We thank Dr. Carl D. Bortner (NIEHS) for his assistance with the FACS analysis. This research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, the National Institutes of Health, Z01-ES-101585 to AMJ.

1. Preparation of hESC-qualified Basement Membrane Matrix-coated Plates
<ol>
	<li>Slowly thaw the hESC-qualified basement membrane matrix (see Table of Materials) solution at 4 &#176;C for at least 2&#8211;3 h or overnight to avoid formation of a gel.</li>
	<li>To prepare basement membrane matrix-coated plates, dilute matrix in cold DMEM/F12 to a 2% final concentration. Mix well and coat each well of a 6 well plate with 1 mL of the diluted matrix solution.</li>
	<li>Incubate the basement membrane matri...

<ol>
	<li>Thomson, J. 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>Rosler, E. 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=Long-term+culture+of+human+embryonic+stem+cells+in+feeder-free+conditions.">Long-term culture of human embryonic stem cells in feeder-free conditions.</a> Developmental Dynamics. 229 (2), 259-274 (2004).</li><li>Mallon, B. S., Park, K. Y., Chen, K. G., Hamilton, R. S., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+xeno-free+culture+of+human+embryonic+stem+cells.">Toward xeno-free culture of human embryonic stem cells.</a> The International Journal of Biochemistry &amp; Cell Biology. 38 (7), 1063-1075 (2006).</li><li>Hoffman, L. M., Carpenter, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+culture+of+human+embryonic+stem+cells.">Characterization and culture of human embryonic stem cells.</a> Nature Biotechnology. 23 (6), 699-708 (2005).</li><li>Yan, 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=Efficient+and+rapid+derivation+of+primitive+neural+stem+cells+and+generation+of+brain+subtype+neurons+from+human+pluripotent+stem+cells.">Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells.</a> Stem Cells Translational Medicine. 2 (11), 862-870 (2013).</li><li>Goncalves, J. T., Schafer, S. T., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Neurogenesis+in+the+Hippocampus:+From+Stem+Cells+to+Behavior.">Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior.</a> Cell. 167 (4), 897-914 (2016).</li><li>Watanabe, 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=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</li><li>International Stem Cell Initiative. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+embryonic+stem+cell+lines+by+the+International+Stem+Cell+Initiative.">Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.</a> Nature Biotechnology. 25 (7), 803-816 (2007).</li><li>Hartung, O., Huo, H., Daley, G. Q., Schlaeger, 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=Clump+passaging+and+expansion+of+human+embryonic+and+induced+pluripotent+stem+cells+on+mouse+embryonic+fibroblast+feeder+cells.">Clump passaging and expansion of human embryonic and induced pluripotent stem cells on mouse embryonic fibroblast feeder cells.</a> Current Protocols in Stem Cell Biology. 14 (1), 10 (2010).</li><li>Chen, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-colony+type+monolayer+culture+of+human+embryonic+stem+cells.">Non-colony type monolayer culture of human embryonic stem cells.</a> Stem Cell Research. 9 (3), 237-248 (2012).</li><li>Chen, G., Hou, Z., Gulbranson, D. R., 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=Actin-myosin+contractility+is+responsible+for+the+reduced+viability+of+dissociated+human+embryonic+stem+cells.">Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.</a> Cell Stem Cell. 7 (2), 240-248 (2010).</li><li>Li, X., Krawetz, R., Liu, S., Meng, G., Rancourt, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+inhibitor+improves+survival+of+cryopreserved+serum/feeder-free+single+human+embryonic+stem+cells.">ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.</a> Human Reproduction. 24 (3), 580-589 (2009).</li><li>Ohgushi, 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=Molecular+pathway+and+cell+state+responsible+for+dissociation-induced+apoptosis+in+human+pluripotent+stem+cells.">Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.</a> Cell Stem Cell. 7 (2), 225-239 (2010).</li><li>Pakzad, 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=Presence+of+a+ROCK+inhibitor+in+extracellular+matrix+supports+more+undifferentiated+growth+of+feeder-free+human+embryonic+and+induced+pluripotent+stem+cells+upon+passaging.">Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging.</a> Stem Cell Reviews and Reports. 6 (1), 96-107 (2010).</li><li>Chambers, S. 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=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> Nature Biotechnology. 27 (3), 275-280 (2009).</li><li>Jeon, 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=GLIS3+Transcriptionally+Activates+WNT+Genes+to+Promote+Differentiation+of+Human+Embryonic+Stem+Cells+into+Posterior+Neural+Progenitors.">GLIS3 Transcriptionally Activates WNT Genes to Promote Differentiation of Human Embryonic Stem Cells into Posterior Neural Progenitors.</a> Stem Cells. 37 (2), 202-215 (2019).</li><li>Emre, 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=The+ROCK+inhibitor+Y-27632+improves+recovery+of+human+embryonic+stem+cells+after+fluorescence-activated+cell+sorting+with+multiple+cell+surface+markers.">The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers.</a> PLoS ONE. 5 (8), e12148 (2010).</li><li>Hanna, 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=Human+embryonic+stem+cells+with+biological+and+epigenetic+characteristics+similar+to+those+of+mouse+ESCs.">Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (20), 9222-9227 (2010).</li><li>Saha, 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=Surface-engineered+substrates+for+improved+human+pluripotent+stem+cell+culture+under+fully+defined+conditions.">Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (46), 18714-18719 (2011).</li><li>Tsutsui, 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=An+optimized+small+molecule+inhibitor+cocktail+supports+long-term+maintenance+of+human+embryonic+stem+cells.">An optimized small molecule inhibitor cocktail supports long-term maintenance of human embryonic stem cells.</a> Nature Communications. 2, 167 (2011).</li><li>Xu, 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=Revealing+a+core+signaling+regulatory+mechanism+for+pluripotent+stem+cell+survival+and+self-renewal+by+small+molecules.">Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (18), 8129-8134 (2010).</li><li>Ungrin, M. D., Joshi, C., Nica, A., Bauwens, C., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible,+ultra+high-throughput+formation+of+multicellular+organization+from+single+cell+suspension-derived+human+embryonic+stem+cell+aggregates.">Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.</a> PLoS ONE. 3 (2), e1565 (2008).</li><li>Kim, D. 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=Highly+pure+and+expandable+PSA-NCAM-positive+neural+precursors+from+human+ESC+and+iPSC-derived+neural+rosettes.">Highly pure and expandable PSA-NCAM-positive neural precursors from human ESC and iPSC-derived neural rosettes.</a> PLoS ONE. 7 (7), e39715 (2012).</li><li>Sun, Y., Hu, J., Zhou, L., Pollard, S. M., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+FGF2+and+BMP+controls+the+self-renewal,+dormancy+and+differentiation+of+rat+neural+stem+cells.">Interplay between FGF2 and BMP controls the self-renewal, dormancy and differentiation of rat neural stem cells.</a> Journal of Cell Science. 124 (Pt 11), 1867-1877 (2011).</li><li>Zhou, J. M., Chu, J. X., Chen, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+protocol+that+induces+human+embryonic+stem+cells+to+differentiate+into+neural+cells+in+vitro.">An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro.</a> Cell Biology International. 32 (1), 80-85 (2008).</li><li>Smith, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Activin/Nodal+signaling+promotes+specification+of+human+embryonic+stem+cells+into+neuroectoderm.">Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm.</a> Developmental Biology. 313 (1), 107-117 (2008).</li></ol>

Scalable and efficient methods for the differentiation of hESCs into various lineages and the generation of sufficient numbers of differentiated cells are important criteria for drug screening and stem cell therapy. Various single-cell passing methods have been published, in which cells are cultured in the presence of ROCK inhibitor or other small molecules to improve survival, but the final products of these culture methods are colony type hESCs17,18,...

Human embryonic stem cells (hESCs) have the potential to differentiate into the three primary germ layers, which then differentiate into various multipotent progenitor cell lineages. These lineages subsequently give rise to all cell types in the human body. In vitro hESC culture systems have transformed the ability to study human embryonic development and have served as a valuable tool for obtaining new insights into how these processes are regulated at the biological and molecular levels. Similarly, studies of induced pluripotent stem cells (iPSCs) generated from reprogramming somatic cells isolated from human patients provide novel insights into various diseases. In addition, progenitor and differentiated cells derived from hESCs can be useful for research involving stem cell therapy and drug screening1,2,3,4.
hESCs can be induced to differentiate into neural progenitor cells (NPCs), which are multipotential cells with an extensive self-renewal capacity. Subsequently, these cells can be differentiated into neurons, astrocytes, and oligodendrocytes5,6. NPCs also offer a cellular system for in vitro studies of neurodevelopmental biology and various neurological diseases. However, current colony type culture methods involving hESCs and their differentiation into NPCs are inefficient and often involve coculture as well as embryoid body (EB) and rosette formation5,7,8,9. These protocols exhibit lower survival rates and spontaneous differentiation and are more time-consuming.
Presented here is an improved and robust culture system that is easily scalable and uses high density single-cell type culture of hESCs10. The inclusion of Roh-kinase (ROCK) inhibitor contributed to significantly enhanced survival efficiency during single cell type culture of hESC10,11,12,13,14. In this culture system, hESCs can be easily maintained and expanded. In addition, the protocol presents an efficient method to generate NPCs from single-cell type culture of hESCs, which allows the production of highly pure NPCs. Inhibition of BMP/TGF&#946;/activin signaling pathways with ALK inhibitors efficiently induce differentiation of single-cell type hESCs into NPCs15,16, which then can be induced to differentiate into functional neural lineages, such as dopaminergic neurons and astrocytes.
In summary, the single-cell type culture protocol using hESCs offers an attractive model to study the differentiation of these cells into various lineages, including NPCs. This protocol is easily scalable and therefore suitable for generating cells for research involving regenerative therapy and drug screening.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm m-dishes</td>
			<td>ibidi</td>
			<td>81156</td>
			<td>Cell culture dish</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104-500</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D system</td>
			<td>338-AC-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement (-Vit A)</td>
			<td>Thermo Fisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Applied Biological Materials</td>
			<td>Z100065</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech</td>
			<td>100-18C</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>DAMON/ICE</td>
			<td>428-6759</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>4110</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning hESC-qulified Matrix (Magrigel)</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Basement membrane matrix (used for most of the protocol here)</td>
		</tr>
		<tr>
			<td>Cryostor CS 10</td>
			<td>Stemcell Technologies</td>
			<td>7930</td>
			<td>Cell freezing solution</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>7923</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Tocris</td>
			<td>3093</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-16A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>SH3007003HI</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF8</td>
			<td>Applied Biological Materials</td>
			<td>Z101705</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Applied Biological Materials</td>
			<td>Z101057</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex matrix</td>
			<td>Thermo Fisher</td>
			<td>A1569601</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Thermo Fisher</td>
			<td>35050061</td>
			<td>Glutamine supplement, 100X</td>
		</tr>
		<tr>
			<td>H9 (WA09) human embryonic stem cell line</td>
			<td>WiCell</td>
			<td>WA09</td>
			<td></td>
		</tr>
		<tr>
			<td>Heregulin b-1</td>
			<td>Peprotech</td>
			<td>100-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Thermo Fisher</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Thermo Fisher</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stemcell Technologies</td>
			<td>85850</td>
			<td>hESC culture medium</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Thermo Fisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Thermo Fisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma Aldrich</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>SHH</td>
			<td>Applied Biological Materials</td>
			<td>Z200617</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemdiff Neural Progenitor medium</td>
			<td>Stemcell Technologies</td>
			<td>5833</td>
			<td>NPC expansion medium</td>
		</tr>
	</tbody>
</table>

efficient neural differentiation using single-cell culture of human embryonic stem cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is an improved protocol for the maintenance and expansion of single-cell type culture of hESCs and their efficient differentiation into neural progenitor cells, which subsequently differentiates into various downstream neural lineages, including dopaminergic neurons and astrocytes.
Representative phase contrast images show cell morphology at different steps during the adaptation of colony type hESCs to the single-cell type culture (Figure 1A...

Watch this Scientific Journal Video about Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells at JoVE.com

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Human embryonic stem cells can be induced to differentiate into neural progenitor cells and subsequently into neuron astrocyte and oligodendrocyte. However, type culture method for human embryonic stem cells and their differentiation into neural progenitor cells are very inefficient and open in vivo co-culture system Here we present an improved and robust culture system that is easily scalable using high-density single cell type culture with human embryonic stem cells. Single cell type culture of human embryonic stem cells provides a rapid and efficient system to study the molecular mechanisms that regulate the multi-step differentiation along various and distinct differentiated lineages, including neural progenitor cells and their subsequent differentiation into additional neural lineages.Begin by preparing human embryonic stem cell qualified basement membrane matrix coated plates. Slowly thaw the basement membrane matrix solution at four degrees Celsius for at least two to three hours or overnight. When thawed, dilute the matrix in cold DMEM/F-12 to 2%mix well, and coat each well of a six-well plate with one milliliter of the diluted solution.Incubate the coated plates at room temperature for at least three hours or at four degrees Celsius overnight. To passage the feeder-free cultures of colony type H9 human embryonic stem cells grown on basement membrane matrix, aspirate the medium from the wells and wash once with one milliliter of DPBS. Add one milliliter of Dispase solution to each well and incubate the plate at 37 degrees Celsius for 20 minutes.Remove the Dispase and gently wash the cells once with two milliliters of DMEM/F-12. After the wash, remove the medium and add two milliliters of DMEM/F-12 to each well. Gently detach the colonies by pipetting up and down and transfer them to a 15 milliliter tube.Centrifuge the tube at 370 times g for two minutes. Aspirate the medium. Then dissociate the cell pellets into single cells by adding two milliliters of cell detachment solution and incubating them at 37 degrees Celsius for 10 minutes.Repeat the centrifugation and remove the detachment solution. Resuspend the cells in mTeSR-1 human ESC medium by gently pipetting up and down. To adapt the colony type human embryonic stem cells to a single cell type culture, plate approximately 1.5 to two million cells into each well of the basement membrane matrix coated plate in two milliliters of mTeSR-1 containing 10 micromolar ROCK inhibitor.After 24 hours, replace the medium with fresh mTeSR-1 without ROCK inhibitor. Allow the cells to grow as a single cell type for three days changing the medium daily. On the fourth day, dissociate the cells in detachment solution and re-plate them as previously described.After resuspending and incubating the cells according to the manuscript directions, transfer the small embryonic bodies to a 15 milliliter tube, let them settle to the bottom, and gently remove the medium with a pipette. Transfer them into EB medium and allow them to expand in low attachment dishes for seven days. To induce neural progenitor cell or NPC differentiation, dissociate the single cell type human embryonic stem cells with one milliliter of detachment solution and incubate them at 37 degrees Celsius for 10 minutes.Centrifuge the cells for two minutes at 370 times g. Remove the detachment solution supernatant. Then resuspend the cells in DMEM/F-12.Plate the cells on a basement membrane matrix coated six-well plate at a density of two times 10 to the fifth cells per well in two milliliters of mTeSR-1 with 10 micromolar ROCK inhibitor. After 24 hours, replace the culture medium with neural induction medium supplemented with one micromolar Dorsomorphin and five micromolar SB431542. Change the medium every other day during the first four days of neural induction, then everyday until confluence is reached at day seven.After seven days of neural induction, dissociate the cells by adding one milliliter of detachment solution and incubating them for 10 minutes at 37 degrees Celsius. Centrifuge the cells at 370 times g and remove the detachment solution supernatant. Add one milliliter of NPC expansion medium and gently pipette the cells up and down to resuspend.Then plate one times 10 to the fifth cells per well in two milliliters of NPC expansion medium on the basement membrane matrix coated six-well plates. Passage the cells as necessary when cultures reach 90%confluency adding 10 micromolar ROCK inhibitor during the first three to four passages. Change the medium every other day during the expansion.To prepare PLO laminin-coated plates, dilute the PLO stock solution in PBS or water to a final concentration of 10 micrograms per milliliter, mix well, then coat each well of a six-well plate with one milliliter of the solution. Incubate the plates for at least two hours at 37 degrees Celsius or overnight at four degrees Celsius. Next, wash each well with PBS and immediately after coat each well with one milliliter of laminin solution prepared according to the manuscript directions.Keep the plates at four degrees Celsius for up to one week. To differentiate NPCs into dopaminergic neurons, plate the cells in PLO laminin-coated dishes in NPC expansion medium at a density of approximately 50%After 24 hours, change the medium to DA1 and change the medium every other day thereafter. To passage cultures when they reach confluency, dissociate the cells with one milliliter of detachment solution and incubate them for 10 minutes at 37 degrees Celsius.Then centrifuge them at 370 times g and remove the detachment solution supernatant. Resuspend the cells in one milliliter of DA1 medium and plate one times 10 to the fifth cells in two milliliters of medium on the PLO laminin-coated six-well plates. To differentiate the NPCs into astrocytes, plate the cells on the PLO laminin plates and NPC expansion medium at a density of 50%change the medium to astrocyte medium after 24 hours, and passage the cells as previously described.When colony type human embryonic stem cells were adapted to the single cell type culture, it was found that the cells were able to be maintained at high density, then easily and efficiently subcultured when confluency was reached. These cells retained the cell cycle characteristics typical of colony type human embryonic stem cells such as a short G1 phase and a high proportion of cells in S phase. Quantitative PCR analysis also confirmed that they express ESC markers at levels comparable to those of colony type cells.Moreover, it was shown that single cell human embryonic stem cells were able to form embryoid bodies containing cells from all three germ layers:endoderm, mesoderm, and ectoderm. It was then demonstrated that single cell type human embryonic stem cells efficiently differentiated into NPCs as indicated by the loss of typical human embryonic stem cell morphology and appearance of NPC morphology. Differentiation was supported by the increased expression of signature NPC markers and confirmed by immunostaining and FACS analysis.The same analysis also showed that more than 90%of the cells stained positive for SOX1, PAX6, and NCAM proteins. Furthermore, the derived NPCs were able to differentiate into dopaminergic neurons and astrocytes as indicated by the appearance of characteristic morphologies and expression of lineage-specific markers. When attempting this procedure, it is essential to plate the human embryonic stem cells at high density in order to maintain a healthy, undifferentiated human embryonic stem cell culture.This protocol provide a platform for simple, robust, and scalable production of progenitor and differentiated cell that will be scalable for basic study, drug screen, and application in neurodegenerative medicine.

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Research

JoVE Journal

Developmental Biology

9.8K 조회수.  National Institutes of Health. 인간 배아 줄기 세포는 신경 전구 세포로 분화하고 뉴런 성상세포 및 올리고드엔드로시테로 분화하도록 유도될 수 있다. 그러나, 인간 배아 줄기세포및 신경전구세포로의 분화에 대한 형배양방법은 매우 비효율적이고 생체내 공동배양 시스템에서 개방되어 인간 배아 줄기세포를 이용한 고밀도 단일세포형 배양을 이용하여 용이하게 확장가능한 개선되고 견고한 배양시스템...