeman to detach all cells</li>
 <li>Triturate cell suspension briefly to break up clumps and transfer 5ml cell suspension to the A2B5 coated bacteriological Petri dishes.</li>
 <li>Incubate 1 hr at room temperature without shaking.</li>
 <li>After 1 hr aspirate media and wash plates 8x with PBS.</li>
 <li>Gently scrape cells with a rubber policeman in 2 ml GRP media then transfer to PLL/ laminin coated plates or flasks with appropriate volume of GRP medium.</li>
</ol>
7. Freezing and Storage of Derived Cells
<ol>
 <li>Cells are harvested from an 85-90% confluent T25 or T75 flask with 2 ml 0.05% trypsin.</li>
 <li>Trypsin is diluted and inactivated with 8ml GRP stock medium and centrifuged at 1000 RPM for 5 min.</li>
 <li>Pellets from T25 flasks are resuspended in 1 ml GRP freezing medium, (GRP stock medium supplemented with 10% (v/v) DMSO and optionally, 20% FBS). The larger pellets from T75 flasks are diluted two-fold.</li>
 <li>Cell suspensions of 1.5 -- 3 x 1010,11,12,13 cells are transferred to cryogenic vials (Nalgene) and stored overnight in a cryogenic container (Nalgene) containing isopropanol at -80 &deg;C to slowly freeze overnight.</li>
 <li>Following overnight freezing cells are transferred to freezer boxes and stored 6 months to 1 yr at -80 &deg;C or longer term in N(l). Thawed cells are typically 60-80% viable depending largely on the quality of the PLL/laminin coating.</li>
</ol>
8. Representative Results
CO2 is not used for anesthetizing the animal because of the possible downstream effect on the viability of the fetuses and ultimately the derived cells. Furthermore, it will be very difficult to totally remove the meninges from the spinal cord during the derivation procedure but the combination of GRP medium and immunopanning will eliminate these fast growing cells. Similarly, the entire spinal cord is harvested despite a greater ventral concentration of the GRP population due to its originating in the ventral spinal cord and migrating dorsally20. However the GRP medium selects for the GRP phenotype and that population is maximized by A2B5 immunopanning. After plating spinal cord tissues, the in vitro cultures are incubated for two passages or about a week. After 2 days in culture, cells of different morphologies can be observed in the tissue culture flasks indicating the heterogeneity of the population but the GRP medium selects for the GRP phenotype. These cells are a combination of A2B5+ GRP, E-NCAM+ neuronal restricted precursors (NRP) and nestin+, A2B5- neuroepithelial cells and possibly fibronectin+ meningeal cells. In addition, more mature phenotypes may be present expressing markers including doublecortin and Tuj1 for neuronal lineage, GFAP for astrocytes and PDGFaR and GalC for oligo lineage In order to maximize a highly GRP enriched population from the starting heterogeneous pool (Figure 1A), cells can be double-immunopanned to select and eliminate the E-NCAM+ cells followed by selection for the A2B5+ GRP population once cell density has increased to about 85-90% confluency in T75 flasks. These GRP cells maintain their ability to become astrocytes despite being in a medium that selects for the oligodendrocyte phenotype so subsequent A2B5 immunopanning may be necessary to maintain a highly enriched GRP population. Immunopanning generally yields up to 95% A2B5+ cells but for even greater yield A2B5 conjugated magnetic beads (Miltenyi Biotec Inc.) can be used to provide a yield of up to 97%. Vigilance in the maintenance GRP cultures such as regular media changes will minimize the proliferation of non-GRP cell types. Even in the absence of BMP-4 astrocytes may still be found and depleted media in particular seems to induce differentiation to an astrocytic phenotype. Although the B27 supplement contains tri-iodothyronine hormone (T3), there has been no observed tendency to differentiate into oligodendrocytes in the GRP population, only when higher levels of T3 are added to the medium does this phenomenon occur. However, a T3 free B27 supplement can be substituted if there is a concern for contamination by mature oligodendrocytes (Figure 1B). These GRP cells can be readily identified by their morphology of small soma with 2 or 3 short processes but to screen for other cell types that may be present, immunocytochemistry can be performed for the previously named common developmental and cell type specific markers. There will commonly be a small persistent population of A2B5- cells that are neuroepithelial (NEP) and capable of becoming either GRP or NRP. Fortunately, the longer cells are maintained in the GRP medium the more likely that medium will select for the GRP phenotype.
<img alt="Figure 1." src="/files/ftp_upload/3462/3462fig1.jpg" />
Figure 1. a) Plated spinal cord cells of heterogeneous morphology are observed after 2 days in culture. b) Immunopanning maximizes a highly GRP-enriched population from the starting heterogeneous pool.

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G., Wang, X., Mayer-Proschel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+novel+glial-restricted+cell+populations+in+the+embryonic+telencephalon+arising+from+unique+origins.">Identification of two novel glial-restricted cell populations in the embryonic telencephalon arising from unique origins.</a> BMC developmental biology. 7, 33-33 (2007).</li><li>Nunes, M. C., Roy, N. S., Keyoung, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+isolation+of+multipotential+neural+progenitor+cells+from+the+subcortical+white+matter+of+the+adult+human+brain.">Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain.</a> Nature. 9, 439-439 (2003).</li><li>Roy, N. S., Wang, S., Harrison-Restelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation,+and+promoter-defined+separation+of+mitotic+oligodendrocyte+progenitor+cells+from+the+adult+human+subcortical+white+matter.">Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter.</a> J. Neurosci. 19, 9986-9986 (1999).</li><li>Chen, Y., Balasubramaniyan, V., Peng, 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+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nature protocols. 2, 1044-1044 (2007).</li></ol>

This study was funded by the National Institute of Health [NICHD P30HD024061 (A.W.P.), NINDS K08NS063956 (A.F.), and R01NS028208 (M.V.J.)] and the Child Neurology Foundation. The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Health, DHHS. The authors have no conflicts of interest relevant to this study.

Disorders of central nervous system white matter include a large number of etiologies, including genetic, inflammatory, ischemic and toxic causes19,21,22,23,24. While multiple sclerosis and vasculopathies are the leading cause of white matter disease in adults, periventricular leukomalacia associated with prematurity is the most common cause of white matter injury in the childhood population. In order to further delineate disease mechanisms further study of cells ofoligodendrocytic lineage, which are greatly affected by the perinatal insult, is required. GRPs were isolated from the rodent embryonic spinal cord and shown to be tripotential in nature, giving rise to oligodendrocytes and two distinct astrocyte populations, but do not differentiate into neurons2,25,26. Likewise,oligodendrocyte precursor cells (OPC) derived from rat optic nerve and late embryonic or early postnatal rat brain have been demonstrated to mature into functioning oligodendrocytes in the appropriate media conditions27,28.Additionally, fetal or adult derived human OPC have been further manipulated into more mature oligodendrocyte phenotypes29,30.
 GRP cells have been isolated from both the spinal cord of E13.5 rats and E12-13.5 in mice. In the rat differences have been reported between dorsal- and ventral-derived GRP cells in their response to conditions that promote generation of oligodendrocytes, or astrocytes with ventral-derived GRPs exhibiting a greater propensity to differentiate into the more mature phenotypes25,26,28. This is may be due to the ventral to dorsal migration of the nascent GRP cells, which could be less receptive to the differentiation cues. Immunopanning allows for the subpopulation that expresses A2B5 to be isolated and collected for maintenance in culture.
 Our mouse GRP cultures are derived from total spinal cord tissue that yields a heterogeneous, pooled population of cells from which GRP cells are selected by chemical (medium) and antibody based techniques. While our studies have utilized the spinal cord derived mouse GRP cells, it has more recently been reported the derivation of brain derived mouse oligodendrocyte progenitor cells that are also capable of developing into mature oligodendrocytes31. Here the derived cells are initially cultured as oligospheres that using the appropriate culture medium are manipulated into an enriched PDGF&alpha;R+, OPC population31. Our studies have focused on the effect of our model of hypoxic-ischemic insult on cells in the oligodendrocyte lineage spectrum from A2B5+, PDGF&alpha;R- GRP precursors to the myelin basic protein expressing (MBP+) mature oligodendrocytes. Indeed our in vitro characterization has shown these immature GRP cells mature into MBP expressing cells either in monocultures or in co-cultures with cortical neurons. These cells can be manipulated in culture into specific differentiation pathways for studying the mechanisms involved in perinatal white matter injury. There are several mouse models of white matter diseases involving cells of oligodendrocyte lineage and the availability of this population allows for thorough investigation into the mechanisms that cause injury as well as to explore the usefulness of possible cell therapy options.

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
		</tr>
		<tr>
			<td>DMEM/ F12 1:1</td>
			<td>Invitrogen</td>
			<td>11320033</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Invitrogen</td>
			<td>17502048</td>
		</tr>
		<tr>
			<td>rH-FGF-basic</td>
			<td>Invitrogen</td>
			<td>PHG0026</td>
		</tr>
		<tr>
			<td>Trypsin (0.05%) EDTA</td>
			<td>Quality Biological, Inc.</td>
			<td>118-087-721</td>
		</tr>
		<tr>
			<td>POLY-L-LYSINE HBr</td>
			<td>Sigma</td>
			<td>P1274-25MG</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
		</tr>
		<tr>
			<td>Dnase 1</td>
			<td>Sigma</td>
			<td>DN25</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Derivation of Glial Restricted Precursors from E13 mice at JoVE.com

Derivation of Glial Restricted Precursors from E13 mice

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 ...

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Neuroscience

11.2K Views.  Johns Hopkins University. Questo protocollo descrive la derivazione di precursori gliali limitati dal midollo spinale del feto e mantenute in vitro sia per trapianto o per lo studio del lignaggio oligodendrocitario.

Video: Derivation of Glial Restricted Precursors from E13 mice

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 ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

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.

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 ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

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Derivation of Glial Restricted Precursors from E13 mice