A method to recapitulate early embryonic spatial patterning in human embryonic stem cells

Journal name:
Nature Methods
Volume:
11,
Pages:
847–854
Year published:
DOI:
doi:10.1038/nmeth.3016
Received
Accepted
Published online

Abstract

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. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned 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 in early development.

At a glance

Figures

  1. hESCs in the pluripotent state show prepatterning in micropatterned culture.
    Figure 1: hESCs in the pluripotent state show prepatterning in micropatterned culture.

    (a,b) Tiled scans of RUES2 hESCs grown under standard (a) and micropatterned (b) conditions. (c) Single image from the tiled scan with all cells identified computationally. (d) Immunofluorescence analysis of pluripotency marker expression in a micropatterned 1000-μm colony. (e) Quantification of expression of markers of the colony shown in d. Each dot represents a single cell, and the color represents the intensity of the indicated marker normalized to the intensity of the DAPI stain. (f) Quantification of average nuclear intensity from immunofluorescence data for the indicated markers. In all cases, nuclei were identified using the DAPI nuclear counterstain, and the intensity of the indicated markers was normalized to the DAPI intensity. In this and all other figures, graphs represent the mean of 25, 144 and 576 colonies for the colonies of diameters 1,000, 500 and 250 μm, respectively. s.d. between colonies is shown in Supplementary Figure 3. Each marker was quantified in two independent experiments. a.u., arbitrary units. Scale bars, 500 μm.

  2. hESCs differentiated on micropatterns form self-organized spatial patterns.
    Figure 2: hESCs differentiated on micropatterns form self-organized spatial patterns.

    (a,b) The plots show quantified immunofluorescence data for the indicated fate markers. Cells were seeded on micropatterned coverslips, grown overnight and then treated with BMP4 for 42 h. In a and b, all plots show data for the same colony. Each dot corresponds to a single cell. The bottom right panel in a and the right panel in b show an immunofluorescence image of the colony quantified in the plots. (c) Quantification of the mean across colonies of immunofluorescence data for germ layer markers. Numbers of colonies are as in Figure 1f. Error bars for these and other markers can be found in Supplementary Figure 5. Each marker was quantified in three independent experiments. a.u., arbitrary units. (d) Schematic of the results of 42 h of BMP4 treatment in micropatterned culture. TE, trophectoderm; PS, primitive streak. Scale bars, 100 μm.

  3. During differentiation, hESCs undergo an epithelial-to-mesenchymal transition in a region expressing markers of the primitive streak.
    Figure 3: During differentiation, hESCs undergo an epithelial-to-mesenchymal transition in a region expressing markers of the primitive streak.

    (ac) Immunofluorescence staining of an hESC colony for the indicated markers. The magnified image in c shows only the E-CAD stain, for clarity. (d) 3D reconstruction of the primitive streak–like region. 3D segmentation of the DAPI image was performed using custom software written in Matlab, and the resulting segmented nuclei were visualized with Imaris. The cells are color coded according to their position in the z direction for ease of visualization. (eg) Phalloidin staining and immunofluorescence staining for the indicated markers in the upper and lower layers of the primitive streak–like region. In e and g, the blue boxes (left) indicate the region expanded in the individual confocal slices (center and right). Each panel in f is an individual confocal slice. EpCAM, epithelial cell adhesion molecule. Scale bars, 50 μm.

  4. Control of cell fate extends from the edge of the colony.
    Figure 4: Control of cell fate extends from the edge of the colony.

    (a) Immunofluorescence for SOX2 and NANOG in a 1,000-μm colony following 42 h of BMP4 treatment. (b) Quantification of single-cell expression of SOX2 and NANOG from immunofluorescence data in colonies of different size. (c) Immunofluorescence of the indicated markers in a 500-μm colony (bottom right) and quantified marker expression with single-cell resolution. (d) Comparison of BRA expression between 500-μm and 1,000-μm colonies. Plots show mean across colonies (n = 25 for 1,000 μm and n = 144 for 500 μm). This experiment was performed three times. Note the distance scale is inverted relative to previous panels to emphasize control from the boundary. a.u., arbitrary units. Scale bars, 100 μm.

  5. Self-organized signaling responses in micropatterned colonies.
    Figure 5: Self-organized signaling responses in micropatterned colonies.

    (a) Immunofluorescence for pSMAD1 and SMAD2 after 24 h of BMP4 treatment in a micropatterned colony. (b) Quantification of average pSMAD1 intensity and SMAD2 nuclear:cytoplasmic (Nuc:cyt) ratio as a function of time after treatment with 50 ng/ml BMP4 in 1,000-μm colonies (n = 25 colonies; this experiment was performed twice). Representative error bars are in Supplementary Figure 2. (c) Quantification of pSMAD1 and SMAD2 responses in the colony shown in a. Each dot represents a single cell. (d) Immunofluorescence of RUES2 colonies treated with 50 ng/ml BMP4 with or without 10 μM SB431542 (SB; right), and quantified data at single-cell resolution. Scale bars, 100 μm. (e) Quantification of intensity of the indicated markers as a function of distance from the colony center, in the presence of either BMP4 or BMP4 + SB (n = 25 colonies; this experiment was performed twice). a.u., arbitrary units.

  6. TGF-[beta] inhibitors are required for pattern formation.
    Figure 6: TGF-β inhibitors are required for pattern formation.

    (a) Immunofluorescence staining of a 1,000-μm micropatterned colony for the indicated markers, when transfected with a nontargeting siRNA (si-NC) or upon specific gene knockdown. Scale bars, 100 μm. (b,c) Quantification of the mean across colonies (n = 25) of BRA (b) and CDX2 (c). This experiment was performed twice. a.u., arbitrary units.

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Author information

  1. These authors contributed equally to this work.

    • Aryeh Warmflash &
    • Benoit Sorre

Affiliations

  1. Center for Studies in Physics and Biology, The Rockefeller University, New York, New York, USA.

    • Aryeh Warmflash,
    • Benoit Sorre,
    • Fred Etoc &
    • Eric D Siggia
  2. Laboratory of Molecular Vertebrate Embryology, The Rockefeller University, New York, New York, USA.

    • Aryeh Warmflash,
    • Benoit Sorre,
    • Fred Etoc &
    • Ali H Brivanlou

Contributions

A.W. designed and performed experiments, performed analysis and wrote the paper. B.S. designed and performed experiments and contributed to writing the paper. F.E. performed experiments and contributed to writing the paper. E.D.S. designed experiments, performed analysis and wrote the paper. A.H.B. designed experiments and wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

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