Work Description

Date: 20 April, 2025

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Dataset Title: Dataset of stem cell colonies differentiating in neural induction medium and code for analysis of resulting fate pattern

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Dataset Contact: Xufeng Xue [email protected]

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Dataset Creators:
Name: Xufeng Xue
Email: [email protected]
Institution: Cincinnati Children's Hospital Medical Center Division of Developmental Biology and UC Department of Pediatrics
ORCID: https://orcid.org/0000-0002-9379-8589

Name: Yubing Sun
Email: [email protected]
Institution: University of Massachusetts (Amherst) Department of Mechanical and Industrial Engineering
ORCID: https://orcid.org/0000-0002-6831-3383

Name: Hayden Nunley
Email: [email protected]
Institution: University of Michigan Biophysics Program
ORCID: https://orcid.org/0000-0002-4634-9422

Name: Agnes M. Resto Irizarry
Email: [email protected]
Institution: University of Michigan Department of Mechanical Engineering
ORCID: https://orcid.org/0000-0002-2292-6029

Name: Ye Yuan
Email: [email protected]
Institution: University of Michigan Department of Mechanical Engineering
ORCID: https://orcid.org/0000-0001-9641-9102

Name: Koh Meng Aw Yong
Email: [email protected]
Institution: University of Michigan Department of Mechanical Engineering
ORCID: https://orcid.org/0000-0001-6295-9645

Name: Yi Zheng
Email: [email protected]
Institution: Syracuse University Department of Biomedical and Chemical Engineering
ORCID: https://orcid.org/0000-0002-2685-3680

Name: Shinuo Weng
Email: [email protected]
Institution: Johns Hopkins University Department of Mechanical Engineering
ORCID: https://orcid.org/0000-0001-7932-913X

Name: Yue Shao
Email: [email protected]
Institution: Tsinghua University Department of Engineering Mechanics
ORCID: https://orcid.org/0000-0001-7548-3551

Name: David K. Lubensky
Email: [email protected]
Institution: University of Michigan Department of Physics and Biophysics Program
ORCID: https://orcid.org/0000-0002-4619-116X

Name: Lorenz Studer
Email: [email protected]
Institution: Memorial Sloan-Kettering Institute Developmental Biology Program and Center of Stem Cell Biology
ORCID: https://orcid.org/0000-0003-0741-7987

Name: Jianping Fu
Email: [email protected]
Institution: University of Michigan Department of Mechanical Engineering, Department of Biomedical Engineering, Department of Cell and Developmental Biology
ORCID: https://orcid.org/0000-0001-9629-6739

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Funding: CMMI 1917304 (NSF), DGE 1256260 (NSF), DMR 2243624 (NSF), 597491-RWC and 1764421 (Simons Foundation/SFARI, NSF), CMMI 1129611 (NSF), CBET 1149401 (NSF), CMMI 1662835 (NSF), 12SDG12180025 (American Heart Association)

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Key Points:
- After optimizing culture conditions, we performed experiments with stem cell colonies on micro-patterned substrates as an in vitro model for an important cell fate decision in vivo.
- We imaged stem cell colonies stained for DAPI (stain for nuclei) and PAX3 (transcription factor for particular fate decision) for different substrate stiffnesses, for different colony diameters, and for different experimental timepoints.
- We developed a method and corresponding code to estimate the concentric width of the outer fate domain (PAX3+) in these images.
- We concluded that the concentric width of the outer fate domain is approximately independent of colony diameter and depends non-monotonically on substrate stiffness.
- Based on a mathematical model of cell-cell mechanical interactions biasing this fate decision, we showed that both findings in the point above are consistent with our model's predictions.

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Research Overview:
Studies of fate patterning during development typically emphasize cell-cell communication via diffusible chemical signals. Recent experiments on stem cell colonies (see Xue et al. Nature Materials 2018), however, suggest that in some cases mechanical stresses, rather than secreted chemicals, enable long-ranged cell-cell interactions that specify positional information and pattern cell fates. The authors of this earlier publication reported a set of in vitro experiments in which uniformly supplied chemical media induced spatially patterned fates in cell colony in a disc geometry. They provided significant evidence that inter-cellular mechanical interactions, as well as mechanical interactions between cells and the substrate, play an important role in this in vitro differentiation process. As part of these experiments, they showed that the concentric width of the outer fate domain is approximately constant as the colony diameter is increased from 300 um to 800 um. In this subsequent publication, we propose a mathematical model for this fate patterning process and explore how the fate pattern depends on substrate stiffness. The experimental images of cell colonies, both for varying cell colony diameter (from Xue et al. Nature Materials 2018) and for varying substrate stiffness (data generated for the publication linked to these data), are provided here. Each example has an image for PAX3 signal (marker for outer fate domain; Paired box gene 3) and an image for DAPI signal (staining nuclei; 4′,6-diamidino-2-phenylindole).

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Methodology:
Embryonic stem cells were seeded on micro-patterned substrates, and uniformly supplied chemical media induced differentiation of these stem cells into one of two cell fates, either neural plate (NP) or neural plate border (NPB), in a spatially patterned way with the NP forming a circular domain near the colony center. To investigate the mechanism of this in vitro pattern formation, we systematically varied the colony size (by micro-patterning), the substrate stiffness (by changing the curing-agent-to-base-monomer-ratio), and the time point at which the colony is imaged relative to the time of cell seeding. For each condition, we assume that the fate pattern is approximately symmetric with respect to rotation about the colony center. To calculate the size of the NPB domain, we extract the average intensity of PAX3 (marker for the NPB fate; Paired box gene 3) as a function of distance from the edge.

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Date Coverage: 2017-2024 (Date range includes dates of experimental data acquisition (2017-2019), and subsequent development and use of analysis code for manuscript preparation (2019-2024)).
Instrument and/or Software specifications: See Methods for experimental instruments, code written in MATLAB

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All folders (except for analyze_pax3_domain) show divisions based on each experimental condition. Each cell colony that is imaged has two TIF images ('DAPI (X).TIF' and 'PAX3 (X).TIF' for each X). The DAPI image is an image of a nuclear stain. The PAX3 image is an image of a stained transcription factor (which is nuclear-localized) for an important cell fate decision. These experimental conditions, including how many images exist for each condition, are outlined below:

- data_glass are images of colonies on a very rigid substrate (glass) at a colony diameter of 500 microns. There are 12 colonies (24 images). Pixel dimensions are [0.6216 um, 0.6216 um].
- 1.5 are images of colonies on a rigid substrate (1:5 is the curing agent to base monomer ratio used in preparing the substrate). There are 19 colonies (38 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns.
- 1.15 are images of colonies on a substrate of intermediate stiffness (1:15 is the curing agent to base monomer ratio used in preparing the substrate). There are 17 colonies (34 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns.
- 1.30 are images of colonies on a substrate of intermediate stiffness (1:30 is the curing agent to base monomer ratio used in preparing the substrate). There are 23 colonies (46 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns.
- 1.45 are images of colonies on a soft, deformable substrate (1:45 is the curing agent to base monomer ratio used in preparing the substrate). There are 17 colonies (34 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns.
- 1.60 are images of colonies on a very soft, deformable substrate (1:60 is the curing agent to base monomer ratio used in preparing the substrate). There are 15 colonies (30 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns.

- 300 um are images of colonies on an intermediate substrate stiffness (1:10 is the curing agent to base monomer ratio used in preparing the substrate). There are 21 colonies (42 images). Pixel dimensions are [0.3115 um, 0.3115 um]. The colony diameter of 300 microns.
- 400 um are images of colonies on an intermediate substrate stiffness (1:10 is the curing agent to base monomer ratio used in preparing the substrate). There are 28 colonies (56 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns.
- 500 um are images of colonies on an intermediate substrate stiffness (1:10 is the curing agent to base monomer ratio used in preparing the substrate). There are 21 colonies (42 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 500 microns.
- 800 um are images of colonies on an intermediate substrate stiffness (1:10 is the curing agent to base monomer ratio used in preparing the substrate). There are 28 colonies (56 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 800 microns.

For the four folders below, the image naming convention is different. 'KSR 20k dayY-XXXX_c2.TIF' is the DAPI image. 'KSR 20k dayY-XXXX_c3.TIF' is the image with a marker for the outer cell fate. Here Y is the day number and XXXX is the index of the colony.
- day2 are images of colonies on an intermediate substrate stiffness (1:10 is the curing agent to base monomer ratio used in preparing the substrate). There are 16 colonies (32 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns. This is at day 2 in the experimental protocol.
- day4 are images of colonies on an intermediate substrate stiffness (1:10 is the curing agent to base monomer ratio used in preparing the substrate). There are 22 colonies (44 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns. This is at day 4 in the experimental protocol.
- day6 are images of colonies on an intermediate substrate stiffness (1:10 is the curing agent to base monomer ratio used in preparing the substrate). There are 15 colonies (30 images). Pixel dimensions are [0.6216 um, 0.6216 um]. The colony diameter of 400 microns. This is at day 6 in the experimental protocol.

The remaining folder is analyze_pax3_domain, which contains the code for analyzing the images above and estimating the concentric width of the outer fate domain.
NOTE: when analyzing the images with this code, make sure to use addpath in MATLAB to add the path to the 'analyze_pax3_domain'
Below is a summary of the main functions and helper functions in this folder:
- pax3_analysis_different_substrates.m is the code for analyzing images in data_glass, 1.5, 1.15, 1.30, 1.45, 1.60 above. The inputs of this function specify the path in which unzipped directory sits, specify the stiffness_number [5, 15, 30, 45, 60, inf], specify whether to generate visualizations (should_I_plot).
- pax3_analysis_different_diameters.m is the code for analyzing images in 300 um, 400 um, 500 um, 800 um. The inputs of this function specify the path in which unzipped directory sits, specify which domain diameter (in um), specify whether to generate visualizations (should_I_plot).
- pax3_analysis_day.m is the code for analyzing images in day2, day4, day6. The inputs of this function specify the path in which unzipped directory sits, specify which day, specify whether to generate visualizations (should_I_plot).

Helper functions include:
- threshold_dapi_image.m for thresholding the dapi image.
- find_boundary_pixels.m extracts which pixels lie at the boundary of the thresholded cell colony
- build_histogram_pixels.m calculates for each pixel a signed distance from the colony boundary, as well as the pax3 and dapi intensities. These are important for estimating the mean pax3 values at different distances from the colony boundary AND/OR colony center
- average_over_vects.m takes the outputs of build_histogram_pixels.m as inputs. It averages pax3 and dapi values at different distances from the colony boundary. These averages are used within the main functions to estimates the concentric width of the outer fate domain.
- ExactMinBoundCircle.m computes exact minimum bounding circle of a 2D point cloud using Welzl's algorithm. See Welzl, E. (1991), 'Smallest enclosing disks (balls and ellipsoids)', Lecture Notes in Computer Science, Vol. 555, pp. 359-370
- FitCircle2Points.m fits a circle to a set of 2 or at most 3 points in 3D space.

- The folder min_circle contains helper functions for fitting the smallest circle in which all segmented nuclei fit.
- - IcosahedronMesh.m generates a triangular surface mesh of an icosahedron.
- - TriQuad.m subdivides triangular mesh using generalized triangular quadrisection.

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Related publication(s):
Nunley H, Xue X, Fu, J, Lubensky, DK. Generation of fate patterns via intercellular forces. BioRxiv 442205 [Preprint]. April 30, 2021 [cited 2025 Feb 20]. Available from: doi: https://doi.org/10.1101/2021.04.30.442205
Xue X, Sun Y, Resto-Irizarry A.M. et al. Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells. Nature Mater 17, 633–641 (2018). https://doi.org/10.1038/s41563-018-0082-9

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Use and Access:
This data set is made available under a Creative Commons Public Domain license (CC BY-NC 4.0).

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To Cite Data:
Nunley, H., Xue, X., Sun, Y., Resto-Irizarry, A. M., Yuan, Y., Yong, K. M. A., Zheng, Y., Weng, S., Shao, Y., Lubensky, D. K., Studer, L., Fu, J. Dataset of stem cell colonies differentiating in neural induction medium and code for analysis of resulting fate pattern [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/axdw-zm41