The authors would like to thank Dr. Y. Sasai for generously providing the FOXG1 antibody. This work was supported by Connecticut Stem Cell Research Grants (08-SCB-UCHC-022 and 11-SCB24) and Spastic Paraplegia Foundation.

1. Generation of Human Pluripotent Stem Cell Aggregates (D1-D4)
<ol>
	<li>Human pluripotent stem cells are cultured on mouse embryonic fibroblast (MEF) feeders in the presence of hESC medium supplemented with basic fibroblast growth factor (bFGF, 4 ng/ml). After 5-7 days in culture, when the colonies are big but still undifferentiated, they are ready for the next step.</li>
	<li>The enzyme solution should first be prepared. In a 50 ml tube, dissolve the dispase (or collagenase) at a 1 mg/ml concentration into DMEM/F12 medium. Since these solutions mix best when warmed, place the tube containing the medium and enzyme into a 37 &deg;C water bath for 10-15 min and then sterilize it using a Steriflip filter.</li>
	<li>Aspirate off the medium from the cells, rinse them with DMEM/F12, and aspirate this off as well. Add 1 ml of dispase to each well and place in the incubator for 3-5 min for hESCs (up to 10 min for iPSCs). Look at the cells under the microscope; when the edges curl up slightly/look a bit darker, the cells are ready for the next step. It is best to check the cells after 3 min and put them back in if necessary. When using this protocol for the first time, it is best to start with only one plate of cells at a time as it is important to remove the cells from the dispase as quickly as possible.</li>
	<li>Aspirate off the dispase from the cells. Gently (the cells are very vulnerable at this stage and will come off of the plate relatively easily) add 1.5 ml of DMEM/F12 medium to each well in order to rinse off the dispase. Aspirate off the DMEM/F12 and add 1.5 ml of hESC medium to each well.</li>
	<li>To detach and break up the cells, place the tip of a 10 ml glass pipette towards the bottom right hand corner of a well (touching the cells) and move the pipette up and down (vertically) - the upward stoke should be in close proximity to the proceeding downward stroke. Once the other side of the well is reached, repeat in the horizontal direction.</li>
	<li>Once all of the cells are in suspension, place them in 15 ml tubes and centrifuge them at 200 x g for 2 min. There should be a pellet of cells at the bottom of the tube. Aspirate off the medium from above the pellet.</li>
	<li>The size of the cell culture flask that should be utilized to keep the aggregates in depends upon the original quantity of cells. For 6 wells (1 plate) of hESCs, one non-tissue culture treated T75 flask with 50 ml of hESC medium should be utilized. Transfer the cells from step 1.6 to the flask and incubate at 37 &deg;C.</li>
	<li>In order to get rid of the cell debris, the cell medium and flask should be replaced approximately 12 hr later. To do this, place all of the medium/cells into a 50 ml tube, spin the cells down (200 x g for 2 min), and aspirate off the old medium. Next, add 50 ml of fresh hESC medium to the cells, and place into a new T75 flask. The cells can then be fed daily following this same procedure (until day 5).</li>
</ol>
2. Induction of Neuroepithelial Cells (D5-D17)
<ol>
	<li>Transfer the cells/medium to a 50 ml tube, and centrifuge the cells for 2 min at 200 x g. Next, aspirate off the supernatant, and transfer the cells to a new (non-tissue culture treated) T75 flask with 50 ml of neural induction medium (NIM).</li>
	<li>Once the cells are in NIM, half of the medium should be replaced every other day. The aggregates should become large enough to where they will sink to the bottom of a 50 ml conical tube on their own after a few minutes (no centrifugation required). After 2-3 days in NIM (D7-D8 total), the cells should be ready to plate. Laminin or fetal bovine serum (FBS) can be utilized to help the cells attach to the plates.</li>
	<li>The number of tissue culture treated 6 well plates that are needed depends upon the density of the cells as each aggregate should be able to grow on the plate for several more days without touching any of the others. On average, the cells from one T75 flask will yield about 4-6 6 well plates worth of cells.</li>
	<li>
	<ol class="ualpha">
		<li>If using laminin, dilute it in NIM to 10-20 &mu;g/ml to coat the 6-well plate (~0.5 ml/well). After coating for 2-3 hr, plate cell aggregates onto 6-well plates (~20-30 aggregates per well). After the aggregates have attached, add 1.5 ml of NIM.</li>
		<li>If using FBS, prepare a mixture of 90% NIM and 10% FBS (1.5 ml/well). Add 1.5 ml of medium with the cells to each well, gently shake the plate back and forth to spread out the aggregates, and allow the cells to attach O/N in an incubator. Be sure to change the medium (2 ml/well of NIM) the following morning to remove the FBS.</li>
	</ol>
	</li>
	<li>After a day or two of being plated, each aggregate will spread out to form a monolayer. Within a couple of days, the centers of most of these cells will morphologically appear similar to columnar cells (elongated). During this period, change the NIM every other day. Some PSC lines (especially iPSC), may form columnar cells less efficiently. If this happens, the addition of BMP and TGF inhibitors (Dorsomorphin29, SB43154219 at 2 &mu;M) during stage 2 can be helpful.</li>
	<li>Around days 14-17, the columnar cells will appear more compact. Sometimes these cells form ridges or rings with visible lumen inside. Besides the columnar morphology, cells at this stage express neuroepithelial markers, such as PAX6 and SOX112,26.</li>
	<li>Before the isolation can begin, one must first examine the cells to determine if there are any non-neuroepithelial cells present. An objective marker can be utilized to indicate the non-neuroepithelial colonies. These can then be eliminated by scraping them off with a pipette tip.</li>
</ol>
3. Generation of Telencephalic Progenitors (D17-D24)
<ol>
	<li>The centers of the colonies contain the neuroepithelial cells, and should be isolated from the rest of the colony. This can be done using a micropipette and a sterile 1 ml filtered pipette tip. When a small amount of pressure is applied to the center portion of the colony, it generally lifts off quite readily. Be cautious to not let the outer portion of the colony detach as well. If the centers are being stubborn and will not come off on their own, they can be removed by excising them using a needle under a microscope in a sterile hood. Transfer the center portions of the colonies to a 15 ml tube and spin at 200 x g for 2 min.</li>
	<li>The medium should be aspirated off of the cells. Two plates worth of cells can then be transferred to a non-tissue culture treated T25 flask containing 10 ml of NIM with the addition of B27 (without vitamin A, 1:50). Use 5-10 ml of medium per one plate of cells.</li>
	<li>When the cells are viewed the following day, they should have a sphere like appearance. It is necessary to transfer these cells into a new flask (in the presence of NIM and B27, 1:50) as it is often the peripheral cells which sneak in and attach to the bottom of the flask.</li>
	<li>The neurospheres should then be cultured in suspension for one week using NIM supplemented with B27 (1:50). In order to promote cell endurance and proliferation, cyclic AMP (cAMP, 1 &mu;M) as well as insulin growth factor (IGF1, 10 ng/ml) can be added into the medium during suspension culture. At this point, the neurospheres contain telencephalic progenitors (FOXG1+) and are ready to be plated onto coverslips for terminal differentiation.</li>
</ol>
4. Further Differentiation into the Telencephalic Neurons (D25+)
<ol>
	<li>
	<ol class="ualpha">
		<li>Prepare the poly-ornithine/laminin coated coverslips (in 24-well plates) for plating the cells. Coverslips are first cleaned, and are then coated with ploy-ornithine (at 0.1 mg/ml, 37 &deg;C, O/N, and stored in the freezer). 
		 Poly-ornithine coating: 
		<ol>
			<li>Sterilize the coverslips: Place the coverslips in a beaker containing nitric acid and gently shake in the fume hood for one hour. Pour out the nitric acid, rinse several times with DI water, and leave the coverslips under running DI water for at least 15 min. Place the coverslips in 95% ethanol and either shake for 30 min (if using immediately) or store in ethanol at RT.</li>
			<li>In a sterile hood, pour out the contents of the 50 ml tube containing sterilized coverslips into the lid of a 6-well plate. Using sterilized forceps, pick up one coverslip at a time and set upright in a single well of a 24-well plate. Repeat so that each well has a coverslip.</li>
			<li>After they dry completely, tap the plates until coverslips have fallen flat in each well. Next add 100 &mu;l poly-ornithine (0.1 mg/ml in sterile dH2O) to each coverslip and incubate the plates O/N at 37 &deg;C.</li>
			<li>The next day, remove plates from the incubator and allow them to cool to RT. Aspirate poly-ornithine off of each coverslip (from the edge of the coverslip) making sure not to touch or scratch the coverslips in the process. Allow the coverslips to dry for approximately 30 min before washing.</li>
			<li>Add 1 ml sterile dH2O to each well, let sit for 10 min, and aspirate off the water from each well. Repeat this wash step two more times. After the plates dry completely, (leave covers off in the hood) place the lids on, cover with aluminum foil, and store at -20 &deg;C.</li>
		</ol>
		</li>
		<li>
		<ol>
			<li>To coat the coverslips with laminin, add 50 &mu;l of a mixture of neural differentiation medium and laminin (final concentration of 20 &mu;g/ml) to each poly-ornithine treated coverslip being careful to only have the liquid touching the coverslip itself (and not spilling off). Incubate them at 37 &deg;C for 1-2 hr before preparing the cells for attachment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Collect and centrifuge the neurospheres at 200 x g for 2 min in a 15 ml tube. Aspirate off the supernatant.</li>
	<li>
	<ol class="ualpha">
		<li>Rinse the cells with DMEM-F12 and centrifuge the cells again. If the neurospheres are too large to attach properly, they can be dissociated with Accutase at this step.</li>
		<li>If using Accutase: After washing the cells with DMEM-F12, the neurospheres are dissociated to small clusters by adding Accutase (~2 ml) and incubating the cells at 37 &deg;C for 3 min. Next, add the same amount of trypsin inhibitor (~2 ml) and centrifuge the cells at 200 x g for 3 min. The cells should then be re-suspended in NDM and broken using a P200 pipette. The small clusters can then be plated onto pre-coated coverslips.</li>
	</ol>
	</li>
	<li>Aspirate off the majority of the medium and place the cells onto a 6 cm Petri dish in a few drops of the medium so that selecting them becomes easier. Add about 4-5 neurospheres to each pre-coated coverslip. There is no need to aspirate off the laminin first.</li>
	<li>Let the neurospheres attach for at least 2-4 hr. Once they have attached well, more medium (0.5 ml/coverslip) should be added. It should consist of neural differentiation medium supplemented with B27 (1:50), cAMP (1 &mu;M), and neurotrophic factors (BDNF, GDNF, and IGF, all at 10 ng/ml). At this point half of the medium can be changed every other day and these cells can be maintained for several months.</li>
</ol>

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D., Yu, J., 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=Induced+pluripotent+stem+cells+from+a+spinal+muscular+atrophy+patient.">Induced pluripotent stem cells from a spinal muscular atrophy patient.</a> Nature. 457, 277-280 (2009).</li><li>Lee, G., Papapetrou, E. P., 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=Modelling+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461, 402-406 (2009).</li><li>Park, I. H., Arora, N., 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=Disease-specific+induced+pluripotent+stem+cells.">Disease-specific induced pluripotent stem cells.</a> Cell. 134, 877-886 (2008).</li><li>Chamberlain, S. J., Chen, P. 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=Induced+pluripotent+stem+cell+models+of+the+genomic+imprinting+disorders+Angelman+and+Prader-Willi+syndromes.">Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 17668-17673 (2010).</li><li>Kiskinis, E., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+toward+the+clinical+application+of+patient-specific+pluripotent+stem+cells.">Progress toward the clinical application of patient-specific pluripotent stem cells.</a> J. Clin. Invest. 120, 51-59 (2010).</li><li>Koch, P., Tamboli, I. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin-1+L166P+mutant+human+pluripotent+stem+cell-derived+neurons+exhibit+partial+loss+of+gamma-secretase+activity+in+endogenous+amyloid-beta+generation.">Presenilin-1 L166P mutant human pluripotent stem cell-derived neurons exhibit partial loss of gamma-secretase activity in endogenous amyloid-beta generation.</a> Am. J. Pathol. 180, 2404-2416 (2012).</li><li>Walsh, R. M., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Rett+syndrome+with+stem+cells.">Modeling Rett syndrome with stem cells.</a> Cell. 143, 499-500 (2010).</li><li>Egawa, N., Kitaoka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+Screening+for+ALS+Using+Patient-Specific+Induced+Pluripotent+Stem+Cells.">Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells.</a> Sci. Transl. Med. 4, (2012).</li><li>Kola, I., Landis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+the+pharmaceutical+industry+reduce+attrition+rates.">Can the pharmaceutical industry reduce attrition rates.</a> Nature Reviews Drug Discovery. 3, 711-716 (2004).</li></ol>

The authors declare that they have no competing financial interests.

There are several critical steps during the neural differentiation process. It is important to ensure that the human PSCs are pluripotent because otherwise the cells may already be biased towards becoming a non-neuronal lineage. This can be confirmed by staining the human PSCs with antibodies against pluripotency markers such as Oct4, Sox2, Nanog, and Tra-1-60 1-3. If the human PSCs do not attach very well after passaging them, ROCK inhibitor (Y27632) can be added to help. For those having difficulty with keep...

Human pluripotent stem cells (PSCs), including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the capacity to generate every cell type in the body, including neurons1-3. Directed differentiation of various neuronal subtypes from human PSCs holds the key for the application of these cells in regenerative medicine. The generation of functional neuronal subtypes during development is a complex process involving the induction of neural lineage, the specification of regional progenitors along the rostro-caudal axis, and the differentiation of post-mitotic neuron types from the regional progenitors4,5. Beginning in 2001, several systems have been established to generate neural lineage from hESCs, which have provided a platform for the subsequent generation of neuronal subtypes6,7. Based upon developmental principles, several neuron types such as spinal motor neurons8-12, midbrain dopaminergic neurons13-15, and neural retinal cells16,17 have been efficiently specified from human PSCs. This was done by applying critical morphogens which are important for the specification of these neuron types during in vivo development. Other protocols have also been developed to promote the differentiation of hESCs into neurons using either additional factors18-20 such as small molecules or by co-culturing with other cell types to help promote differentiation21.
The human neocortex is highly developed and contains many cell types, including glutamatergic neurons which play an important role in learning, memory, and cognitive function22,23. The first step in generating glutamatergic neurons in culture is to specify telencephalic progenitor cells. Yoshiki Sasai's group first reported the directed differentiation of telencephalic precursors from mouse ESCs (mESCs) using a serum-free suspension culture in the presence of DKK1 (which inhibits Wnt signaling) as well as LeftyA (which inhibits nodal signaling)24. Subsequently, several groups including ours have also reported the specification of telencephalic precursors from human PSCs in serum free medium 25-27. The generation of telencephalic precursors from human PSCs does not require the use of exogenous morphogens and the efficiency in generating these precursors is much higher than that from mESCs 26,27. Here, a chemically defined system for neural induction which was well established by Zhang's group7 has been described. Without the addition of exogenous caudalizing factors, this protocol efficiently generates telencephalic precursors from human PSCs27. These progenitors can then be differentiated into dorsal or ventral progenitors by regulating the signaling of Wnt and sonic hedgehog (SHH).The dorsal progenitors can further differentiate into glutamatergic neurons efficiently27. In addition, this protocol also works well for the generation of glutamatergic neurons from human iPSCs28, which allows for the generation of patient-specific neurons that can be utilized to explore the mechanism of action as well as potential therapies for a large array of diseases. Moreover, our system also provides a platform to explore the development and specification of diverse neuronal types in the telencephalon.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11330-032</td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>10828-028</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>1140-050</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol (14.3 M)</td>
			<td>Sigma</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine)</td>
			<td>Sigma</td>
			<td>116K5103</td>
		</tr>
		<tr>
			<td>Laminin (human)</td>
			<td>Sigma</td>
			<td>L-6274</td>
		</tr>
		<tr>
			<td>Laminin (mouse)</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini</td>
			<td>100-106</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
		</tr>
		<tr>
			<td>ROCK Inhibitor</td>
			<td>Stemgent</td>
			<td>04-0012</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Stemgent</td>
			<td>04-0024</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075</td>
		</tr>
		<tr>
			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
		</tr>
		<tr>
			<td>Foxg1 antibody</td>
			<td>Dr. Y. Sasai</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoxb4 antibody (1:50)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>I12</td>
		</tr>
		<tr>
			<td>Pax6 antibody (1:5000)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>PAX6</td>
		</tr>
		<tr>
			<td>Nkx2.1 antibody (1:200)</td>
			<td>Chemicon</td>
			<td>MAB5460</td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
		</tr>
		<tr>
			<td>vGLUT1 antibody (1:100)</td>
			<td>Synaptic Systems</td>
			<td>135302</td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-02</td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (GDNF)</td>
			<td>PrepoTech Inc.</td>
			<td>450-10</td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>PrepoTech Inc.</td>
			<td>100-11</td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma</td>
			<td>D-0260</td>
		</tr>
		<tr>
			<td>Sonic hedgehog (SHH)</td>
			<td>R&amp;D</td>
			<td>1845-SH</td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Becton Dickinson (BD)</td>
			<td>352098</td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352097</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>BD</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>BD</td>
			<td>353047</td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>BD</td>
			<td>353009</td>
		</tr>
		<tr>
			<td>T75 flasks (untreated)</td>
			<td>BD</td>
			<td>353133</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>BD</td>
			<td>353004</td>
		</tr>
		<tr>
			<td>9'' glass pipetes</td>
			<td>Fisher</td>
			<td>13-678-20D</td>
		</tr>
		<tr>
			<td>Steriflip filters (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
		</tr>
		<tr>
			<td>Stericup filters 1,000 ml (0.22 &mu;M)</td>
			<td>Millipore</td>
			<td>SCGPU10RE</td>
		</tr>
		<tr>
			<td>Phase contrast microscope (Observer A1)</td>
			<td>Zeiss</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator (Hera Cell 150)</td>
			<td>Thermo Electron Corporation</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biosafety hood (Sterilgard III Advance)</td>
			<td>The Baker Company</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge (5702 R)</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

the specification of telencephalic glutamatergic neurons from human pluripotent stem cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

Here, a protocol to differentiate human PSCs into telencephalic glutamatergic neurons through several critical steps: the formation of PSC aggregates, the induction of neuroepithelial cells, the generation of telencephalic progenitors, and the terminal differentiation of these progenitors into telencephalic neurons (Figure 1) has been described. This system is robust and efficient in the generation of telencephalic progenitors and glutamatergic neurons. As an example (Figure 2), without ...

Watch this Scientific Journal Video about The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells at JoVE.com

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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Neuroscience

10.9K Views.  The University of Connecticut Health Center. This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Video: The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

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 ...

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

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

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

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

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

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

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

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

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

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

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

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

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

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...