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 ...

<ol>
	<li>Takahashi, K., Tanabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Thomson, J. A., Itskovitz-Eldor, 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Yu, J., Vodyanik, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+specification+in+the+spinal+cord:+inductive+signals+and+transcriptional+codes.">Neuronal specification in the spinal cord: inductive signals and transcriptional codes.</a> Nat. Rev. Genet. 1, 20-29 (1038).</li><li>Wilson, S. I., Edlund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction:+toward+a+unifying+mechanism.">Neural induction: toward a unifying mechanism.</a> Nat. Neurosci. 4, 1161-1168 (2001).</li><li>Reubinoff, B. E., Itsykson, 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=Neural+progenitors+from+human+embryonic+stem+cells.">Neural progenitors from human embryonic stem cells.</a> Nat. Biotechnol. 19, 1134-1140 (2001).</li><li>Zhang, S. C., Wernig, M., Duncan, I. D., Brustle, O., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+transplantable+neural+precursors+from+human+embryonic+stem+cells.">In vitro differentiation of transplantable neural precursors from human embryonic stem cells.</a> Nat. Biotechnol. 19, 1129-1133 (2001).</li><li>Hu, B. Y., Weick, J. 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=Neural+differentiation+of+human+induced+pluripotent+stem+cells+follows+developmental+principles+but+with+variable+potency.">Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 4335-4340 (2010).</li><li>Singh Roy, N., Nakano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancer-specified+GFP-based+FACS+purification+of+human+spinal+motor+neurons+from+embryonic+stem+cells.">Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells.</a> Exp. Neurol. 196, 224-234 (2005).</li><li>Lee, H., Shamy, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+and+transplantation+of+human+embryonic+stem+cell-derived+motoneurons.">Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons.</a> Stem Cells. 25, 1931-1939 (2007).</li><li>Boulting, G. L., Kiskinis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functionally+characterized+test+set+of+human+induced+pluripotent+stem+cells.">A functionally characterized test set of human induced pluripotent stem cells.</a> Nat. Biotechnol. 29, 279-286 (2011).</li><li>Li, X. J., Du, Z. W., 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=Specification+of+motoneurons+from+human+embryonic+stem+cells.">Specification of motoneurons from human embryonic stem cells.</a> Nat. Biotechnol. 23, 215-221 (2005).</li><li>Perrier, A. L., Tabar, V., 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=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 12543-12548 (2004).</li><li>Roy, N. S., Cleren, C., 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=Functional+engraftment+of+human+ES+cell-derived+dopaminergic+neurons+enriched+by+coculture+with+telomerase-immortalized+midbrain+astrocytes.">Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes.</a> Nat Med. 12, 1259-1268 (2006).</li><li>Yan, Y., Yang, D., 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=Directed+differentiation+of+dopaminergic+neuronal+subtypes+from+human+embryonic+stem+cells.">Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.</a> Stem Cells. 23, 781-790 (2005).</li><li>Meyer, J. S., Shearer, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+early+retinal+development+with+human+embryonic+and+induced+pluripotent+stem+cells.">Modeling early retinal development with human embryonic and induced pluripotent stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 16698-16703 (2009).</li><li>Osakada, F., Ikeda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+generation+of+rod+and+cone+photoreceptors+from+mouse,+monkey+and+human+embryonic+stem+cells.">Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.</a> Nat Biotechnol. 26, 215-224 (2008).</li><li>Carpenter, M. K., Inokuma, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+neurons+and+neural+precursors+from+human+embryonic+stem+cells.">Enrichment of neurons and neural precursors from human embryonic stem cells.</a> Exp. Neurol. 172, 383-397 (2001).</li><li>Chambers, S. M., Fasano, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nat. Biotechnol. 27, 275-280 (2009).</li><li>Chambers, S. M., Qi, 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=Combined+small-molecule+inhibition+accelerates+developmental+timing+and+converts+human+pluripotent+stem+cells+into+nociceptors.">Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors.</a> Nat. Biotechnol. 30, 715-720 (2012).</li><li>Kawasaki, H., Mizuseki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28, 31-40 (2000).</li><li>Rubenstein, J. L., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+of+the+embryonic+forebrain.">Patterning of the embryonic forebrain.</a> Curr. Opin. Neurobiol. 8, 18-26 (1998).</li><li>Hevner, R. F., Hodge, R. D., Daza, R. A., Englund, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+in+glutamatergic+neurogenesis:+conserved+programs+in+neocortex,+cerebellum,+and+adult+hippocampus.">Transcription factors in glutamatergic neurogenesis: conserved programs in neocortex, cerebellum, and adult hippocampus.</a> Neurosci. Res. 55, 223-233 (2006).</li><li>Watanabe, K., Kamiya, D., 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=Directed+differentiation+of+telencephalic+precursors+from+embryonic+stem+cells.">Directed differentiation of telencephalic precursors from embryonic stem cells.</a> Nat. Neurosci. 8, 288-296 (2005).</li><li>Watanabe, K., Ueno, M., 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=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Pankratz, M. T., Li, X. 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=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Li, X. J., Zhang, X., 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=Coordination+of+sonic+hedgehog+and+Wnt+signaling+determines+ventral+and+dorsal+telencephalic+neuron+types+from+human+embryonic+stem+cells.">Coordination of sonic hedgehog and Wnt signaling determines ventral and dorsal telencephalic neuron types from human embryonic stem cells.</a> Development. 136, 4055-4063 (2009).</li><li>Zeng, H., Guo, M., 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=Specification+of+region-specific+neurons+including+forebrain+glutamatergic+neurons+from+human+induced+pluripotent+stem+cells.">Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells.</a> PLoS One. 5, e11853 (2010).</li><li>Kim, D. S., Lee, J. 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=Robust+enhancement+of+neural+differentiation+from+human+ES+and+iPS+cells+regardless+of+their+innate+difference+in+differentiation+propensity.">Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity.</a> Stem Cell Rev. 6, 270-281 (2010).</li><li>Zhang, X., Huang, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6+is+a+human+neuroectoderm+cell+fate+determinant.">Pax6 is a human neuroectoderm cell fate determinant.</a> Cell Stem Cell. 7, 90-100 (2010).</li><li>Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+patterning+of+the+central+nervous+system:+how+many+organizers.">Initial patterning of the central nervous system: how many organizers.</a> Nat. Rev. Neurosci. 2, 92-98 (2001).</li><li>Ma, L., Hu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+GABA+neurons+correct+locomotion+deficits+in+quinolinic+acid-lesioned+mice.">Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice.</a> Cell Stem Cell. 10, 455-464 (2012).</li><li>Li, W., Wei, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Lowry, W. E., Richter, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Dimos, J. T., Rodolfa, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Ebert, A. 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>

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 ...

the-specification-telencephalic-glutamatergic-neurons-from-human

Research

JoVE Journal

Neuroscience

10.8K visualizações.  The University of Connecticut Health Center. Este procedimento gera os neurónios telencefálicos atravessando checkpoints que são semelhantes aos observados durante o desenvolvimento humano. As células são deixadas a diferenciar-se espontaneamente, são expostas a factores que os empurram no sentido da linhagem neural, são isolados, e são plaqueadas em lamelas de cobertura para permitir a diferenciação terminal e maturação.