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

Summary

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.

Abstract

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.

Introduction

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 novo^1,2,3,4,5. This is due, in large part, to a limited understanding of the mechanisms that drive the formation ....

Protocol

 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

1. 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 patterning onto 18 mm diameter coverslips, group features for each experimental condition into 10 x 10 mm areas to ensure the stencils generated in Step....

Results

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

Discussion

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.......

Materials

List of materials used in this article

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