TITLE: A method to recapitulate early embryonic spatial patterning in human embryonic stem cells
AUTHORS: Aryeh Warmflash, Benoit Sorre, Fred Etoc, Eric D. Siggia, Ali H. Brivanlou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture, however, differentiation is typically heterogeneous and spatially disordered. Here we show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, these colonies reproducibly differentiate to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates are defined relative to the boundary with a fixed length scale: small colonies correspond to the outer layers of larger ones. Inhibitory signals limit the range of BMP4 signaling to the colony edge and induce a gradient of Activin/Nodal signaling that patterns mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries, and provide a quantitative assay for studying paracrine signaling.</div></div>

[STEPS]
?. [Cell culture]
For routine culture for maintenance, grow RUES2 cells in HUESM medium that was conditioned by mouse embryonic fibroblasts (MEF-CM) and supplement with  bFGF.
All experiments are performed with the RUES2 hESC line derived in our laboratory and described previously.
?. [Cell culture]
Test cells for mycoplasma prior to beginning experiments and then again at two-month intervals.
?. [Cell culture]
Grow cells on tissue culture dishes coated with Matrigel (BD Biosciences 1:40 dilution).
?. [Cell culture]
Coat dishes in Matrigel at and then incubate at  for  immediately prior to seeding the cells on the surface.
4 °C
37 °C
?. [Cell culture]
For micropatterned cell culture, coat micropatterned glass coverslips (CYTOO) with  Poly-D-Lysine in H2O (PDL; Millipore) for .
?. [Cell culture]
Remove the PDL by serial dilutions without allowing the coverslip to dry (dilution 1:4 in H2O, six times), before performing two complete washes with H2O.
?. [Cell culture]
Incubate coverslips with Matrigel (1:100 dilution in DMEM-F12) at .
4 °C
?. [Cell culture]
Before cell seeding, remove the Matrigel with serial dilutions in ice-cold PBS (dilution 1:4, six times) before 2 complete washes in ice cold PBS.
?. [Cell culture]
Seed the cells which already resuspended in growth medium onto the coverslips immediately following the removal of the PBS.
We found it is important to take care to keep the coverslips at   at all times when in Matrigel solution and to ensure that the coverslips are not allowed to dry at any time after the application of the Matrigel. Both polymerization and drying of the Matrigel lead to inconsistent cell adhesion with cells more likely to detach from the surface during the experiment.
?. [Cell culture]
Perform cell seeding onto micropatterned coverslips as follows:
?. [Cell culture]
Pretreat cells growing in MEF-CM and FGF with the Rock-inhibitor Y27632 (Rock-I; 10uM) for  , wash once with PBS, and dissociate with Trypsin.
?. [Cell culture]
Centrifuge cells and resuspend 5×105cells in  growth medium contain Rock-I and place the entire solution over the coverslip in a  tissue culture dish.
2.5 mL
35
?. [Cell culture]
After , replace the medium with MEF-CM without Rock-I and incubate cells .
?. [Cell culture]
Test the effects of using the chemically defined mTeSR1 culture medium rather than MEF-CM and obtain similar patterns upon treatment with BMP4 to those presented here (Supplementary Fig. 9).
However, we found that MEF-CM better promoted adhesion to the micropatterned surface and it was therefore used in all subsequent experiments.
?. [Cell culture]
Finally test the effects of growing cells on recombinant Laminin521 (Biolamina) rather than Matrigel, and again obtain similar patterns (Supplementary Fig. 9).
?. [Cell culture]
Perform Laminin521 coating of coverslips as follows – Coat coverslips with  of Laminin521 diluted in PBS with calcium and magnesium for  at.
37 °C
?. [Cell culture]
Remove laminin with six serial dilutions in warm PBS (dilution 1:4) before two complete washes in PBS.
?. [Cell culture]
Seed cells as described above for the PDL-Matrigel coated coverslips.
?. [siRNA Experiments]
Passage cells as single cells in Rock-I into  dishes at a density of 200000 cells/dish.
35
?. [siRNA Experiments]
The next day, transfect cells with siRNA (Ambion Silencer select) using RNAiMax (Invitrogen).
?. [siRNA Experiments]
Use the final concentration of siRNA  and  of RNAiMax for each dish.
2.5 µl
?. [siRNA Experiments]
The following day, seed cells onto micropatterned coverslips and perform differentiation experiments as described above.
?. [PDMS microwells]
Design molds to create PDMS wells of controlled diameter and depth using a 3D CAD software (Autodesk Inventor) and then 3D printed (3D Systems Projet 3510 HD Plus printer).
The smallest wells this technique allowed us to make reliably were   in diameter and   in depth. We found that boiling the 3D printed parts in water containing 1% Triton X100 for four hours was necessary to allow the PDMS to cure on the 3D printed parts.
?. [PDMS microwells]
Fill molds with PDMS (10:1 base:reticulant ratio) and degase under vacuum.
?. [PDMS microwells]
In order to create opened wells, place the mold between 2 glass slides on which pressure was applied, typically with a large paper clip.
?. [PDMS microwells]
After PDMS curing at  for several hours, unmold the PDMS wells and boil in distilled water to ensure sterility and that all PDMS was cured.
80 °C
?. [PDMS microwells]
Wash PDMS wells with ethanol and dry them in a cell culture cabinet to keep them sterile.
?. [PDMS microwells]
Stuck wells on the dry cell culture substrate (either glass coverslips, regular tissue culture dish, or optically clear plastic dishes (ibidi)).
?. [PDMS microwells]
Coat the wells with cell adhesion promoting proteins either in a one-step protocol (Laminin521) or a two-step protocol (PDL followed by Matrigel).
?. [PDMS microwells]
To remove bubbles trapped in wells, centrifuge the PDMS well filled with the coating solution (, ).
Centrifuge: 2000 33
?. [Immunofluorescence]
Rinse coverslips once with PBS, fix with 4% paraformaldehyde, rinse twice with PBS, then block and permiabilize with 3% Donkey Serum, 0.1% Triton X-100 in PBS for .
?. [Immunofluorescence]
When performing immunofluorescence for pSmad1, pretreat cells with 1% SDS in PBS for  at  before blocking.
37 °C
?. [Immunofluorescence]
Incubate coverslips with primary antibodies at  (for primary antibodies and dilutions see Supplementary Table 1), wash three times in PBS for  each wash, incubate with secondary antibodies (Alexa488, Alexa555 or Alex647 conjugated (Molecular probes); dilution 1:500) and DAPI nuclear counterstain for , and then wash twice with PBS.
4 °C
?. [Immunofluorescence]
Mount coverslips on slides using Fluoromount-G mounting medium (Southern Biotech).
?. [Imaging]
Acquire all widefield images on an Olympus IX71 inverted microscope with a 20X, 0.75 Na lens.
?. [Imaging]
Utilize tiled image acquisition to acquire images of the entire coverslip (approximately 2500 stage positions/coverslip) in four channels corresponding to DAPI and Alexa488, Alexa555, and Alexa647 conjugated antibodies.
?. [Imaging]
Acquire all confocal images on a Leica SP8 inverted confocal microscope with a 40X, 1.1 Na water immersion objective.
?. [Imaging]
Perform three-dimensional visualization and rendering using Imaris software.
?. [Image analysis]
Perform all image analysis using custom software written in MATLAB.
Since we imaged the entire coverslip, we utilized the fact that we also imaging the regions without cells to create background and normalization images as follows. We first took the pixel-by-pixel minimum and average across all images:m= minj(Ij); a= meanj(Ij)where IJrepresents thejth image. We then usedmas a background imaged and defined a normalization image according to:n= (a- m)/max (a- m)where the max is across all pixels in the image. The mean and normalization images were then smoothed with a Gaussian filter several times the size of a cell diameter and each image was corrected according to:Ij → (Ij- m)./nWhere the “./” represents pixel-wise division. In general, subtractingmremoves the (typically spatially homogenous) camera noise, while dividing byncorrects for any inhomogeneities in the image due to the illumination or other factors.
?. [Image analysis]
After each image is corrected in this way, identify and quantify the cells in each image using an algorithm which we described previously.
?. [Image analysis]
Because larger colonies will span multiple images, perform image alignment and use the resulting overlap between images to put the coordinates of each cell in “coverslip coordinates” resulting in a list of the position of every cell on the coverslip.
?. [Image analysis]
Separate these cells into colonies by computing the alphavol of the points using the MATLAB function alphavol (http://www.mathworks.com/matlabcentral/fileexchange/28851-alpha-shapes) with a radius (rv) of 100 pixels.
This function is similar in concept to a convex hull except that it will form separate boundaries for sets A and B if all points in set A are greater than rv away from all points in set B.
?. [Image analysis]
Having identified colonies, classify them based on their radius. For quantification of immunofluorescence, normalize the intensity to the DAPI intensity in the same cell, or, for proteins that translocate to the nucleus upon activation,  normalize the nuclear intensity to the cytoplasmic intensity in the same cell. Both of these normalizations serve to remove imaging artifacts.
?. [Marker quantification]
Identify individual cells in images and quantify for markers as described previously.
?. [Marker quantification]
Normalize all marker intensities to the DAPI intensity in the same cell with the exception of SMAD2 where we normalized the nuclear SMAD2 intensity to the cytoplasmic intensity in the same cell.
We have found this nuclear to cytoplasmic ratio to be a sensitive metric for signaling activity.
?. [Marker quantification]
Perform radial averages over all colonies on the micropatterned coverslip (n=25 for  colonies, n=144 for  colonies, and n=576 for  colonies).
1000
500
250
?. [Marker quantification]
Perform each marker quantification in at least two different independent experiments.
For   and   colonies, we manually excluded those colonies in which cell seeding was uneven (for example, large empty areas within the micropatterned patch).