Name: patterning the geometry of human embryonic stem cell colonies on compliant substrates to control tissue-level mechanics
Uploaded: 2019-09-28T15:00:00
Description: Watch this Scientific Journal Video about Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics at JoVE.com

We would like to acknowledge funding from CIRM grant RB5-07409. J.M.M. would like to thank FuiBoon Kai, Dhruv Thakar, and Roger Oria for various discussions that guided the generation and troubleshooting of this method. J.M.M. also thanks the UCSF Discovery Fellowship for the ongoing support of his work.

&#160;All methods described here pertaining to the use of hESCs have been approved by the Human Gamete, Embryo and Stem Cell Research (GESCR) Committee at the University of California San Francisco.
1. Preparation of silicon wafer with geometric features
<ol>
	<li>Create a photomask with desired geometric features. Use computer-aided drafting software to design the features. For use with negative photoresist, make features opaque and the remainder of the mask transparent. 
	NOTE: For pa...

<ol>
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To simplify a long and detailed protocol, this method consists of three critical stages: 1) generating patterns of ECM ligand on glass coverslips, 2) transferring the patterns to polyacrylamide hydrogels during polymerization of the gel, and 3) seeding hESCs on the patterned hydrogel. There are critical steps that must be considered at each of these three stages. In order to generate high-fidelity patterns on the glass coverslips, the stencil must be firmly pressed onto the coverslip to prevent leaking of the ligand solu...

Human embryonic stem cells (hESCs) hold great promise for use in regenerative medicine and tissue engineering applications. The pluripotent nature of these cells gives them the ability to differentiate into any adult cell type. While great strides have been made in directing the fate of hESCs to particular cell types, it has remained very difficult to generate whole tissues or organs de novo1,2,3,4,5. This is due, in large part, to a limited understanding of the mechanisms that drive the formation of these tissues during human development. In order to fill this gap in knowledge, a number of methods have emerged in recent years to model the early embryo and subsequent stages of development with embryonic stem cells6,7,8,9,10,11,12,13.
Shortly after the derivation of the first hESC lines14, it was demonstrated that embryoid bodies formed from hESCs were capable of spontaneously producing cells of the three primary germ layers6. However, due to the inherent lack of control over the size and morphology of embryoid bodies, the organization of germ layers varied significantly and failed to match the organization of the early embryo. More recently, Warmflash et al. developed a method to confine colonies of hESCs on glass substrates via micropatterning, providing control and consistency over the size and geometry of the colonies8. In the presence of BMP4, an important morphogen in early development, these confined colonies were capable of self-organizing reproducible patterns of specification to fates representing the primary germ layers. Although this provided a useful model for studying the mechanisms by which primary germ layers are established, the patterns of fate specification did not precisely match the organization and morphogenesis observed during embryogenesis15. A more faithful recapitulation of early embryonic development was achieved by embedding hESCs in a three-dimensional extracellular matrix (ECM) of matrigel11, providing the strongest evidence to date for the ability of hESCs to self-organize and model the early stages of embryogenesis ex vivo. However, this method yields inconsistent results and is thus incompatible with a number of assays that could be used to reveal the underlying mechanisms of self-organization and fate specification.
Given these existing methods and their respective limitations, we sought to develop a method for reproducibly culturing hESC colonies of defined geometries in conditions that model the extracellular environment of the early embryo. To achieve this, we used polyacrylamide hydrogels of tunable elasticity to control the mechanical properties of the substrate. Using atomic force microscopy on gastrulation-stage chicken embryos, we found that the elasticity of the epiblast ranged from hundreds of pascals to a few kilopascals. Thus, we focused on generating polyacrylamide hydrogels with elasticity in this range to serve as the substrate for hESC colonies. We modified our previous methods for culturing hESCs on polyacrylamide hydrogels7,9 to provide robust control over the geometry of the colonies. We achieved this by first patterning ECM ligands, namely matrigel, onto glass coverslips through microfabricated stencils, as previously reported16. We then designed a novel technique to transfer the patterned ligand to the surface of polyacrylamide hydrogels during polymerization. The method we describe here involves using photolithography to fabricate a silicon wafer with the desired geometric patterns, creating stamps of theses geometric features with polydimethylsiloxane (PDMS), and using these stamps to generate the stencils that ultimately allow patterning of ligand onto the surface of glass coverslips and transfer to polyacrylamide.
In addition to recapitulating the mechanical environment of the early embryo, confining hESC colonies on polyacrylamide enables the measurement of cell-generated forces with traction force microscopy (TFM), as reported in our previous method9. In brief, fluorescent beads can be embedded in the polyacrylamide and used as fiducial markers. Cell-generated forces are calculated by imaging the displacement of these beads after seeding hESCs onto the patterned substrate. Furthermore, the resulting traction force maps can be combined with traditional assays, such as immunostaining, to examine how the distribution of cell-generated forces in confined hESC colonies may regulate or modulate downstream signaling. We expect these methods will reveal that mechanical forces play a critical role in the patterning of cell fate specification during early embryonic development that is currently overlooked.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm glass petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-747B</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm plastic petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical-bottom tubes</td>
			<td>Corning</td>
			<td>352095</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm plastic petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875714</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm diameter #1 coverslips</td>
			<td>Thermo Scientific</td>
			<td>18CIR-1</td>
			<td></td>
		</tr>
		<tr>
			<td>2% bisacrylamide</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>3-aminopropyltrimethoxysilane</td>
			<td>ACROS Organics</td>
			<td>313251000</td>
			<td></td>
		</tr>
		<tr>
			<td>40% acrylamide</td>
			<td>Bio-Rad</td>
			<td>161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>01-213-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor</td>
			<td>Sigma-Aldrich</td>
			<td>F0291</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge with swing-buckets</td>
			<td>Eppendorf</td>
			<td>22623508</td>
			<td>Model: 5804 R</td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Dessicator</td>
			<td>Fisher Scientific</td>
			<td>08-642-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>AC615095000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microspheres</td>
			<td>Thermo Scientific</td>
			<td>F8821</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (for coverslips)</td>
			<td>Fisher Scientific</td>
			<td>16-100-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (for wafers)</td>
			<td>Fisher Scientific</td>
			<td>17-467-328</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel holders</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Gel holders are custom 3D-printed, CAD drawing available on request</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-261-94</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Scientific</td>
			<td>J16926A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>HP88854100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Fisher Scientific</td>
			<td>A416-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes (delicate task wipes)</td>
			<td>Kimberly-Clark Professional</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout serum replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout-DMEM</td>
			<td>Gibco</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Mask aligner (for photolithography)</td>
			<td>Karl Suss America, Inc.</td>
			<td>Karl Suss MJB3 Mask Aligner</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope for traction force</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>Model: Eclipse TE200 U</td>
		</tr>
		<tr>
			<td>Motorized positioning stage</td>
			<td>Prior Scientific</td>
			<td>N/A</td>
			<td>Model: HLD117</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>Airgas</td>
			<td>NI 250</td>
			<td></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 74 (UV-curable polymer)</td>
			<td>Norland Products</td>
			<td>NOA 74</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Thermo Scientific</td>
			<td>PR305225G</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm (laboratory film)</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS (Sylgard 184)</td>
			<td>Fisher Scientific</td>
			<td>NC9285739</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomask</td>
			<td>CAD/Art Services, Inc.</td>
			<td>N/A</td>
			<td>Photomasks are custom made. CAD drawing for our designs available upon request</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Fisher Scientific</td>
			<td>NC9332171</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic for gasket</td>
			<td>Marian Chicago</td>
			<td>HT6135</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic for spacer</td>
			<td>TAP Plastics</td>
			<td>N/A</td>
			<td>Polycarbonate sheet, .01 inch thickness</td>
		</tr>
		<tr>
			<td>Potassium chloride (for making PBS)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (for making PBS)</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottassium persulfate</td>
			<td>ACROS Organics</td>
			<td>424185000</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fisher Scientific</td>
			<td>14-840-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>Electron Microscopy Sciences</td>
			<td>71893-06</td>
			<td>Type P, 3 inch, silicon wafers</td>
		</tr>
		<tr>
			<td>Sodium chloride (for making PBS)</td>
			<td>Fisher Scientific</td>
			<td>S271-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic dihydrate (for making PBS)</td>
			<td>Fisher Scientific</td>
			<td>S472-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8-3050 Photoresist</td>
			<td>MicroChem</td>
			<td>SU8-3000</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8-Developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-sterilization box</td>
			<td>Bio-Rad</td>
			<td>N/A</td>
			<td>Bio-Rad GS Gene Linker UV Chamber</td>
		</tr>
		<tr>
			<td>Y27632 (Rho kinase inhibitor)</td>
			<td>StemCell Technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
	</tbody>
</table>

patterning the geometry of human embryonic stem cell colonies on compliant substrates to control tissue-level mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

The main challenge to overcome in attempting to culture hESCs in colonies of controlled geometry on compliant substrates is to generate a homogenous pattern of ECM-ligand on the surface of the substrate. The strategy presented in this method involves first generating the desired pattern on the surface of a glass coverslip and then subsequently transferring that pattern to the surface of a polyacrylamide hydrogel during polymerization of the gel (Figure 1<stro...

Watch this Scientific Journal Video about Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics at JoVE.com

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

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Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...