This work was supported by grants from the Stanford University School of Medicine and a Siebel Fellowship awarded to S. C.

1. Stem Cell Maintenance
NOTE: The cell maintenance protocol described below applies to pluipotent stem cells (PSCs) maintained in an adherent monolayer. Media, other reagents, and cell culture plates used prior to DMSO treatment can be adjusted as needed. For all the following protocols in this manuscript, cells should be handled under a biological safety cabinet.
<ol>
	<li>Coat sterile, 6 well, tissue culture-treated plates with a pluripotent stem cell-qualified matrix or substrate prepared per the manufacturer&#8217;s instructions and incubate for at least 1 h in a CO2 incubator (5% CO2, humid atmosphere). Coated plates can be film wrapped and stored at 4 &#176;C for up to one week.</li>
	<li>Thaw the cryopreserved PSCs in a 37 &#176;C water bath. Sterilize vial with ethanol prior to introduction to the biological safety cabinet, then immediately transfer the cells by pipetting to a sterile conical tube containing 5-10 volumes of prewarmed stem cell media.</li>
	<li>Centrifuge the cells at 300 x g for 5 min at room temperature (RT).</li>
	<li>Aspirate the media and gently resuspend the cell pellet in 1 mL of stem cell media supplemented with a 10 &#181;M ROCK inhibitor, such as Y-27632.</li>
	<li>Aspirate the culture matrix from the plate and seed the cells at the desired density, typically 0.5-1 x 106 cells per well in at least 2 mL of stem cell media per well. 
	NOTE: The plating density can vary across different cell lines, and clones and should be optimized accordingly.</li>
	<li>Maintain the cells by replacing with prewarmed stem cell media daily. Split the cells at roughly 70%-80% confluency or when the cell colonies begin to make contact.</li>
	<li>For splitting cells, aspirate the media and wash the cells once with sterile PBS. Incubate the cells with 1 mL of a dissociation enzyme solution per well for 5-10 minutes at 37 &#176;C.</li>
	<li>Wash and resuspend the cells with prewarmed stem cell media and transfer to a sterile conical tube with 5-10 volumes of stem cell media. Follow steps 1.3-1.7 to plate the cells.</li>
</ol>
2. DMSO Pretreatment
NOTE: When plating the cells for DMSO pretreatment prior to differentiation, the starting plating cell density should be optimized with consideration of the typical growth rate of the stem cell line as well as the differentiation protocol being used. Validate pluripotency using conventional markers, as necessary. Cells should be passaged at least 1x-2x after initial thawing prior to differentiation.
<ol>
	<li>2D culture differentiation
	<ol>
		<li>When the cells reach an appropriate confluency, prepare coated plates, dissociate the cells, and prepare a single-cell suspension as described above.</li>
		<li>Count the live cells using a hemocytometer or automatic cell counter including trypan blue or another viability marker.</li>
		<li>Plate the cells onto a coated 6 well plate at 0.5-1 x 106 cells per well in stem cell media with the 10 &#181;M ROCK inhibitor. 
		NOTE: For the cell lines tested in our laboratory, these densities typically resulted in 80%-90% confluent cells within the 24 h DMSO pretreatment.</li>
		<li>Allow cells to incubate for 24 h at 37 &#176;C in a CO2 incubator (5% CO2, humid atmosphere).</li>
		<li>Prepare 1%-2% DMSO in prewarmed stem cell media (e.g., 100 &#181;L of DMSO in 10 mL of media = 1% DMSO solution, or 200 &#181;L DMSO in 10 mL of media = 2% DMSO solution).</li>
		<li>After 24 h incubation, aspirate the media from cells and replace it with DMSO solution.</li>
		<li>Allow the cells to incubate for 24 h to 48 h at 37 &#176;C in a CO2 incubator (5% CO2, humid atmosphere) prior to differentiation. 
		NOTE: Typically, a 24 h DMSO treatment is sufficient across a majority of human ESC and iPSC lines. Cell lines with very slow growth rates (long doubling times) can benefit from the 48 h incubation with DMSO. For a 48 h incubation with DMSO, media can be replaced with fresh stem cell media with 1%-2% DMSO after the first 24 h of treatment.</li>
	</ol>
	</li>
	<li>3D culture differentiation:
	<ol>
		<li>When the cells reach an appropriate confluency, dissociate and collect the cells in a cell suspension as described above.</li>
		<li>Count the live cells using a hemocytometer or automatic cell counter including a viability marker.</li>
		<li>Plate the cells in an uncoated, low-attachment 6 well plate at 0.5-1 x 106 cells per well in stem cell media with 10 &#181;M ROCK inhibitor. 
		NOTE: For the cell lines tested in our laboratory, these densities typically resulted in 3D hPSC sphere formation within 24 h of setting cells.</li>
		<li>Allow cells to incubate for 24 h at 37 &#176;C in a CO2 incubator (5% CO2, humid atmosphere).</li>
		<li>Prepare 1%-2% DMSO in prewarmed stem cell media (e.g., 100 &#181;L of DMSO in 10 mL of media = 1% DMSO solution, or 200 &#181;L of DMSO in 10 mL of media = 2% DMSO solution).</li>
		<li>Replace the media following standard procedures (e.g., tilt the plate at a 30&#176;-45&#176; angle to allow cell spheres to settle at the bottom of the well; transfer cells to a sterile conical tube and allow cell spheres to settle at the bottom of the tube; or gently collect cells in suspension using a 5 or 10 mL pipette into a sterile conical tube and centrifuge cells at 300 x g for 5 min at RT).</li>
		<li>Aspirate the media from cells and replace it with DMSO solution, pipetting gently.</li>
		<li>Allow cells to incubate for 24 h to 48 h at 37 &#176;C in a CO2 incubator (5% CO2, humid atmosphere) prior to differentiation. 
		NOTE: Typically, a 24 h DMSO treatment is sufficient across a majority of human ESC and iPSC lines. Cell lines with very slow growth rates (long doubling times) can benefit from the 48 h incubation with DMSO. For a 48 h incubation with DMSO, media can be replaced with fresh stem cell media with 1%-2% DMSO after the first 24 h of treatment.</li>
	</ol>
	</li>
</ol>
3. Differentiation to Primary Germ Layers
NOTE: The following describes methods previously shown to be effective in our laboratory for PSCs grown in a monolayer on 6 well plates. Any differentiation protocol of choice should be used after the DMSO treatment to promote differentiation into desired lineages. Remove DMSO solution after a 24-48 h treatment and proceed with differentiation following standard protocols.
<ol>
	<li>Endoderm differentiation (adapted from Kroon et al.30)
	<ol>
		<li>Pretreat cells with DMSO as described above for 2D cultures.</li>
		<li>Prepare Wnt3a and Activin A stock solutions.</li>
		<li>Prepare Day 1 endodermal differentiation media by adding Wnt3a to a final concentration of 20 ng/mL and Activin A to a final concentration of 100 ng/mL to the appropriate volume of prewarmed RPMI media.</li>
		<li>After DMSO pretreatment, aspirate media from the cells and replace it with Day 1 media (e.g., 2 mL per well of a 6 well plate).</li>
		<li>Allow cells to incubate for 24 h at 37 &#176;C in a CO2 incubator (5% CO2, humid atmosphere).</li>
		<li>Prepare Days 2 and 3 endodermal differentiation media by adding Activin A to a final concentration of 100 ng/mL to the appropriate volume of prewarmed RPMI media.</li>
		<li>Aspirate media from the cells and replace it with Day 2 media (e.g., 2 mL per well of a 6 well plate).</li>
		<li>Allow the cells to incubate for 24 h at 37 &#176;C in a CO2 incubator (5% CO2, humid atmosphere).</li>
		<li>Aspirate media from the cells and replace with Day 3 media (e.g., 2 mL per well of a 6 well plate).</li>
	</ol>
	</li>
	<li>Mesoderm differentiation (adapted from Zhang et al.31)
	<ol>
		<li>Pretreat the cells with DMSO as described above for 2D cultures.</li>
		<li>Prepare Wnt3a and Activin A stock solutions.</li>
		<li>Prepare mesodermal differentiation media by adding Wnt3a to a final concentration of 20 ng/mL and Activin A to a final concentration of 100 ng/mL to the appropriate volume of prewarmed advanced RPMI media.</li>
		<li>After DMSO pretreatment, aspirate media from cells and replace with differentiation media (e.g., 2 mL per well of a 6 well plate).</li>
		<li>Allow cells to incubate for 24 h at 37 &#176;C in a CO2 incubator (5% CO2, humid atmosphere).</li>
	</ol>
	</li>
	<li>Ectoderm differentiation (adapted from Chambers et al.32)
	<ol>
		<li>Pretreat the cells with DMSO as described above for 2D cultures.</li>
		<li>Prepare Noggin and SB431542 stock solutions.</li>
		<li>Prepare ectodermal differentiation base media by dissolving knockout serum replacement (KOSR) to a final concentration of 10% in knockout DMEM. 
		NOTE: Prepare enough base media for 3-4 days of media change.</li>
		<li>Prepare ectodermal differentiation media by adding Noggin to a final concentration of 500 ng/mL and SB431542 to a final concentration of 10 &#181;M to the appropriate volume of prewarmed KOSR/knockout DMEM.</li>
		<li>After DMSO pretreatment, aspirate media from cells and replace with differentiation media (e.g., 2 mL per well of a 6 well plate).</li>
		<li>Allow cells to incubate for 3-4 days at 37 &#176;C in a CO2 incubator (5% CO2, humid atmosphere), replacing media daily with freshly added differentiation factors.</li>
	</ol>
	</li>
</ol>
4. Differentiation to Progenitor Cell Types
The following describes methods previously shown to be effective in our laboratory for PSCs grown in a 2D or 3D cultures. Any differentiation protocol of choice should be used after the DMSO treatment to promote differentiation into desired lineages. Remove DMSO solution after a 24-48 h treatment and proceed with differentiation following standard protocols.
<ol>
	<li>Neural progenitor cell differentiation (adapted from Tchieu et al.33)
	<ol>
		<li>Prepare 6 well plates by coating with a pluripotent stem cell-qualified reduced growth factor matrix or substrate, per the manufacturer&#8217;s instructions, for at least 1 h in a CO2 incubator (5% CO2, humid atmosphere). Coated plates can be film wrapped and stored at 4 &#176;C for up to 1 week.</li>
		<li>Plate PSCs as described above at density of 0.5-1 x 106 cells per well in stem cell media containing a ROCK inhibitor.</li>
		<li>Pretreat cells with DMSO as described above for 2D cultures.</li>
		<li>Prepare small chemical inhibitors LDN193189, SB431542, and XAV939 stock solutions.</li>
		<li>Prepare Days 1-3 neuroectoderm differentiation media by supplementing Essential 6 Media with 500 nM LDN193189, 10 &#181;M SB431542, and 2 &#181;M XAV939.</li>
		<li>After DMSO pretreatment, aspirate the media and replace with Days 1-3 neuroectoderm media (e.g., 2 mL per well of a 6 well plate). Change media daily.</li>
		<li>Prepare Days 4-12 neuroectoderm differentiation media by supplementing Essential 6 Media with 500 nM LDN193189 and 10 &#181;M SB431542.</li>
		<li>On day 4 of differentiation, aspirate the media and replace with Days 4-12 neurodectoderm media. Change the media daily.</li>
		<li>After 12 days of differentiation, differentiated cells should express appropriate markers of neural progenitor cells (NPCs). NPCs can be further maintained in neural media containing DMEM/F-12, 2% B-27, 1% N-2, and supplemented with 10 &#181;g/mL basic fibroblast growth factor (bFGF). Passage the NPCs when confluent using a cell detachment solution, plating NPCs at 0.5-1 x 106 cells per well.</li>
	</ol>
	</li>
	<li>Oligodendrocyte progenitor cell differentiation (adapted from Douvaras and Fossati34)
	<ol>
		<li>Plate the PSCs as described above at a density of 1 x 105 per well on coated 6 well plates in stem cell media containing a ROCK inhibitor.</li>
		<li>Pretreat cells with DMSO as described above for 2D cultures.</li>
		<li>Prepare SB431542, LDN193189, all-trans retinoic acid (RA), and smoothened agonist (SAG) stock solutions.</li>
		<li>Prepare Days 0&#8211;8 differentiation media by supplementing DMEM/F-12 with 10 &#956;M SB431542, 250 nM LDN193189, and 100 nM RA.</li>
		<li>After DMSO pretreatment, incubate cells with differentiation media for 8 days, changing media daily with freshly added differentiation factors (e.g., 2 mL per well of a 6 well plate).</li>
		<li>On day 8, replace media with DMEM/F-12 containing 1x MEM non-essential amino acids (NEAA) solution, 1X L-glutamine, 2-mercaptoethanol, penicillin/streptomycin, and 1x N-2 supplemented 100 nM RA and 1 &#181;M SAG. Change the media daily.</li>
		<li>After 12 days of differentiation, differentiated cells should express appropriate markers of oligodendrocyte progenitor cells (OPCs).</li>
	</ol>
	</li>
	<li>Endocrine progenitor cell differentiation (adapted from Pagliuca et al.35)
	<ol>
		<li>Seed PSCs at 6 x 105 cells/mL in stem cell media plus 10 &#181;M ROCK inhibitor in 500 mL spinner flasks placed on a 9-position stir plate set at rotation rate of 70 rpm in a 37 &#176;C incubator, 5% CO2, and 100% humidity.</li>
		<li>Allow clusters to settle at the bottom of the flask, aspirate the media, then pretreat with 1%-2% DMSO.</li>
		<li>Prepare Activin A, Chir99021, KGF, Sant1, all-trans retinoic acid (RA), LDN193189, PdBU, XXI, Alk51, T3, and Betacelluin stock solutions.</li>
		<li>Prepare differentiation base media based on formulation in Table 3.</li>
		<li>After DMSO pretreatment, aspirate media and replace with S1 media supplemented with 100 ng/mL Activin A and 3 mM Chir99021 (e.g., 500 mL per flask). Allow incubation for 24 h.</li>
		<li>On day 2, replace media with S1 media supplemented with 100 ng/mL Activin A. Allow incubation for 2 days.</li>
		<li>On day 4, replace media with S2 media supplemented with 50 ng/mL KGF. Allow incubation for 3 days, changing media after the first 2 days (Day 6).</li>
		<li>On day 7, replace media with S3 media supplemented with 50 ng/mL KGF, 0.25 mM Sant1, 2 mM RA, and 200 nM LDN193189. Allow incubation for 24 h.</li>
		<li>On day 8, replace media with S3 media supplemented with 50 ng/mL KGF, 0.25 mM Sant1, 2 mM RA, 200 nM LDN193189, and 500 nM PdBU. Allow incubation for 24 h.</li>
		<li>On day 9, replace media with S3 media supplemented with 50 ng/mL KGF, 0.25 mM Sant1, and 100 nM RA. Allow incubation for 5 days, changing media every 2 days (day 11 and 13).</li>
		<li>On days 14 and 16, replace media with S5 media supplemented with 0.25 mM Sant1, 100 nM RA, 1 mM XXI, 10 mM Alk5i II, 1 mM T3, and 20 ng/mL betacellulin (4 days total incubation).</li>
		<li>On days 18 and 20, replace media with S5 media supplemented with 25 nM RA, 1 mM XXI, 10 mM Alk5i II, 1 mM T3, and 20 ng/mL betacellulin.</li>
	</ol>
	</li>
</ol>
5. Immunocytochemical Validation of Differentiation
NOTE: The following methods describes a general immunocytochemical protocol that can be adjusted as needed. Primary antibodies are those that have been previously validated in our laboratory. Other techniques for validation of differentiation can also be used (e.g., flow cytometry, qPCR, RNA sequencing, western blotting, functional assays, etc.).
<ol>
	<li>Immunolabeling cells
	<ol>
		<li>For 3D cultures in suspension, plate whole cell clusters or clusters dispersed into single-cell suspension onto coated plates for 18-24 h prior to fixation.</li>
		<li>Aspirate media from adherent cells and wash briefly with PBS at RT on a shaker.</li>
		<li>For cell fixation, aspirate PBS and incubate cells with 4% paraformaldehyde (PFA) in PBS for 20 min at RT on shaker. 
		CAUTION: PFA stock should be prepared under a fume hood due to its toxicity. Do not inhale and wear proper personal protective equipment.</li>
		<li>Remove PFA and discard in the proper chemical waste container.</li>
		<li>Wash cells 3x with PBS for at least 5 min per wash at RT on a shaker.</li>
		<li>For cell permeabilization and blocking, incubate cells with 5% donkey serum prepared in 0.3% triton-x 100/PBS for 1 h at RT on a shaker.</li>
		<li>Prepare primary antibody solution in the same solution used for permeabilization/blocking.</li>
		<li>Incubate in primary antibody solution overnight at 4 &#176;C on shaker.</li>
		<li>After overnight incubation, wash cells 3x with PBS for at least 5 min per wash at RT on a shaker.</li>
		<li>Prepare secondary antibody solution in permeabilization/blocking solution.</li>
		<li>Allow to incubate in secondary antibody solution for 1 h at RT on a shaker.</li>
		<li>Aspirate secondary antibody solution and wash cells 3x with PBS for at least 5 min per wash at RT on a shaker.</li>
		<li>Incubate cells with DAPI or another preferred marker for appropriate incubation time, and rinse in PBS.</li>
	</ol>
	</li>
	<li>Image quantification
	<ol>
		<li>Acquire a minimum of three images per condition on a fluorescent microscope and/or with a high content screening platform.</li>
		<li>Quantify the percent of positive cells for each marker by counting the total number of antibody stained cells and total cell numbers (based on DAPI/Hoechst nuclei staining) using unbiased imaging software (e.g., ImageJ) or an automated screening platform for analyses.</li>
	</ol>
	</li>
</ol>

The authors having nothing to disclose.

In summary, this protocol describes a simple and inexpensive tool to enhance the differentiation capacity of pluripotent stem cells (PSCs) to all primary germ layers, various types of specialized progenitor cells, and even functional, mature cell types in in vitro and in vivo settings. Illustrated are specific differentiation protocols that have been effectively reproduced in our laboratory as well as others, but any differentiation protocol of choice can be used following the DMSO treatment. As shown in Table 1, a number of laboratories have also demonstrated an enhancement of PSC differentiation after transient DMSO treatment using different paradigms to generate various other terminal cell types. Furthermore, although the methods here describe the use of human PSCs, the DMSO pretreatment can be utilized across species and has been shown to be effective in mouse, rabbit, and primate PSCs.
Although higher doses of DMSO are known to be cytotoxic, the low doses used in this method (1%-2%) for a transient period result in minimal cell death. While overall cell numbers immediately after DMSO treatment may decrease due to DMSO promotion of cell cycle arrest in the G1 phase of the cell cycle, previous studies show that cells are able to reach the same level of confluency as control cultures after removal of DMSO3.
The percent and duration of DMSO pretreatment should be optimized per cell line. The treatment time should be adjusted with consideration for the cycling/doubling time of the cells. For example, mouse PSCs typically have much shorter cycling times of about 15 h; thus, DMSO treatment for 15 h for these cells is sufficient. Some labs have also found the DMSO treatment to be beneficial when continued during the differentiation protocol or at lower concentrations (see Table 1). It should be noted that some PSC lines are more amendable to differentiation to specific lineages. For example, HUES6 cells have been shown to be less permissive to differentiation and thus had marked improvement with DMSO treatment (Figure 2). Alternatively, HUES8 cells used in Figure 4 and Figure 5 have been shown to have a higher propensity towards endodermal differentiation; thus, fewer differences were shown between control and DMSO for differentiation at the initial stages towards definitive endoderm. Nonetheless, the enhancement of DMSO pretreatment is observed in later stages of differentiation in this cell line (Figure 4B). The DMSO treatment is also versatile in that it is effective in both 2D and 3D cell cultures systems, it can be used with various types of coating material on cell culture plates, and it works in different types of maintenance medium that promote growth and expansion of hPSCs (e.g., mTeSR, E8, MEF conditioned media, etc).
More generally, these results suggest that the starting state of pluripotent stem cells has a strong influence on the propensity for initial differentiation as well as terminal differentiation into functional cell types. We have previously shown that the DMSO treatment functions through Rb in hPSCs3,5. Rb plays an important role in promoting terminal differentiation, cell survival, and the genetic stability of cells41,42,43,44, and it may therefore explain the persistent effects on cells differentiated from DMSO-treated hPSCs. Targeting these early modes of regulation may place hPSCs on a better trajectory for differentiation and ultimately improve their utility for regenerative medicine.

The use of pluripotent stem cells has led to numerous advancements in biomedical research, including the fields of regenerative medicine and stem-cell based therapies, disease modeling, and drug screening. It has also led to the overall prospect of more translatable research and personalized medicine. The advent of induced pluripotent stem cell (iPSC) technology over 20 years ago has allowed researchers to develop pluripotent stem cells from somatic tissues and differentiate them into functional cell types to study a variety of pathologies, including cardiovascular, neurological, and immunological diseases. Although significant strides have been made in stem cell differentiation technology, challenges in effectively differentiating human embryonic stem cells (hESCs) and iPSCs still persist, limiting the widespread use of stem cell technology across different research programs. Inherent variability across different cell lines and clones continues to pose obstacles for differentiating stem cell lines to desired linages1. Furthermore, deriving mature, terminally differentiated functional cells from hPSCs remains a tedious and inefficient process across many lineages. In fact, cells differentiated from hPSCs often fail to terminally differentiate into functional cells2. In further moving stem cell-based therapies to use in patients, there is a need to improve and ensure the efficacy of cells that are generated from hPSCs.
Our lab has established a quick, inexpensive tool to significantly enhance the efficiency of differentiating both iPSCs and ESCs into mature cell types. We found that pretreatment of hiPSCs and hESCs with the commonly used reagent dimethyl sulfoxide (DMSO) for 24 h to 48 h prior to directed differentiation results in a marked improvement in stem cell differentiation capacity. Treatment with DMSO increases the proportion of hiPSCs and hESCs in the early G1 phase of the cell cycle and activates the retinoblastoma protein (Rb)3, a critical regulator of cell proliferation, survival, and differentiation4. In more recent work, it has been found that Rb and its family members are required for the pro-differentiation effects of DMSO, such that transient inactivation of Rb suppresses the effects of DMSO, while constitutive activation of Rb in a transient manner enhances DMSO&#8217;s effects5. Analogous to the cell cycle during embryonic development, the cell cycle of ESCs and iPSCs is characterized by an abbreviated G1 phase that promotes self-renewal6,7,8. This abbreviated G1 phase allows for more unrestricted proliferation but limits the potential for differentiation4,9. By promoting growth arrest in G1 and activating checkpoint controls in the cell cycle of hESCs and iPSCs, the DMSO treatment primes cells for cell fate changes following directed differentiation.
To date, DMSO pretreatment has been shown to improve the differentiation capacity to all three germ layers in over 30 control and disease-specific human ESC and iPSC cell lines3,5 as well as the differentiation of stem cells and other cell lines to a variety of other mature cell types in subsequent studies10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28 (Table 1). Furthermore, DMSO treatment has been shown to be effective in enhancing differentiation of non-human primary cells21,23 (e.g., mouse, primate, rabbit), suggesting shared mechanisms across species. Lastly, DMSO pretreatment has also been extended to gene editing technology, with one particular study showing that 24 h DMSO pretreatment of hESCs/iPSCs significantly increased the ability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 (Cas9)-mediated editing efficiency of non-coding DNA without incorporating unintended mutations29. Provided here is a detailed methodology of the DMSO pretreatment of hESCs and iPSCs for applications in stem cell biology and directed differentiation.

<table border="0" cellpadding="0" cellspacing="0" style="width:651px;" width="651">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">2-mercaptoethanol</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:135px;">21985023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:215px;">6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td style="width:135px;">Corning</td>
			<td style="width:135px;">353046</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">9-Position stir plate&#160;</td>
			<td style="width:135px;">Chemglass</td>
			<td style="width:135px;">CLS-4100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Accutase</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:135px;">11105-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Activin A</td>
			<td style="width:135px;">R&#38;D Systems</td>
			<td>338-AC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Advanced RPMI</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:135px;">12633012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alk5i II</td>
			<td style="width:135px;">Axxora</td>
			<td style="width:135px;">ALX-270-445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">all-trans retinoic acid&#160;</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:135px;">R2625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Brachyury</td>
			<td>R&#38;D Systems</td>
			<td style="width:135px;">AF2085</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">anti-C-peptide</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td style="width:135px;">GN-ID4</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">anti-FoxA2</td>
			<td style="width:135px;">Millipore</td>
			<td>07-633</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">anti-Nkx2.2</td>
			<td>University of Iowa, Developmental Hybridoma Bank</td>
			<td>74.5A5</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">anti-Nkx6.1</td>
			<td>University of Iowa, Developmental Hybridoma Bank;&#160;</td>
			<td style="width:135px;">F55A12-supernatant</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">anti-Olig2</td>
			<td style="width:135px;">EMD Millipore</td>
			<td style="width:135px;">MABN50</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Pax-6</td>
			<td>Biolegend</td>
			<td style="width:135px;">901301</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">anti-Pdx1</td>
			<td style="width:135px;">R&#38;D Systems</td>
			<td>AF2419</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-SOX1&#160;</td>
			<td>R&#38;D Systems</td>
			<td style="width:135px;">AF3369</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-SOX17</td>
			<td>R&#38;D Systems</td>
			<td style="width:135px;">AF1924</td>
			<td>No variablity observed across different lot numbers</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:215px;">B-27 Supplement, minus Vitamin A</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:135px;">12587010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">basic fibroblast growth factor</td>
			<td style="width:135px;">Gibco</td>
			<td>PHG0264</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Betacellulin</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>50932345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chir99021</td>
			<td style="width:135px;">Stemgent</td>
			<td>04-000-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">CMRL 1066</td>
			<td style="width:135px;">Corning</td>
			<td style="width:135px;">99-603-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:215px;">Countess II FL Automated Cell Counter</td>
			<td style="width:135px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:135px;">AMQAF1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">D-(+)-Glucose</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:135px;">G7528&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">DAPI</td>
			<td style="width:135px;">Invitrogen&#160;</td>
			<td style="width:135px;">D1306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Spinner Flasks</td>
			<td style="width:135px;">Corning, VWR</td>
			<td style="width:135px;">89089-814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">DMEM/F-12</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:135px;">11320033</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">DMSO&#160;</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Essential 6 Media</td>
			<td style="width:135px;">Gibco</td>
			<td>A1516501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">FAF-BSA</td>
			<td style="width:135px;">Proliant&#160;</td>
			<td style="width:135px;">68700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">FGF7</td>
			<td style="width:135px;">PeproTech</td>
			<td style="width:135px;">100-19</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Geltrex</td>
			<td style="width:135px;">Gibco</td>
			<td>A1413202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">GlutaMAX&#160;</td>
			<td style="width:135px;">Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Heparin&#160;</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:135px;">H3149&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Ultrasensitive Insulin ELISA</td>
			<td>ALPCO Diagnostics</td>
			<td>80-INSHUU-E01.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">ITS-X</td>
			<td style="width:135px;">Invitrogen&#160;</td>
			<td style="width:135px;">51500056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KGF</td>
			<td style="width:135px;">Peprotech</td>
			<td style="width:135px;">AF-100-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Knockout DMEM</td>
			<td style="width:135px;">Gibco</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">KnockOut Serum Replacement</td>
			<td style="width:135px;">Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-3,3&#8242;,5-Triiodothyronine (T3)</td>
			<td style="width:135px;">EMD Millipore</td>
			<td style="width:135px;">642245</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">LDN193189</td>
			<td style="width:135px;">Stemgent</td>
			<td>04-0074</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Matrigel Matrix</td>
			<td style="width:135px;">Corning</td>
			<td style="width:135px;">354277</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MCDB-131&#160;</td>
			<td style="width:135px;">Cellgro&#160;</td>
			<td style="width:135px;">15-100-CV&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">MEM NEAA</td>
			<td style="width:135px;">Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">mTeSR 1</td>
			<td style="width:135px;">StemCell Technologies</td>
			<td style="width:135px;">5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">N2 Supplement</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:135px;">17502048</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaHCO3</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:135px;">S3817&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Noggin Fc Chimera Protein</td>
			<td style="width:135px;">R&#38;D Systems</td>
			<td style="width:135px;">3344-NG-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PdBU</td>
			<td style="width:135px;">EMD Millipore</td>
			<td style="width:135px;">524390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin&#160;</td>
			<td style="width:135px;">Mediatech&#160;</td>
			<td style="width:135px;">30-002-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">RPMI</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:135px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sant1</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:135px;">S4572</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB431542</td>
			<td style="width:135px;">Stemgent</td>
			<td>04-0010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Smoothened Agonist, SAG</td>
			<td style="width:135px;">EMD Millipore</td>
			<td>566660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">StemPro Accutase</td>
			<td style="width:135px;">Gibco</td>
			<td>A1110501&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">TrypLE</td>
			<td style="width:135px;">Gibco</td>
			<td style="width:135px;">12604013</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:215px;">Ultra-Low Attachment Microplates</td>
			<td style="width:135px;">Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Vitamin C</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:135px;">A4544&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">Wnt3a</td>
			<td style="width:135px;">R&#38;D Systems</td>
			<td>5036-WN</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:215px;">XAV 939</td>
			<td style="width:135px;">Tocris</td>
			<td style="width:135px;">3748</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">XXI</td>
			<td style="width:135px;">EMD Millipore</td>
			<td style="width:135px;">565790</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Y-27632</td>
			<td style="width:135px;">StemCell Technologies</td>
			<td style="width:135px;">72302</td>
			<td></td>
		</tr>
	</tbody>
</table>

transient treatment of human pluripotent stem cells with dmso to promote differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Watch this Scientific Journal Video about Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation at JoVE.com

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

transient-treatment-human-pluripotent-stem-cells-with-dmso-to-promote

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

14.3K Views.  Stanford University School of Medicine. Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Video: Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

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 ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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 ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&amp;#239;ve-like State with Improved Multilineage Differentiation Potency

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

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 ...