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Developmental Biology

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

Published: June 17th, 2016



1Department of Biomedical Engineering, National University of Singapore, 2Singapore Institute of Neurotechnology (SINAPSE), National University of Singapore
* These authors contributed equally

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 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.

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 ....

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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-.......

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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.......

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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 .......

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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.


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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
mTeSR1 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

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