This work is supported by NUS Start up grant (R-397-000-192-133) and ETPL Gap Fund (R-397-000-198-592). G.S. is a NUS Research scholar. Authors would like to thank Dr. Jiangwa Xing for her technical support on cell micropatterning.

NOTE: This protocol describes the fabrication of PDMS stencil with 1,000 &#181;m patterns by laser-cutting and micropatterning of the hESC line, H9 using the PDMS stencil.
1. Design and Fabrication of PDMS Stencil for Micropatterning
<ol>
	<li>Design the stenciling sheet with through-holes of the desired geometry and size (e.g., 1,000 &#181;m circles) and the stencil gasket using computer-aided design software10.</li>
	<li>Laser-cut the stenciling sheet and gasket on 120-150 &#181;m and 2 mm thick polydimethylsiloxane (PDMS) sheets respectively using a CO2 laser-cutter10.</li>
	<li>Bond the PDMS gasket with the PDMS stencil sheet using liquid uncured PDMS and bake at 60 &#176;C for 3-4 hr to obtain the PDMS stencil for micropatterning.</li>
	<li>Sterilize the PDMS stencil by autoclaving at 120 &#176;C for 30 min and drying in an oven before use.</li>
</ol>
2. hESCs Maintenance
<ol>
	<li>Culture the hESC lines in a feeder-free maintenance medium on 1&#215; hESC-qualified basement membrane matrix coated cell culture plates at 37 &#176;C and 5% CO2.</li>
	<li>Passage the hESCs when they are approximately 70% confluent by 3 min incubation at 37 &#176;C with dispase treatment and mechanically dissociating the hESC colonies into 100-200 &#181;m clumps. Plate the hESCs clumps at a density of 30-50 clumps per 9.6 cm2 of growth area on basement membrane matrix-coated tissue culture polystyrene at 1:6 splitting ratio.</li>
</ol>
3. Stencil Micropatterning of Human Embryonic Stem Cells
<ol>
	<li>Preparation prior to cell patterning
	<ol>
		<li>Seal a PDMS stencil onto a 60 mm Petri dish by dispensing 700 &#181;l 70% analytical grade ethanol in ultrapure water into the Petri dish and placing the stencil on top.</li>
		<li>Place the Petri dish inside biosafety cabinet overnight to let the ethanol dry.</li>
		<li>Check no bubble exists underneath the stencil to ensure that the stencil forms a good seal with the Petri dish. This prevents leakages of ECM coating solution. Seal the Petri dish and store under sterile conditions until it is ready for cell seeding.</li>
		<li>Prepare aliquots of hESC-qualified basement membrane matrix according to the Certificate of Analysis. Ensure that each aliquot yields 1&#215; hESC-qualified basement membrane matrix solution when diluted in 25 ml of Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) according to manufacturer&#39;s product sheet.</li>
		<li>Prepare the ECM coating solution by adding one aliquot of hESC-qualified basement membrane matrix into 16.7 ml of DMEM/F12 to make 1.5&#215; hESC-qualified basement membrane matrix solution. Keep all the ECM coating solution on ice to prevent gelation.</li>
		<li>Supplement hESC maintenance medium with 10 &#181;M ROCK inhibitor (Y27632).</li>
	</ol>
	</li>
	<li>ECM coating on the stenciled substrate
	<ol>
		<li>Treat the Petri dish with 100 W O2 plasma for 90 sec. To ensure sterility, only open the Petri dish cover inside the plasma chamber prior to plasma treatment, and quickly cover back the Petri dish after completion of treatment. This facilitates surface wetting and prevents air bubble formation in the micropattern through-holes during the addition of ECM coating solution.</li>
		<li>Add 450 &#181;l of 1.5&#215; hESC-qualified basement membrane matrix solution to cover the whole stencil. Seal the Petri dish with self-sealable film, such as Parafilm, to prevent the solution from drying out, and incubate for 5 hr at 37 &#176;C before use.</li>
	</ol>
	</li>
	<li>Seeding hESCs onto the stenciled substrate
	<ol>
		<li>Examine hESC colonies in 6-well plate to identify the differentiated cell areas showing loss of typical hESC morphology (e.g., loss of rounded, tightly packed epithelial morphology, high nucleus/cytoplasm ratio with prominent nucleoli). Remove differentiated regions using a vacuum aspirator.</li>
		<li>Wash twice with 2 ml DMEM/F12 per well.</li>
		<li>Add 1 ml of digestive enzymes, such as Accutase, per well of 6-well plate and incubate at 37 &#176;C for 8 min. Tap the plate gently to detach all colonies from substrate.</li>
		<li>Rinse each well with at least 4 ml of DMEM/F12 per 1 ml of digestive enzymes and collect the cell suspension into 15 ml conical tube.</li>
		<li>Centrifuge the cell suspension at 200 &#215; g for 3 min at room temperature.</li>
		<li>Aspirate to remove the supernatant and add 400 &#181;l of hESC maintenance medium supplemented with ROCK inhibitor (ROCKi) to re-suspend the cells. Pipette the cell suspension up and down 3 times gently to break clumps into single cells.</li>
		<li>Mix the single cell suspension well and dilute 10 &#181;l of cell samples into 190 &#181;l of DMEM/F12 (1:20 dilution). Use a hemocytometer to determine the cell density in the stock cell suspension.</li>
		<li>Calculate the required cell seeding density for a given stencil. 
		NOTE: For example, we have experimentally determined the cell seeding density to obtain a confluent monolayer of single cells is approximately 4,444 cells/mm2. Thus, a stencil with an area of 450 mm2 and seeding volume of 400 &#181;l will require a cell suspension to be at a density of 2 million cells / 400 &#181;l.</li>
		<li>Dilute the stock cell suspension to the required seeding density (see step 3.3.8 NOTE) with hESC culture medium supplemented with ROCKi.</li>
		<li>Add a designated volume of cell suspension containing the required number of cells into each stencil and leave the Petri dish undisturbed in hood for 5 min at room temperature to allow cells to settle.</li>
		<li>Transfer the Petri dish into incubator and incubate for 1 hr to allow for cell attachment. Take care to keep the Petri dish level during the transfer process so that cells remain as a monolayer in the stencil.</li>
	</ol>
	</li>
	<li>Stencil removal and passivation of unpatterned substrate
	<ol>
		<li>Examine the Petri dish under a microscope to check if cells are properly attached onto the underlying substrate.</li>
		<li>Aspirate away cell suspension from the stencil.</li>
		<li>Add 2 ml/dish of 0.5% cell culture compatible non-ionic surfactant in DMEM/F12 to the area surrounding the stencil.</li>
		<li>Use a pair of autoclaved forceps to gently peel off the stencil. Swirl the non-ionic surfactant solution around as the stencil is peeled off to prevent the cells from drying out. Visually observe the micropatterned-cells in the Petri dish.</li>
		<li>Incubate the micropatterned-cells in 0.5% non-ionic surfactant-DMEM/F12 solution for 10 min at 37 &#176;C.</li>
		<li>Aspirate away the non-ionic surfactant-DMEM/F12 solution. Wash 3 times with 2 ml/dish of DMEM/F12.</li>
		<li>Add 2 ml/dish of hESC maintenance medium supplemented with ROCKi and incubate at 37 &#176;C overnight.</li>
		<li>After overnight incubation, aspirate hESC maintenance medium supplemented with ROCKi, wash once with DMEM/F12 and induce mesoendoderm differentiation by adding 2 ml/dish of differentiation medium supplemented with 100 ng/ml Activin, 25 ng/ml BMP4 and 10 ng/ml FGF2.</li>
		<li>Evaluate the micropatterns using phase contrast imaging and compute the average Brachyury (T) intensity profiles for each pattern as described previously8.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests.

Fabrication of micropatterning stencils
Stencil micropatterning provides an ideal method to generate hPSC micropatterns for investigating niche-mediated differentiation patterning. The key advantage of stencil patterning over other micropatterning techniques, such as microcontact printing and photopatterning, is that it does not require surface modification and can be implemented on conventional TCPS substrates. Therefore, optimized culture media and ECM coatings for ...

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are widely exploited in regenerative medicine as well as experimental modeling of normal and diseased organogenesis because of their differentiation potential into cell lineages of all three germ layers1,2. The differentiation fates of hPSCs are highly sensitive to local environmental factors that can modulate autocrine or paracrine signaling1 as well as mechanotransduction processes mediated by physical cues3-5. Cell micropatterning encompasses a set of techniques that have been developed to spatially organize the geometry and location of a cell population as a mean to control the local cellular microenvironment, such as cell-cell interactions6 and cell-matrix interactions3. In the context of hPSCs, cell micropatterning has been employed to gain significant insights into how niche-dependent autocrine signaling modulates hESC pluripotency-differentiation decisions7 and organization into early embryonic differentiation patterns6. 2D and 3D micropatterned hPSCs have been used to control the colony size of multicellular patterns, which in turn influenced differentiation decisions into the three germ layers8,9. We have employed multicellular hPSC micropatterns to modulate the extent of cell-cell and cell-matrix interactions within a hPSC colony to probe how integrin-E-cadherin crosstalk can give rise to cell fate heterogeneity10. The demonstrations from the above reports open new avenues towards the application of multicellular micropatterns of hPSCs as experimental models for drug toxicity screening for developmental diseases11, to study the effect of growth factors and hormones during tissue or organ development, and to unravel the formation of tissue patterns.
A myriad of cell micropatterning techniques have been developed as reviewed by Falconnet et. al.12 but only a handful, such as micro-contact printing7,8,13, microwell culture14,15, photopatterning6 and microstencils16 have been successfully implemented with hPSCs. The challenge with micropatterning hPSCs lies in their vulnerability and a stringent requirement of specific extracellular matrices (ECM) and growth conditions for cell attachment and survival. For 2D hPSC patterns, micro-contact printing is one of the most common methods to generate hPSC micropatterns on tissue culture and glass substrates13. The method can be used to pattern common ECM used in hPSC culture, including laminin and basement membrane matrices, such as Matrigel. However, it typically requires a two-step coating process aided by Poly-D-Lysine, and needs specific inert atmospheric and humidity conditions to make stable ECM micropatterns for hPSCs to attach on6,13. The foremost consideration of each micropatterning method is whether the surface modification regime can generate hPSC-adhesive ECM patterns at the desired geometrical resolution while minimizing unspecific cell attachment to the surrounding areas.
Here, we report the use of stencil micropatterning as a simple method to generate hPSC micropatterns without additional surface modification steps prior to the generation of adhesive ECM patterns for hPSCs to attach on. The cell stencil comprises of a thin membrane, e.g., polydimethylsiloxane (PDMS) sheet, with micron to millimeter size through-holes sealed onto a cell culture substrate to physically contain ECM coatings and subsequently seeded hPSCs. As stencil patterning works by physically restraining the location where hPSC can access and attach directly to the underlying ECM coated substrate, this method is compatible with various substrates that can support hPSC cultures. The only requirement is that the choice of stencil material can form a reversible seal with the substrate. These substrates include conventional tissue culture polystyrene (TCPS)17, ligand conjugated substrates18, as well as elastomeric substrates with tunable stiffness (e.g., PDMS)19. This method also allows coating of different ECM, such as vitronectin (or VTN protein), laminin and basement membrane matrices (e.g., Matrigel and Geltrax) to allow for proper attachment and differentiation of hPSCs. Therefore, we can transfer optimized ECM-substrate configurations for a specific hPSC line to stencil micropatterning for optimal cell-matrix adhesion, survival and differentiation. Recently, a similar method has also been reported to direct hepatic differentiation by micropatterning hESCs using poly(methyl methacrylate) (PMMA) micro-stencil arrays16.
Cell stencils can be fabricated from different materials, including metals20,21, poly(p-xylylene) polymers22,23, PMMA16 and most commonly, PDMS24-28. Silicon and poly(p-xylylene) polymers stencils require direct etching of the through-holes with specialized equipment20-23, which limits their accessibility to biological users. PDMS stencils can be fabricated by different methods depending on the feature size required, which typically ranges from 3 &#181;m to 2,000 &#181;m11,26-29. If small features are desired, thin stenciling sheets can be produced by press molding PDMS pre-polymer on a microfabricated silicon template containing reliefs of the micropatterns28. For features &#62; 1,000 &#181;m, a CO2 laser cutter provides an easy and low cost method to directly cut the patterns on a pre-casted PDMS sheet during stencil fabrication. The recyclability of PDMS stencils also makes them cost-effective to conduct a series of experiments with sufficient consistency.
Here, we present the detailed methodology for the fabrication of a PDMS stencil with 1,000 &#181;m features by laser cutting and the generation of hESC micropatterns. These hESC micropatterns were used to modulate the extent of integrin and E-cadherin mediated adhesions within a cohesive hESC colony so as to investigate how spatial polarization of cell adhesion resulted in cell fate heterogeneity10.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#160;2 mm thick PDMS sheet</td>
			<td style="width:191px;">Specialty Silicone Products Inc., USA</td>
			<td style="width:119px;">SSPM823-.005</td>
			<td style="width:423px;">Used to form reservoir for stencil</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">120-150 &#956;m thick PDMS sheet</td>
			<td style="width:191px;">Specialty Silicone Products Inc., USA</td>
			<td style="width:119px;">SSPM823-.040</td>
			<td style="width:423px;">Used to form stencil&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">60 mm petri dish</td>
			<td style="width:191px;">&#160;Nunc Nunclon Delta</td>
			<td style="width:119px;">150326</td>
			<td style="width:423px;">Substrate for micropatterning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase</td>
			<td style="width:191px;">Accutase, Merck Millipore, Singapore</td>
			<td style="width:119px;">SCR005</td>
			<td style="width:423px;">Enzyme to break H9 Cells into single cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Activin&#160;&#160;</td>
			<td style="width:191px;">R&#38;D Systems, Singapore</td>
			<td style="width:119px;">338-AC-010</td>
			<td style="width:423px;">Growth factor for H9 differentiation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BMP4&#160;</td>
			<td style="width:191px;">R&#38;D Systems, Singapore</td>
			<td style="width:119px;">338-BP-010</td>
			<td style="width:423px;">Growth factor for H9 differentiation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plasma system&#160;</td>
			<td style="width:191px;">&#160;Femto Science, Korea</td>
			<td style="width:119px;">CUTE-MP</td>
			<td style="width:423px;">For plasma oxidation of stencil</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dispase</td>
			<td style="width:191px;">StemCell&#8482; Technologies, Singapore</td>
			<td style="width:119px;">7923</td>
			<td style="width:423px;">Enzyme used to weaken the cell-ECM adhesion during passaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:191px;">GIBCO, USA</td>
			<td style="width:119px;">11330032</td>
			<td style="width:423px;">Basal medium for H9 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FGF2</td>
			<td style="width:191px;">&#160;R&#38;D Systems, Singapore</td>
			<td style="width:119px;">233&#8211;FB&#8211;025</td>
			<td style="width:423px;">Growth factor for H9 differentiation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">H9 Cell line</td>
			<td style="width:191px;">WiCell Research Institute, Inc., USA</td>
			<td style="width:119px;">WA09</td>
			<td style="width:423px;">Human embryonic stem cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">hESC-qualified basement membrane matrix</td>
			<td style="width:191px;">Matrigel, BD Biosciences, Singapore</td>
			<td style="width:119px;">354277</td>
			<td style="width:423px;">Extra-cellular matrix coating to support growth of H9 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope</td>
			<td style="width:191px;">Leica Microsystems, Singapore</td>
			<td style="width:119px;">DMi1</td>
			<td style="width:423px;">For capturing bright-field images</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser cutter</td>
			<td style="width:191px;">Epilog Helix 24 Laser System</td>
			<td style="width:119px;"></td>
			<td style="width:423px;">Used to generate through holes in PDMS sheet</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">mTeSR&#8482;1 medium&#160;</td>
			<td style="width:191px;">StemCell&#8482; Technologies, Singapore</td>
			<td style="width:119px;">5850</td>
			<td style="width:423px;">Maintainence medium for H9 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PDMS&#160;</td>
			<td style="width:191px;">SYLGARD&#174; 184, Dow Corning Co., USA</td>
			<td style="width:119px;">3097358-1004</td>
			<td style="width:423px;">Used for sticking the PDMS stencil and reservior</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ROCKi Y27632</td>
			<td style="width:191px;">Calbiochem, Merck Millipore, Singapore</td>
			<td style="width:119px;">688000</td>
			<td style="width:423px;">Maintains H9 cells as single cells&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">STEMdiff&#8482; APEL&#8482; medium&#160;</td>
			<td style="width:191px;">StemCell&#8482; Technologies, Singapore</td>
			<td style="width:119px;">5210</td>
			<td style="width:423px;">Differentiation medium for H9 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethylene terephthalate film</td>
			<td style="width:191px;">SureMark Singapore</td>
			<td style="width:119px;">SQ-6633</td>
			<td style="width:423px;">Used to form stencil&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell culture compatible non-ionic surfactant</td>
			<td style="width:191px;">Pluronic acid F-127, Sigma, Singapore</td>
			<td style="width:119px;">P2443</td>
			<td style="width:423px;">Passivating reagent to repel cell adhesion in non-micropatterned substrates</td>
		</tr>
	</tbody>
</table>

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.

In this paper, we describe the fabrication of a cell stencil by using a laser cutter to generate 1,000 &#181;m features. The stencil was composed of 2 parts: a thin stenciling sheet (approximately 100-200 &#181;m thick) containing the micropattern through-holes, and a PDMS gasket to contain the ECM coating solution or cell suspension. Here, 127 &#181;m and 2 mm thick commercially available PDMS sheets were used as the stenciling sheet and gasket respectively. Other methods for preparing P...

Watch this Scientific Journal Video about Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates at JoVE.com

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

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 ...

stencil-micropatterning-human-pluripotent-stem-cells-for-probing

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JoVE Journal

Developmental Biology

8.4K Views.  National University of Singapore. 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.

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

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 ...

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 ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...