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-...

<ol>
	<li>Graf, T., Stadtfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+embryonic+and+adult+stem+cells.">Heterogeneity of embryonic and adult stem cells.</a> Cell Stem Cell. 3 (5), 480-483 (2008).</li><li>Nishikawa, S., Goldstein, R. A., Nierras, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+human+induced+pluripotent+stem+cells+for+research+and+therapy.">The promise of human induced pluripotent stem cells for research and therapy.</a> Nat Rev Mol Cell Biol. 9 (9), 725-729 (2008).</li><li>Guilak, F., Cohen, D. M., Estes, B. T., Gimble, J. M., Liedtke, W., Chen, C. 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H., Peerani, R., Ungrin, M., Joshi, C., Kumacheva, E., Zandstra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+of+human+embryonic+stem+cells+dissects+the+mesoderm+and+endoderm+lineages.">Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages.</a> Stem Cell Res. 2 (2), 155-162 (2009).</li><li>Hwang, Y. S., Chung, B. G., Ortmann, D., Hattori, N., Moeller, H. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+stiffness+affects+early+differentiation+events+in+embryonic+stem+cells.">Substrate stiffness affects early differentiation events in embryonic stem cells.</a> Eur Cell Mater. 18, 1-13 (2009).</li><li>Carter, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haptotactic+islands:+a+method+of+confining+single+cells+to+study+individual+cell+reactions+and+clone+formation.">Haptotactic islands: a method of confining single cells to study individual cell reactions and clone formation.</a> Exp Cell Res. 48 (1), 189-193 (1967).</li><li>Jimbo, Y., Robinson, H. 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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 ...

The overall goal of this stencil-based micropatterning method, for human pluripotent stem cells, is to provide a simple and robust means to generate spatial environmental gradients that control stem cell differentiation patterns. This method can help you answer key questions in tissue patterning using a human stem cell model, such as how spatial gradients in cell-cell and cell matrix adhesion forces can help to organize stem cell differentiation fates. The advantage of this method is that it does not require additional surface modification to generate cell adhesive ECM patterns for cells to attach to.This means the method is compatible with various ECM and substrate configurations, specific for each cell line. Visual demonstration of this method is critical, as the micropatterning steps are difficult to learn. Because it requires precise and careful handling of the stencil and the cells.For this procedure, the PTMS stencils must already be designed and fabricated. Likewise, have human EFC cells growing on basement membrane matrix, such that the colonies will be ready for seeding later in the procedure. To begin, load a dish with 700 microliters of 70%analytical-grade ethanol, in ultra-pure water.Then, place the PDMS stencil on the solution, and set the dish into a bio-safety cabinet to dry out overnight. The next day, check that no bubbles have formed under the stencil. Then, field the dish until the cells can be seeded onto the stencil.Prepare an aliquot of human ESC-quality basement membrane matrix by adding 16.7 milliliters of DMF12, to make a 1.5x solution and keep it on ice. Treat the substrate with 100 watts of oxygen plasma for 90 seconds. Don't open the dish until it is inside the plasma chamber, and quickly cover it up after the treatment.This treatment will prevent bubbles from forming when the substrate is immersed in solution. Now, cover the stencil with 450 microliters of prepared 1.5x basement membrane matrix solution. Then, seal the dish with parafilm and incubate it for five hours.Before proceeding, check over the six well plates of prepared HESC colonies. Identify and aspirate off colonies of differentiated cells that have lost their typical HESC morphology. They should all appear rounded, have a tightly-packed epithelial morphology, and have a high nucleus-to-cytoplasm ratio with prominent nucleoli.Then, wash each well of plated cells with two milliliters of DMF12 two times. After the second wash, add one milliliter of digestive enzymes to each well, such as accutase and incubate the plate for eight minutes at 37 degrees Celsius. After the incubation, tap the plate gently to detach all the colonies from the substrate.Next, rinse each well with at least four milliliters of media for every milliliter of digestive enzyme applied to the well, and collect the cell suspensions into a single 15 milliliter conical tube. Centrifuge the cells at 200 g for three minutes at room temperature to form a cell pellet. Then aspirate the supernatant, and re-suspend the cells in 400 microliters of HESC maintenance media, supplemented with 10 micromolar of rock inhibitor.Triturate the cells gently three times to break up the clumps. Next, correct the suspensions'density. Mix it well, and make a 200 microliter 1:20 dilution in media.Count the cells in the dilution using a hemocytometer. Then, dilute the main suspension as needed, using media with rock inhibitor. To proceed, add the required number of cells onto each prepared stencil.Only a mono-layer of cells should be deposited, so spread them out evenly. Let the stencil sit undisturbed for five minutes, so the cells can settle. Then, carefully transfer the loaded stencils to a incubator, keeping the dishes level throughout the transfer.Let the cells attach to the stencils over an hour-long incubation. After the cells have been allowed to attach, examine them under a microscope to ensure that they did so properly. Transfer the stencil back to the hood, and aspirate the unattached cells off the stencil.Next, add two milliliters of 0.5%compatible non-ionic surfactant in DMF12 to the area surrounding the stencil. Swirl the solution around to cover the stencil. Then, use a pair of autoclaved forceps to gently peel the stencil off the dish.After removing the stencil, inspect the micropatterend cells under a microscope. The non-ionic surfactant prevents these cells from drying out. Let the micropatterend cells incubate in the surfactant-supplemented media solution for 10 minutes at 37 degrees Celsius.After 10 minutes, aspirate away the solution, and wash the plate with two milliliters of normal DMF12 medium, three times. After the washes, add two milliliters of HESC maintenance medium with rock inhibitor. Once again, inspect the cells to check that the micropatterns have formed properly.Then, incubate the cells at 37 degrees Celsius overnight. The next day, aspirate the medium, wash the cells once with DMF12, and then, add two milliliters of supplemented differentiation medium, to induce the cells to undergo mesoendoderm differentiation. Later, analyze the results.The fabrication of the cell stencil using a laser cutter, provided 1000 micron features. The stencil was composed of a thin stenciling sheet with micropatterned through holes, and a PDMS gasket used to contain the cells. Using this tool, micropatterened HESCs were investigated during mesoendoderm differentiation.H9-HESC colony peripheries have higher integrin adhesions than cells at the colony interior. Thus, the cells differentiate into brachiary T-positive mesoendoderm cells, when induced with activan, BMP4, and FGF2. When such cells were patterned into monolayers of different geometries, geometric and isotropiate right angles and at convex curvatures, led to a local concentration of integrin mediated traction forces, and resulted in increased mesoendoderm differentiation.T expression intensity at different localities within a single colony showed that the extent of meso endoderm induction was indeed higher in sharp corners of square and rectangular colonies, and at the convex curvatures of a semi-circular arc. This demonstrates that cell adhesion mediated mechanical forces can be modulated using the described method. Before attempting the procedure, it is important to optimize the matrix coating time, the cell digestion and attachment time, for the specific human pluripotent stem cell line that you are working with.

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Research

JoVE Journal

Developmental Biology

8.4K Views.  National University of Singapore. The overall goal of this stencil-based micropatterning method, for human pluripotent stem cells, is to provide a simple and robust means to generate spatial environmental gradients that control stem cell differentiation patterns. This method can help you answer key questions in tissue patterning using a human stem cell model, such as how spatial gradients in cell-cell and cell matrix adhesion forces can help to organize stem cell differentiation fates. The advantage of this method is that it ...