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Engineered signaling centers for the spatially controlled patterning of human pluripotent stem cells

Abstract

Signaling centers, localized groups of cells that secrete morphogens, play a key role in early development and organogenesis by orchestrating spatial cell fate patterning. Here we present a microfluidic approach that exposes human pluripotent stem cell (hPSC) colonies to spatiotemporally controlled morphogen gradients generated from artificial signaling centers. In response to a localized source of bone morphogenetic protein 4 (BMP4), hPSC colonies reproducibly break their intrinsic radial symmetry to produce distinct, axially arranged differentiation domains. Counteracting sources of the BMP antagonist NOGGIN enhance this spatial control of cell fate patterning. We also show how morphogen concentration and cell density affect the BMP response and germ layer patterning. These results demonstrate that the intrinsic capacity of stem cells for self-organization can be extrinsically controlled through the use of engineered signaling centers.

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The authors declare no competing interests.

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Peer review information: Nina Vogt and Tal Nawy were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Acknowledgements

We thank A.G. Elefanty (The Royal Children Hospital) for providing the HES3 MIXL1GFP/+ hESC line and A.H. Brivanlou (The Rockefeller University) for providing the RUES2 hESC line. We thank N. Brandenberg (École Polytechnique Fédérale de Lausanne (EPFL)) for help with diffusion studies and O. Mashinchian (EPFL and Nestlé Institute of Health Sciences) for suggestions on HYS0103 hiPSC line culture. We thank L. Manfrin for helping with tools for the syringe pumps setup. We thank G. Rossi and all the members of the Laboratory of Stem Cell Bioengineering (EPFL) for helpful discussions. A.M. is supported by the EPFL Fellows program co-funded by the Marie Skłodowska-Curie and Horizon2020 (grant agreement 665667). E.R.P. was supported by a Canadian Institute of Health Research (CIHR 358808) and a SystemsX.ch Transition Postdoc Fellowship (51FSP0163584). A.R.V. and F.N. were supported by StoNets, a grant from the Swiss SystemsX.ch (https://www.systemsx.ch) initiative evaluated by the Swiss National Science Foundation. This work was funded by the EU Framework7 HEALTH research program Plurimes (http://plurimes.eu/), the European Union’s Horizon 2020 research and innovation program (INTENS 668294), the Swiss National Science Foundation (grant 310030_179447), the National Center of Competence in Research (NCCR) ‘Bio-Inspired Materials’, and the Personalized Health and Related Technologies Initiative from the ETH Board and EPFL.

Author information

A.M. and M.P.L. conceived the study and designed the experiments; A.M. and Y.T. performed the experiments; Y.T. designed the original diffusion device; A.M., Y.T. and F.R.R. produced the PDMS microfluidic devices; M.P.L. and A.M. wrote the manuscript; E.R.P. performed the fluorescence-intensity-based image analysis; A.M. performed the image analysis for the percentage of marker-positive cells; A.R.V. and F.N. analyzed the mathematical model; and all authors read and approved the final manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Matthias P. Lutolf.

Integrated supplementary information

Supplementary Figure 1 Schemes and characterization of the microfluidic device.

(a) Detail of the two layers for the SU8 mold of the device. In the final device the white parts will be occupied by the PDMS and the colored parts will be empty. (b) Bright-field picture of the gel compartments loaded with colored ink to show how the liquid is retained by the phase-guiding features inside the compartments. (c) Schematic representation of the casting of the PEG-hydrogel (here represented in green) in the gel compartments. (d) Computational simulation of the diffusion of a reference molecule from the source side of the cell chamber. The graphs (right) is the calculated distribution at different time points of a reference molecule at the source edge of the chamber along the blue line reported in the left panel. (e) Computational simulation of the diffusion of a reference molecule from the source side of the cell chamber. The graphs (right) is the calculated distribution at different time points of a reference molecule at the sink edge of the chamber along the blue line reported in the left panel. (f) Schematic representation of the microfluidic device for alternating perfusion. By alternating perfusion through inlet A and inlet B it is possible to temporally control the composition at the source. Inlet C is perfused continuously. (g) Visualization of TexasRed-40kDa Dextran at the specified time points. The device has been perfused intermittently with “medium + TexasRed-40kDa Dextran” through inlet A (6hr), and “medium only” through inlet B (6hr). Inlet C has been perfused continuously with “medium only”. The scheme represents the period of perfusion through inlet A and inlet B. (h) Quantification of the fluorescent signal of TexasRed-40kDa Dextran within the red rectangles reported in “g” for different time-points. Repetition on eight samples showed similar results.

Supplementary Figure 2 Characterization of HES3 MIXL1GFP/+ cells exposed to uniform BMP4 concentration in the microfluidic device.

(a) Combined graph of the quantification of MIXL1, T, SOX17 and CDX2 after 48 hours of constant and uniform exposure of HES3 MIXL1GFP/+ cells to 50ng/ml BMP4 (from Fig. 1k). (b, c, d, e) Fluorescence pictures of indicated markers for HES3 MIXL1GFP/+ cells after 48hours of exposure to constant and uniform 50ng/ml BMP4 in the microfluidic device. These experiments were repeated independently two times (with eight samples per repetition) and showed similar results. (b) Red arrows indicate CDX2-positive MIXL1-negative cells located at the edges of the colony. White arrows indicate MIXL1/CDX2 double positive cells. (c) The vast majority of the SOX17-positive cells co-express CDX2. White arrows indicate CDX2-positive/SOX17-negative cells. Red arrows indicate SOX17-positive/CDX2-negative cells. (d) Red arrows indicate MIXL1/SOX17 double negative cells present in patches at the edges of the colony. White arrows indicate MIXL1/SOX17 double-positive cells. (e) White arrows indicate cells negative for MIXL1, T and SOX2 with wider internuclear distances located at the edges of the colony in patches of various sizes. (f) Scheme that approximates the distribution of markers in a colony of HES3 MIXL1GFP/+ cells after 48hours of constant and uniform exposure to 50ng/ml BMP4. (g) Computational simulation showing the local percentage of phospho-SMAD1 positive cells in a colony under a uniform and constant concentration of BMP4 at different time points. Top, distribution of exogenously provided BMP4. Bottom, local percentage of phospho-SMAD1 positive cells. Scale bar 200μm.

Supplementary Figure 3 Characterization of HES3 MIXL1GFP/+ cells exposed to a localized source of 50ng/ml BMP4.

(a) Time-points of computational simulation showing the local concentration of exogenously provided BMP4 diffusing from a 50ng/ml source located at the top of the colony. Refer to Fig. 2a for the associated simulated response of BMP signaling (local percentage of pSMAD1 in the colony). (b) Boxplots of the distribution of HES3 MIXL1GFP/+ cells positive for the reported markers along the axis of the cell chamber after 48 hours of exposure to a localized 50ng/ml BMP4 source (n=8 independent samples). (c) Combined graph of the quantification of MIXL1, T, SOX17 and CDX2 in HES3 MIXL1GFP/+ cells after 48 hours of exposure to a localized 50ng/ml BMP4 source (from Fig. 2e). In the boxplots the center line represents the median, the box limits represent the first and third quartiles, the whiskers represent the upper and lower extremes of the distribution, while white circles represent outliers (defined as a data point located more than 1.5 times outside the interquartile range above the upper quartile or below the lower quartile). Single data points are also shown in red. (d) Scheme that approximates the distribution of markers in a colony of HES3 MIXL1GFP/+ cells after 48 hours of exposure to a localized 50ng/ml source of BMP4.

Supplementary Figure 4 Characterization of H1, RUES2 and HYS0103 cells exposed to a localized source of 50ng/ml BMP4

(a) Representative fluorescence pictures for indicated markers in H1 cells after 48 hours of exposure to a localized 40ng/ml source of BMP4 in the microfluidic device. This experiment was repeated independently two times (with four samples per repetition) and showed similar results. (b) Fluorescence pictures for indicated markers in RUES2 cells after 48 hours of exposure to a localized 80ng/ml source of BMP4 in the microfluidic device. This experiment was repeated independently two times and showed similar results. (c) Mean (line) and standard error of the mean (light-colored area) of normalized fluorescence intensity for the indicated markers along the source-sink axis of multiple cell chambers of RUES2 cells after 48 hours of exposure to a localized 80ng/ml BMP4 source (n=7 independent samples). (d) Fluorescence pictures for indicated markers in HYS0103 hiPSC cells after 48 hours of exposure to a localized 20ng/ml source of BMP4 in the microfluidic device. This experiment was repeated independently two times and showed similar results. (e) Mean (line) and standard error of the mean (light-colored area) of normalized fluorescence intensity for the indicated markers along the source-sink axis of multiple cell chambers of HYS0103 hiPSC cells after 48 hours of exposure to a localized 20ng/ml BMP4 source (n=7 independent samples). Scale bar 200μm.

Supplementary Figure 5 Characterization of HES3 MIXL1GFP/+ cells exposed to a localized BMP4 source in a smaller cell chamber

(a) Design of the two layers for the SU8 mold of the device with 500μm-diameter cell chambers; detail of a single unit. In the final device the white parts will be occupied by the PDMS and the colored parts will be empty. (b) Computational simulation of the diffusion of a reference molecule from the source side of the 500μm-diameter cell chamber after 48 hours of perfusion. (c) Calculated distribution of the reference molecule along the red line reported in Supplementary Fig. 5b at the indicated time points. (d) Fluorescence pictures for indicated markers in HES3 MIXL1GFP/+ cells after 48 hours of exposure to a localized 40ng/ml source of BMP4 in the 500μm-diameter chambers microfluidic device. This experiment was repeated independently two times and showed similar results. (e) Mean (line) and standard error of the mean (light-colored area) of normalized fluorescence intensity for the indicated markers along the source-sink axis of multiple cell chambers of HES3 MIXL1GFP/+ cells after 48 hours of exposure to a localized 40ng/ml BMP4 source in 500μm-diameter chambers (n=8 independent samples).

Supplementary Figure 6 Characterization of HES3 MIXL1GFP/+ cells exposed to a localized source of 250ng/ml BMP4.

(a) Boxplots of the distribution of HES3 MIXL1GFP/+ cells positive for the reported markers along the axis of the cell chamber after 48 hours of exposure to a localized 250ng/ml BMP4 source (n=8 independent samples). In the boxplots the center line represents the median, the box limits represent the first and third quartiles, the whiskers represent the upper and lower extremes of the distribution, while white circles represent outliers (defined as a data point located more than 1.5 times outside the interquartile range above the upper quartile or below the lower quartile). Single data points are also shown in red. (b) Orthogonal projection of DAPI signal for HES3 MIXL1GFP/+ cells at the source side after 48 hours of exposure to a localized 250ng/ml BMP4 source. This experiment was repeated three times and showed similar results.

Supplementary Figure 7 Effect of cell density on the patterning of hESC induced by a localized BMP4 source.

(a) Computational simulation showing the local percentage of phospho-SMAD1 positive cells in a colony with an initially high cell density exposed to a localized BMP4 source of 50ng/ml. (b) Representative pictures (DAPI) of HES3 MIXL1GFP/+ cell suspensions at different concentrations 30 hours after the loading in the cell chambers. (c) Boxplots of the measured cell density for the different cell suspension concentrations (n=8 independent samples per condition). For simplicity the conditions are named “Low”, “Intermediate” and “High” density. ANOVA and subsequent multiple two-tailed t-test Benjamini-Hochberg corrected were performed. (d) Boxplots of the distribution of HES3 MIXL1GFP/+ cells positive for the reported markers along the axis of the cell chamber after 48 hours of exposure to a localized 50ng/ml BMP4 source starting with high cell density (n=8 independent samples). (e) Boxplots of the distribution of HES3 MIXL1GFP/+ cells positive for the reported markers along the axis of the chamber after 48 hours of exposure to a localized 250ng/ml BMP4 source starting with high cell density (n=6 independent samples). For all the boxplots the center line represents the median, the box limits represent the first and third quartiles, the whiskers represent the upper and lower extremes of the distribution, while white circles represent outliers. Single data points are also shown in red. (f) Time points from a computational simulation showing the local percentage of phospho-SMAD1 positive cells in a colony with an initially high cell density exposed to a localized BMP4 source of 250ng/ml. (g) Representative fluorescence pictures for indicated markers in HES3 MIXL1GFP/+ cells exposed 48 hours to a localized 50ng/ml source of BMP4 in the microfluidic device starting with low cell density. This experiment was repeated two times on a total of 8 samples and showed similar results. Scale bar 200μm.

Supplementary Figure 8 Effect of counteracting BMP4 and NOGGIN sources on the patterning of HES3 MIXL1GFP/+ cells.

(a) Boxplots of the distribution of HES3 MIXL1GFP/+ cells positive for the reported markers along the axis of the cell chamber after 48 hours of exposure to counteracting sources of 50ng/ml BMP4 and 200ng/ml NOGGIN (n=8 independent samples). In the boxplots the center line represents the median, the box limits represent the first and third quartiles, the whiskers represent the upper and lower extremes of the distribution, while white circles represent outliers (defined as a data point located more than 1.5 times outside the interquartile range above the upper quartile or below the lower quartile). Single data points are also shown in red. (b) Combined graph of the quantification of MIXL1, T, SOX17 and CDX2 for of HES3 MIXL1GFP/+ cells after 48 hours of exposure to counteracting sources of 50ng/ml BMP4 and 200ng/ml NOGGIN (from Fig. 6d). (c) Scheme that approximates the distribution of markers in a colony of HES3 MIXL1GFP/+ cells after 48 hours of exposure to counteracting sources of 50ng/ml BMP4 and 200ng/ml NOGGIN.

Supplementary Figure 9 Effect of counteracting BMP4 and NOGGIN sources on the patterning of H1, RUES2 and HYS0103 cells.

(a) Representative fluorescence pictures for indicated markers in H1 cells after 48 hours of exposure to counteracting sources of 40ng/ml BMP4 and 200ng/ml NOGGIN in the microfluidic device. This experiment was repeated independently two times (with four samples per repetition) and showed similar results. (b) Fluorescence pictures for indicated markers in RUES2 cells after 48 hours of exposure to counteracting sources of 80ng/ml BMP4 and 200ng/ml NOGGIN in the microfluidic device. This experiment was repeated independently two times and showed similar results. (c) Mean (line) and standard error of the mean (light-colored area) of normalized fluorescence intensity for the indicated markers along the source-sink axis of multiple cell chambers of RUES2 cells after 48 hours of exposure to counteracting sources of 80ng/ml BMP4 and 200ng/ml NOGGIN (n=7 independent samples). (d) Fluorescence pictures for indicated markers in HYS0103 hiPSC cells after 48 hours of exposure to counteracting sources of 20ng/ml BMP4 and 200ng/ml NOGGIN in the microfluidic device. This experiment was repeated independently two times and showed similar results. (e) Mean (line) and standard error of the mean (light-colored area) of normalized fluorescence intensity for the indicated markers along the source-sink axis of multiple cell chambers of HYS0103 hiPSC cells after 48 hours of exposure to counteracting sources of 20ng/ml BMP4 and 200ng/ml NOGGIN (n=6 independent samples). Scale bar 200μm.

Supplementary Figure 10 Effect of counteracting BMP4 and NOGGIN sources on the patterning of HES3 MIXL1GFP/+ cells in smaller chamber.

(a) Fluorescence pictures for indicated markers in HES3 MIXL1GFP/+ cells after 48 hours of exposure to counteracting sources of 40ng/ml BMP4 and 200ng/ml NOGGIN in the 500μm-diameter chambers microfluidic device. This experiment was repeated independently two times on a total of 8 samples and showed similar results. (b) Mean (line) and standard error of the mean (light-colored area) of normalized fluorescence intensity for the indicated markers along the source-sink axis of multiple cell chambers of HES3 MIXL1GFP/+ cells after 48 hours of exposure counteracting sources of 40ng/ml BMP4 and 200ng/ml NOGGIN in 500μm-diameter chambers (n=8 independent samples).

Supplementary information

Supplementary Information

Supplementary Figures 1–10

Reporting Summary

Supplementary Video 1

Dynamics of MIXL1 pattern formation upon 10 ng/ml BMP4 localizedsource (3–48 h). Time-lapse of HES3 MIXL1GFP/+ from 3 h until 48 h of exposure to localized BMP4 with a source concentration of 10 ng/ml. The source is at the top. BF on the left and GFP (MIXL1) is on the right. Pictures were acquired every hour. This experiment was performed on four samples that showed similar results.

Supplementary Video 2

Dynamics of MIXL1 pattern formation upon 10 ng/ml BMP4 localized source (24–65 h). Time-lapse of HES3 MIXL1GFP/+ from 24 h until 65 h of exposure tolocalized BMP4 with a source concentration of 10 ng/ml. The source is at the top. BF on the left and GFP (MIXL1) is on the right. Pictures were acquiredevery hour. This experiment was performed on four samples that showed similar results.

Supplementary Video 3

Supplementary Video 3 Dynamics of MIXL1 pattern formation upon 50 ng/ml BMP4 localized source (3–48 h). Time-lapse of HES3 MIXL1GFP/+ from 3 h until 48 h of exposure tolocalized BMP4 with a source concentration of 50 ng/ml. The source is at the top. BF on the left and GFP (MIXL1) is on the right. Pictures were acquired every hour. This experiment was performed on four samples that showed similar results.

Supplementary Video 4

Dynamics of MIXL1 pattern formation upon 50 ng/ml BMP4 localized source (24–65 h). Time-lapse of HES3 MIXL1GFP/+ from 24 h until 65 h of exposure to localized BMP4 with a source concentration of 50 ng/ml. The source is at the top. BF on the left and GFP (MIXL1) is on the right. Pictures were acquired every hour. This experiment was performed on four samples that showed similar results.

Supplementary Video 5

Dynamics of MIXL1 pattern formation upon 250 ng/ml BMP4 localized source (3–48 h). Time-lapse of HES3 MIXL1GFP/+ from 3 h until 48 h of exposure to localized BMP4 with a source concentration of 250 ng/ml. The source is at the top. BF on the left and GFP (MIXL1) is on the right. Pictures were acquired every hour. This experiment was performed on four samples that showed similar results.

Supplementary Video 6

Dynamics of MIXL1 pattern formation upon 250 ng/ml BMP4 localized source (24–65 h). Time-lapse of HES3 MIXL1GFP/+ from 24 h until 65 h of exposure to localized BMP4 with a source concentration of 250 ng/ml. The source is at the top. BF on the left and GFP (MIXL1) is on the right. Pictures were acquired every hour. This experiment was performed on four samples. A certain variability in the behavior of the four samples was observed: two of them showed MIXL1 expression extending at the center of the colony (as the one shown in this video), while the center of the colony was preserved from MIXL1 expression in the other two samples.

Supplementary Data

Layout of the design of the microfluidic devices used in this study for the production of the photolithography masks (.cif file format). The masks can be used to fabricate the two-layer SU8 mold for the production of the PDMS device.

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Fig. 1: Design and characterization of the microfluidic device.
Fig. 2: A localized source of BMP4 biases the positioning of hPSC-derived germ layers.
Fig. 3: Effects of the BMP4 source concentration on hPSC patterning.
Fig. 4: Effects of the BMP4 source concentration on hPSC patterning dynamics.
Fig. 5: The balance between cell density and BMP4 source concentration is relevant for the patterning outcome.
Fig. 6: Counteracting sources of BMP4 and its inhibitor NOGGIN reinforce the spatial control on hPSC patterning.
Supplementary Figure 1: Schemes and characterization of the microfluidic device.
Supplementary Figure 2: Characterization of HES3 MIXL1GFP/+ cells exposed to uniform BMP4 concentration in the microfluidic device.
Supplementary Figure 3: Characterization of HES3 MIXL1GFP/+ cells exposed to a localized source of 50ng/ml BMP4.
Supplementary Figure 4: Characterization of H1, RUES2 and HYS0103 cells exposed to a localized source of 50ng/ml BMP4
Supplementary Figure 5: Characterization of HES3 MIXL1GFP/+ cells exposed to a localized BMP4 source in a smaller cell chamber
Supplementary Figure 6: Characterization of HES3 MIXL1GFP/+ cells exposed to a localized source of 250ng/ml BMP4.
Supplementary Figure 7: Effect of cell density on the patterning of hESC induced by a localized BMP4 source.
Supplementary Figure 8: Effect of counteracting BMP4 and NOGGIN sources on the patterning of HES3 MIXL1GFP/+ cells.
Supplementary Figure 9: Effect of counteracting BMP4 and NOGGIN sources on the patterning of H1, RUES2 and HYS0103 cells.
Supplementary Figure 10: Effect of counteracting BMP4 and NOGGIN sources on the patterning of HES3 MIXL1GFP/+ cells in smaller chamber.