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 matrix-coated plates at room temperature (RT) for at least 3 h or at 4 &#176;C overnight. 
	NOTE: Plates with basement membrane matrix can be stored at 4 &#176;C for 1 week before the matrix solution is removed and plates are used.</li>
</ol>
2. Adaptation of Colony type hESCs to Single-cell hESC Culture
<ol>
	<li>To passage the feeder-free cultures of colony type H9 (WA09) hESCs grown on basement membrane matrix, aspirate the medium from the wells (Figure 1A)9.</li>
	<li>Wash 1x with 1 mL of DPBS. Add 1 mL of dispase solution (1 U/mL) per well and incubate at 37 &#176;C for 20 min.</li>
	<li>Remove the dispase, wash the cells 1x gently with 2 mL of DMEM/F12, remove the medium, and add 2 mL of DMEM/F12 to each plate.</li>
	<li>Gently detach colonies by gently pipetting up and down and transfer to a 15 mL tube.</li>
	<li>Centrifuge the pellets for 2 min at 370 x g and aspirate the medium.</li>
	<li>To dissociate the cell pellets into single cells, add 2 mL of cell detachment solution (1x concentration, see Table of Materials) and incubate at 37 &#176;C for 10 min.</li>
	<li>Centrifuge cells for 2 min at 370 x g and remove the detachment solution.</li>
	<li>Add fresh mTeSR1 human ESC medium and dissociate the cells into single cells by gentle pipetting up and down.</li>
	<li>To adapt colony type hESCs to a single-cell type culture, plate approximately 1.5&#8211;2.0 x 106 hESCs into each well of the basement membrane matrix-coated 6 well plate in 2 mL of mTeSR1 containing 10 &#956;M ROCK inhibitor for 24 h (Figure 1A).</li>
	<li>After 24 h, replace the hESC medium with fresh mTeSR1 without ROCK inhibitor and allow the hESCs to grow as a single-cell type for 3 days. Change the medium daily.</li>
	<li>On day 4, when cultures reach nearly 100% confluency, dissociate cells in detachment solution, then replate as described in step 2.6. 
	NOTE: The ROCK inhibitor improves cell survival during the initial 24 h of single-cell type hESC culture.</li>
</ol>
3. Embryoid Body Formation and Differentiation into Three Germ Layers (Figure 2)
<ol>
	<li>To form EBs, first resuspend cells in 3 mL of mTeSR1 medium with 10 &#956;M ROCK inhibitor during the first 24 h, then incubate overnight into 60 mm low attachment dishes in a 37 &#176;C incubator to allow aggregation.</li>
	<li>After 24 h, the small EBs are transferred to a 15 mL tube. Let the EBs settle to the bottom of the tube and gently remove the medium with a pipette. Transfer EBs into EB medium (knockout-DMEM supplemented with 20% knockout serum replacement, 1x glutamine supplement [see Table of Materials], 1% NEAA [non-essential amino acids; see Table of Materials], and 0.2% &#946;-mercaptoethanol) and allow them to expand in low attachment dishes for 7 days. The medium can be changed every other day as described above (Figure 2A).</li>
	<li>On day 7, collect the EBs from the dishes and transfer them into a 15 mL tube. Gently remove the medium with a pipette and transfer the EBs to a basement membrane matrix-coated 6 well plate.</li>
	<li>Allow the EBs to attach to the plate and incubate for 12 days in EB medium, during which they will differentiate into the three germ layers (Figure 2B). Change the medium every other day. 
	NOTE: During aggregation of cells, the low attachment dish helps avoid EB attachment.</li>
</ol>
4. Differentiation of Single-cell Type hESCs Into NPCs (Figure 3)
<ol>
	<li>To induce NPC differentiation, dissociate single-cell type hESCs with 1 mL of detachment solution (1x) and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge the cells for 2 min at 370 x g and remove the detachment solution supernatant. Add 1 mL of DMEM/F12 and resuspend cells by gentle pipetting.</li>
	<li>Plate cells on a basement membrane matrix-coated 6 well plate at a density of 2 x 105 cells/well in 2 mL of mTeSR1 containing 10 &#956;M ROCK inhibitor.</li>
	<li>After 24 h, replace the culture medium with neural induction medium (DMEM with 1% B27 minus vitamin A) supplemented with 1 &#956;M dorsomorphin and 5 &#956;M SB431542.</li>
	<li>Change the medium every other day during the first 4 days of neural induction, then every day until reaching confluence at day 7 (Figure 3B). 
	NOTE: (1) Dorsomorphin inhibits the BMP pathway by targeting ALK2,3,6 receptors and also inhibits AMPK; SB431542 is an inhibitor of the TGF&#946;/Activin pathway by targeting ALK5,7. (2) The same protocol was tested with another ESC line (WA01), which yielded similar results as WA0916. (3) We have generally used Matrigel for the basement membrane matrix; however, cells can be cultured on Geltrex and then differentiated into NPCs with very similar results as indicated by the expression of several NPC markers (Figure 4D,E). Handle Geltrex in the same manner as Matrigel (see section 1).</li>
</ol>
5. NPC Expansion and Cryopreservation
<ol>
	<li>After 7 days of neural induction, dissociate cells with 1 mL of detachment solution (1x) and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge the cells for 2 min at 370 x g and remove the detachment solution supernatant. Add 1 mL of NPC expansion medium (see Table of Materials) and resuspend cells by gentle pipetting.</li>
	<li>Plate 1 x 105 cells in 2 mL of NPC expansion medium on basement membrane matrix-coated 6 well plates.</li>
	<li>Passage NPCs multiple times, as necessary, when cultures reach nearly 90% confluency. During the first 3&#8211;4 passages, add 10 &#956;M ROCK inhibitor during the initial 24 h to prevent cell death. After these passages, cells can be passaged and cultured without ROCK inhibitor. Change the medium every other day during the expansion.</li>
	<li>Cryopreserve NPCs when cultures reach confluency.
	<ol>
		<li>For cryopreservation, dissociate cells in 1 mL of detachment solution (1x) for 5 min at 37 &#176;C, then centrifuge suspension for 2 min at 370 x g to remove the detachment solution.</li>
		<li>Add cell freezing solution (see Table of Materials) to dissociated NPCs at 1 x 106 cells/mL and distribute 1 mL aliquots to cryovials.</li>
		<li>Place the cryovials into freezing container and store them overnight in a -80 &#176;C freezer.</li>
	</ol>
	</li>
	<li>Store the cryovials in the liquid nitrogen.</li>
</ol>
6. Preparation of poly-L-ornithine (PLO) and Laminin Coated Plates
<ol>
	<li>To prepare PLO/laminin coated plates, dilute the PLO stock solution into PBS or water to a final concentration of 10 &#956;g/mL. Mix well and coat each well of a 6 well plate with 1 mL of diluted PLO solution.</li>
	<li>Incubate for at least 2 h at 37 &#176;C or overnight at 4 &#176;C.</li>
	<li>Wash each well with PBS.</li>
	<li>Dilute laminin stock solution in cold PBS or water at 10 &#956;g/mL. Mix the laminin solution well. Remove the PBS and immediately coat each well with 1 mL of the laminin solution. Keep plates at 4 &#176;C and use within 1 week. Wash with PBS and immediately add culture medium. 
	NOTE: Do not let the PLO/laminin coated plates dry out.</li>
</ol>
7. Differentiation of hESC-derived NPCs Into Dopaminergic Neurons (Figure 6A)
<ol>
	<li>Plate NPCs in PLO/laminin-coated dishes in NPC expansion medium at a density of approximately 50%.</li>
	<li>After 24 h, change the medium to DA1 medium (neurobasal medium containing 2 mM glutamine supplement, 1x NEAA, 1x B27, 200 ng/mL SHH, and 100 ng/mL FGF8) and change the medium every other day.</li>
	<li>Passage cultures when they reach confluency. Dissociate cells with 1 mL of detachment solution (1x) and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge the cells for 2 min at 370 x g and remove the detachment solution supernatant. Add 1 mL of DA1 medium and resuspend the cells by gentle pipetting.</li>
	<li>Plate 1 x 105 cells in 2 mL of DA1 medium on PLO/laminin-coated 6 well plates.</li>
	<li>After 10 days, change to DA2 medium (neurobasal medium containing 2 mM glutamine supplement, 1x NEAA, 1x B27, 200 &#956;M ascorbic acid, 20 ng/mL BDNF, and 20 ng/mL GNDF) and change medium every other day for an additional 20 days (Figure 6A). 
	NOTE: Add fresh ascorbic acid daily to the DA2 medium. Within the first 14 days, the differentiated cells should be passed when they reach 90% confluency, but no longer after that.</li>
</ol>
8. Differentiation of ESC-derived NPCs into Astrocytes
<ol>
	<li>Plate neural progenitor cells on PLO/laminin-coated plates in NPC expansion medium at a density of approximately 50%.</li>
	<li>After 24 h, change the medium to astrocyte medium (DMEM/F12 medium including 1:100 N2, 1:100 B27, 200 ng/mL IGF, 10 ng/mL activin A, 10 ng/mL heregulin &#946;-1, and 8 ng/mL bFGF) and change the medium every other day.</li>
	<li>Passage cultures when they reach confluency. Dissociate cells with 1 mL of detachment solution (1x) and incubate for 10 min at 37 &#176;C.</li>
	<li>Centrifuge the cells for 2 min at 370 x g and remove the detachment solution supernatant. Add 1 mL of astrocyte medium and resuspend the cells by gentle pipetting.</li>
	<li>Plate 1 x 105 cells in 2 mL of astrocyte medium on PLO/laminin-coated 6 well dishes.</li>
	<li>Allow differentiation to continue for a total of 5&#8211;6 weeks (Figure 6B).</li>
</ol>
9. Immunofluorescence Staining
<ol>
	<li>Culture cells in 35 mm &#956;-dishes as described above for each cell type. Aspirate the medium and add 1 mL of 4% PFA solution per dish and incubate for 20 min at RT.</li>
	<li>Wash 2x for 5 min with PBS. 
	NOTE: Once the cells are fixed, the assay plates can be sealed with parafilm and stored at 4 &#176;C. Stain with an antibody within 1 week after fixation.</li>
	<li>Add 300 &#956;L of blocking solution (goat or donkey serum in PBS) per dish and incubate at RT for 30&#8211;60 min.</li>
	<li>Wash the dish 2x with PBS for 5 min.</li>
	<li>Add 300 &#956;L of primary antibody (Table 1) solution (diluted in blocking solution) and incubate at RT for 1.5 h or overnight at 4 &#176;C.</li>
	<li>Wash 2x with PBS for 10 min.</li>
	<li>Add 300 &#956;L of fluorescent-conjugated secondary antibody (Table 1) in blocking solution and incubate in the dark for 1 h at RT.</li>
	<li>Wash the cells 2x for 10 min with PBS.</li>
	<li>Add Hoechst staining solution (1 &#956;M final concentration in PBS) to stain nuclei. Incubate the cells in the dark for 5 min at RT.</li>
	<li>Wash cells 1x with PBS for 5 min and then add 500 &#956;L of PBS. Keep dishes in the dark, then observe fluorescence with a confocal microscope (Figure 2B, Figure 5C, Figure 6A, and Figure 7B).</li>
</ol>

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The authors declare no conflicts of interest.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.9K Views.  National Institutes of Health. 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.

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

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 ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

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 ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

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

Summary

Explore More Videos

Chapters in this video

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

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

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

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

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

Single-cell Photoconversion in Living Intact Zebrafish