Spatial patterning of liver progenitor cell differentiation mediated by cellular contractility and Notch signaling
- University of Illinois at Urbana-Champaign, United States
Figures
Figure 1 with 3 supplements
Localized differentiation of liver progenitors in arrayed patterns.
(A) Immunolabeling of BMEL cells for the biliary marker OPN and hepatocyte marker ALB on arrayed collagen I patterns with control IgG or Fc-recombinant Notch ligands DLL1, DLL4, and JAG1. (B) …
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Figure 1—source data 1
Summary table for OPN data in Figure 1B.
- https://doi.org/10.7554/eLife.38536.007
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Figure 1—source data 2
Summary table for ALB data in Figure 1C.
- https://doi.org/10.7554/eLife.38536.008
Figure 1—figure supplement 1
Immunolabeling and quantification of CK19.
(A) Immunolabeling of BMEL cells presented with IgG for the biliary marker CK19. (B) Quantification of CK19 intensity in BMEL cells presented with IgG as a function of radial distance from the …
Figure 1—figure supplement 2
Immunolabeling of OPN and CK19 at and cell density with radius at .
(A) Immunolabeling at of BMEL cells on 30 kPa substrates presented with IgG and DLL4 for the biliary markers OPN and CK19. Scale bar indicates 75 µm. (B) Measurement of cell density with radius …
Figure 1—figure supplement 3
Immunolabeling for OPN with 300, 600, and 1000 µm diameter patterns.
(A) Immunolabeling for OPN of BMEL cells presented with DLL4 on 30 kPa substrates at . Both 300 µm and 1000 µm pattern diameters were included in this experiment in addition to the 600 µm pattern …
Figure 2 with 3 supplements
Peripheral biliary differentiation is dependent on both Notch signaling and substrate stiffness.
(A) Immunolabeling for OPN of BMEL cells presented with DLL4 on 30 kPa and 4 kPa substrates. Cells were treated with vehicle control (DMSO) or an inhibitor of Notch signaling (-secretase inhibitor …
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Figure 2—source data 1
Summary table for OPN data in Figure 2B and Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.38536.013
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Figure 2—source data 2
Summary table for SOX9 and HNF4A data in Figure 2D.
- https://doi.org/10.7554/eLife.38536.014
Figure 2—figure supplement 1
Quantification of OPN+ cell counts in arrayed patterns.
Cells were cultured on 30 kPa and 4 kPa substrates and presented with IgG, DLL1, DLL4, and JAG1. Treatments included vehicle control (DMSO) or an inhibitor of Notch signaling (-secretase inhibitor …
Figure 2—figure supplement 2
Regression analysis of OPN+ and ALB+ cell counts.
Data in Figure 2B were separated into peripheral and central subsets for which dimensionless radius was greater than 0.75 () and less than 0.75 (). Separate multiple regression models were …
Figure 2—figure supplement 3
Quantification of ALB+ cell counts in arrayed patterns.
Cells were cultured on 30 kPa substrates and presented with IgG, DLL1, DLL4, and JAG1. Treatments included vehicle control (DMSO) or an inhibitor of Notch signaling (-secretase inhibitor X, GSI, 5 …
Figure 3 with 2 supplements
TGFβ signaling and cell–cell interaction strength modulate pattern formation.
(A) Immunolabeling for OPN and HNF4A of BMEL cells presented with IgG and DLL4 on 30 kPa substrates. Cultures were treated with vehicle control (DMSO), inhibitor of TGFβ signaling (SB-431542, 10 …
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Figure 3—source data 1
Summary table for OPN+ probability density data in Figure 3B.
- https://doi.org/10.7554/eLife.38536.029
Figure 3—figure supplement 1
Quantification of OPN+ cell counts and HNF4A intensity with SB-431542 and DECMA treatment.
(A) Quantification of OPN+ cell counts on 30 kPa substrates presented with DLL4 after treatment with vehicle control (DMSO) or inhibitor of TGFβ signaling (SB-431542, 10 µm). (B) Quantification of …
Figure 3—figure supplement 2
TGFβ1 induces Notch ligand and receptor expression uniformly.
BMEL cells were cultured on 30 kPa substrates, presented with IgG or DLL4, and treated with TGFβ1 (1.5 ng/ml). Scale bar is 150 µm.
Figure 4 with 2 supplements
Liver progenitors in arrayed patterns generate gradients of traction stress independent of ligand presentation.
(A) Simulated finite element modeling (FEM) stress profile of arrayed patterns. (B) Stress from FEM as a function of radius. (C) Experimental stress profiles obtained by traction force microscopy …
Figure 4—figure supplement 1
Effect of Notch inhibition on experimental stress profiles.
Experimental stress profiles were obtained by TFM of BMEL cells presented with DLL4 on 30 kPa and 4 kPa substrates and treated with vehicle control (DMSO) or GSI (10 µM). Mean ± 95% CI.
Figure 4—figure supplement 2
Effect of TGFβ and E-cadherin inhibition on experimental stress profiles.
Experimental stress profiles were obtained by TFM of BMEL cells on 30 kPa and 4 kPa substrates presented with ligand (IgG and DLL4) and treated with vehicle control (DMSO), inhibitor of TGFβ …
Figure 5
Peripheral differentiation is dependent on a gradient of actomyosin contractility.
(A) Simulated effect of stress gradients of increasing steepness on Notch signaling activity via trans-activation. Darker shades of blue represent increased Notch signaling activity as measured by …
Figure 6
Mechanotransduction by ERK and ROCK modulate peripheral biliary fate.
(A) BMEL cells on 30 kPa substrates were presented with DLL4 and treated with vehicle control (DMSO) and inhibitors of ERK signaling (FR180204, 10 µM) and ROCK (Y-27632, 10 µM). Samples were …
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Figure 6—source data 1
Summary table for OPN+ percentage data in Figure 5D and Figure 6B.
- https://doi.org/10.7554/eLife.38536.020
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Figure 6—source data 2
Summary table for TFM data in Figure 4D, Figure 4—figure supplement 1, Figure 4—figure supplement 2, Figure 5D, and Figure 6C.
- https://doi.org/10.7554/eLife.38536.021
Figure 7 with 1 supplement
Notch ligands Jag1 and Dll1 are both required for segregation of hepatocytic fate centrally and biliary fate peripherally.
(A) Immunolabeling for OPN and HNF4A of BMEL cells presented with DLL4 on 30 kPa substrates. Control cells were transduced with an shRNA vector coding for a non-mammalian target. shJag1 and shDll1 …
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Figure 7—source data 1
Summary table for OPN data in Figure 7C.
- https://doi.org/10.7554/eLife.38536.024
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Figure 7—source data 2
Summary table for SOX9 and HNF4A data in Figure 7D.
- https://doi.org/10.7554/eLife.38536.025
Figure 7—figure supplement 1
Regression analysis of OPN+ cell counts.
Data in Figure 7B were separated into peripheral and central subsets for which dimensionless radius was greater than 0.75 () and less than 0.75 (). Separate multiple regression models were …
Tables
Table 1
List of growth factors and drugs.
https://doi.org/10.7554/eLife.38536.030| Factor or drug | Stock | Target | Manufacturer | Catalog # |
|---|---|---|---|---|
| (–)-Blebbistatin | 1 mg/ml | 25 μM | Cayman Chemical | 13013 |
| DECMA | 1 mg/ml | 10 μg/ml | Fisher Scientific | 50-245-625 |
| FR180204 | 10 mg/ml | 10 μM | Sigma-Aldrich | SML0320 |
| L-685,458 (GSI) | 1 mM | 5 μM | Tocris | 2627 |
| SB-431542 | 10 mM | 10 μM | Sigma-Aldrich | S4317 |
| TGFβ1 | 5 μg/ml | 1.5 ng/ml | R&D Systems | 240-B-002 |
| Y-27632 | 5 mg/ml | 10 μM | Enzo Life Sciences | 270–333-M005 |
Table 2
List of primary antibodies.
https://doi.org/10.7554/eLife.38536.031| Antibody target | Dilution | Manufacturer | Catalog # |
|---|---|---|---|
| ALB | 1/100 | Bethyl | A90-134A |
| CK19 | 1/200 | Abcam | ab52625 |
| Digoxigenin | 1/500 | Roche | 11 093 274 910 |
| HNF4A | 1/200 | Abcam | ab41898 |
| OPN (SPP1) | 1/50 | R&D Systems | AF808 |
| SOX9 | 1/200 | EMD Millipore | AB5535 |
| YAP1 | 1/50 | ProteinTech | 13584–1-AP |
Additional files
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Source code 1
MATLAB function to process TFM images.
- https://doi.org/10.7554/eLife.38536.032
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Source code 2
MATLAB function to analyze and plot TFM data.
- https://doi.org/10.7554/eLife.38536.033
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Source code 3
MATLAB function to retrieve plane data.
- https://doi.org/10.7554/eLife.38536.034
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Source code 4
MATLAB function to retrieve the reader for an image.
- https://doi.org/10.7554/eLife.38536.035
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Source code 5
MATLAB function to draw boundaries around cells automatically.
- https://doi.org/10.7554/eLife.38536.036
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Source code 6
MATLAB function to find the best fit of an ellipse for a given set of points.
- https://doi.org/10.7554/eLife.38536.037
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Source code 7
MATLAB function to rotate and center cell boundaries for averaging.
- https://doi.org/10.7554/eLife.38536.038
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Source code 8
COMSOL FEM simulation of cells on 30 kPa and 4 kPa substrates.
- https://doi.org/10.7554/eLife.38536.039
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Source code 9
MATLAB Notch simulation for no stress (b=0).
- https://doi.org/10.7554/eLife.38536.040
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Source code 10
MATLAB Notch simulation for intermediate stress (b=0.5).
- https://doi.org/10.7554/eLife.38536.041
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Source code 11
MATLAB Notch simulation for high stress (b=5).
- https://doi.org/10.7554/eLife.38536.042
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Transparent reporting form
- https://doi.org/10.7554/eLife.38536.043
