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

Summary

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.

Abstract

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.

Introduction

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 layers^1,2. 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 cues^3-5. 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 layers^8,9. 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 printing^7,8,13, microwell culture^14,15, 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 on^6,13. 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 metals^20,21, poly(p-xylylene) polymers^22,23, PMMA¹⁶ and most commonly, PDMS^24-28. Silicon and poly(p-xylylene) polymers stencils require direct etching of the through-holes with specialized equipment^20-23, 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 µm to 2,000 µm^11,26-29. 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 > 1,000 µm, 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 µm 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.

Protocol

NOTE: This protocol describes the fabrication of PDMS stencil with 1,000 µm patterns by laser-cutting and micropatterning of the hESC line, H9 using the PDMS stencil.

1. Design and Fabrication of PDMS Stencil for Micropatterning

1. Design the stenciling sheet with through-holes of the desired geometry and size (e.g., 1,000 µm circles) and the stencil gasket using computer-aided design software¹⁰.
2. Laser-cut the stenciling sheet and gasket on 120-150 µm and 2 mm thick polydimethylsiloxane (PDMS) sheets respectively using a CO₂ laser-cutter¹⁰.
3. Bond the PDMS gasket with the PDMS stencil sheet using liquid uncured PDMS and bake at 60 °C for 3-4 hr to obtain the PDMS stencil for micropatterning.
4. Sterilize the PDMS stencil by autoclaving at 120 °C for 30 min and drying in an oven before use.

2. hESCs Maintenance

1. Culture the hESC lines in a feeder-free maintenance medium on 1× hESC-qualified basement membrane matrix coated cell culture plates at 37 °C and 5% CO₂.
2. Passage the hESCs when they are approximately 70% confluent by 3 min incubation at 37 °C with dispase treatment and mechanically dissociating the hESC colonies into 100-200 µm 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.

3. Stencil Micropatterning of Human Embryonic Stem Cells

1. Preparation prior to cell patterning
   1. Seal a PDMS stencil onto a 60 mm Petri dish by dispensing 700 µl 70% analytical grade ethanol in ultrapure water into the Petri dish and placing the stencil on top.
   2. Place the Petri dish inside biosafety cabinet overnight to let the ethanol dry.
   3. 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.
   4. Prepare aliquots of hESC-qualified basement membrane matrix according to the Certificate of Analysis. Ensure that each aliquot yields 1× hESC-qualified basement membrane matrix solution when diluted in 25 ml of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) according to manufacturer's product sheet.
   5. 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× hESC-qualified basement membrane matrix solution. Keep all the ECM coating solution on ice to prevent gelation.
   6. Supplement hESC maintenance medium with 10 µM ROCK inhibitor (Y27632).
2. ECM coating on the stenciled substrate
   1. 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.
   2. Add 450 µl of 1.5× 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 °C before use.
3. Seeding hESCs onto the stenciled substrate
   1. 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.
   2. Wash twice with 2 ml DMEM/F12 per well.
   3. Add 1 ml of digestive enzymes, such as Accutase, per well of 6-well plate and incubate at 37 °C for 8 min. Tap the plate gently to detach all colonies from substrate.
   4. 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.
   5. Centrifuge the cell suspension at 200 × g for 3 min at room temperature.
   6. Aspirate to remove the supernatant and add 400 µl 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.
   7. Mix the single cell suspension well and dilute 10 µl of cell samples into 190 µl of DMEM/F12 (1:20 dilution). Use a hemocytometer to determine the cell density in the stock cell suspension.
   8. 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 µl will require a cell suspension to be at a density of 2 million cells / 400 µl.
   9. Dilute the stock cell suspension to the required seeding density (see step 3.3.8 NOTE) with hESC culture medium supplemented with ROCKi.
   10. 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.
   11. 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.
4. Stencil removal and passivation of unpatterned substrate
   1. Examine the Petri dish under a microscope to check if cells are properly attached onto the underlying substrate.
   2. Aspirate away cell suspension from the stencil.
   3. Add 2 ml/dish of 0.5% cell culture compatible non-ionic surfactant in DMEM/F12 to the area surrounding the stencil.
   4. 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.
   5. Incubate the micropatterned-cells in 0.5% non-ionic surfactant-DMEM/F12 solution for 10 min at 37 °C.
   6. Aspirate away the non-ionic surfactant-DMEM/F12 solution. Wash 3 times with 2 ml/dish of DMEM/F12.
   7. Add 2 ml/dish of hESC maintenance medium supplemented with ROCKi and incubate at 37 °C overnight.
   8. 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.
   9. Evaluate the micropatterns using phase contrast imaging and compute the average Brachyury (T) intensity profiles for each pattern as described previously⁸.

Results

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

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
2 mm thick PDMS sheet	Specialty Silicone Products Inc., USA	SSPM823-.005	Used to form reservoir for stencil
120-150 μm thick PDMS sheet	Specialty Silicone Products Inc., USA	SSPM823-.040	Used to form stencil
60 mm Petri dish	Nunc Nunclon Delta	150326	Substrate for micropatterning
Accutase	Accutase, Merck Millipore, Singapore	SCR005	Enzyme to break H9 cells into single cells
Activin	R&D Systems, Singapore	338-AC-010	Growth factor for H9 differentiation
BMP4	R&D Systems, Singapore	338-BP-010	Growth factor for H9 differentiation
Plasma system	Femto Science, Korea	CUTE-MP	For plasma oxidation of stencil
Dispase	StemCell™ Technologies, Singapore	7923	Enzyme used to weaken the cell-ECM adhesion during passaging
DMEM/F12	GIBCO, USA	11330032	Basal medium for H9 cells
FGF2	R&D Systems, Singapore	233–FB–025	Growth factor for H9 differentiation
H9 cell line	WiCell Research Institute, Inc., USA	WA09	Human embryonic stem cells
hESC-qualified basement membrane matrix	Matrigel, BD Biosciences, Singapore	354277	Extra-cellular matrix coating to support growth of H9 cells
Inverted microscope	Leica Microsystems, Singapore	DMi1	For capturing bright-field images
Laser cutter	Epilog Helix 24 Laser System		Used to generate through holes in PDMS sheet
mTeSR™1 medium	StemCell™ Technologies, Singapore	5850	Maintainence medium for H9 cells
PDMS	SYLGARD® 184, Dow Corning Co., USA	3097358-1004	Used for sticking the PDMS stencil and reservior
ROCKi Y27632	Calbiochem, Merck Millipore, Singapore	688000	Maintains H9 cells as single cells
STEMdiff™ APEL™ medium	StemCell™ Technologies, Singapore	5210	Differentiation medium for H9 cells
Polyethylene terephthalate film	SureMark Singapore	SQ-6633	Used to form stencil
Cell culture compatible non-ionic surfactant	Pluronic acid F-127, Sigma, Singapore	P2443	Passivating reagent to repel cell adhesion in non-micropatterned substrates