Name: efficient neural differentiation using single-cell culture of human embryonic stem cells
Uploaded: 2020-01-18T13:00:00
Description: Watch this Scientific Journal Video about Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells at JoVE.com

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.

efficient-neural-differentiation-using-single-cell-culture-human

Research

JoVE Journal

Developmental Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...