Endpoint gastruloid-on-microraft analysis (EGOMRA)
Data files
Jun 04, 2025 version files 12.76 GB
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EGOMRA_Images.zip
12.76 GB
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EGOMRA.zip
481.53 KB
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README.md
17.85 KB
Abstract
Although adherent two-dimensional human gastruloids are highly reproducible and provide insights into early embryogenesis, no available automated technology can screen and sort large numbers of these near-millimeter-sized complex structures for large-scale assays. Consequently, we developed a microraft array-based technology to perform image-based assays of large numbers of fixed or living gastruloids and sort individual gastruloids for downstream assays, such as gene expression analysis. We created datasets with metrics describing the microraft arrays and the templates used to make the arrays, micropatterning of extracellular matrix (ECM) to form gastruloids, gastruloid formation, and gastruloids formed on both multi-well plates and microraft arrays. The microraft array platform was used to assay individual euploid and aneuploid (possessing abnormal numbers of chromosomes) gastruloids with clear phenotypic and gene expression differences. Both noggin (NOG) and keratin 7 (KRT7), two genes involved in spatial patterning within gastruloids, were upregulated in aneuploid relative to that in the euploid gastruloids. Moreover, relative NOG and KRT7 expressions were negatively correlated with DNA/area. The microraft arrays will empower novel screens of single gastruloids for a better understanding of key mechanisms underlying phenotypic differences between gastruloids.
This repository hosts datasets describing metrics for microraft arrays, micropatterning, and two-dimensional human gastruloid culture for the following manuscript in APL Bioengineering: Development of Large Scale Gastruloid Array to Identify Aberrant Developmental Phenotypes.
We submitted raw data (EGOMRA.zip) and images of both fixed/living gastruloids on microrafts (EGOMRA Images.zip).
Files and variables
File: EGOMRA.zip
Automated Microraft Image Analysis Metrics
Metrics describing the accuracy of automated grading of gastruloid placement on microrafts.
Automated Microraft Image Analysis Metrics.csv
Array Type: fixed or living gastruloid array
Replicate: replicate number
Positive: number of identified centered gastruloids
Negative: number of identified not centered gastruloids
False Positive: number of falsely identified centered gastruloids
False Negative: number of falsely identified not centered gastruloids
True Positive: number of correctly identified centered gastruloids
True Negative: number of correctly identified not centered gastruloids
Positive: number of true centered gastruloids
Negative: number of true not centered gastruloids
Sensitivity: percentage of correctly identified out of true centered gastruloids
Specificity: percentage of correctly identified out of true not centered gastruloids
Automated Microraft Image Analysis Metrics_Means.csv
Array Type: fixed or living gastruloid array
Summary: mean or standard deviation (stdev) across replicates for array type
Sensitivity: percentage of correctly identified out of true centered gastruloids
Specificity: percentage of correctly identified out of true not centered gastruloids
Automated Microraft Imaging Times
Time durations of array imaging.
Automated Microraft Imaging Times.csv
Array Type: fixed or living gastruloid array
Num Channels: total number of channels
Replicate: replicate number
Scan_Time (s): time to scan entire array in s
Scan_Time (min): time to scan entire array in min
Scan_Time Per Channel (min): time to scan entire array per channel in min
Automated Microraft Imaging Times_Means.csv
Array Type: fixed or living gastruloid array
Num Channels: total number of channels
Summary: mean or standard deviation (stdev) across replicates
Scan_Time (min): time to scan entire array in min
Scan_Time Per Channel (min): time to scan entire array per channel in min
Automated Microraft Isolation Successes
Success rates of microraft release & collection.
Automated Microraft Isolation Successes.csv
Variable: microraft release or collection
Replicate/Summary: replicate number or summary [mean or standard deviation (stdev)] across replicates
Count: number of microrafts attempted to be isolated
Success %: percent success rate
Automated Microraft Isolation Times
Time durations of microraft isolations.
Automated Microraft Isolation Times.csv
Replicate: replicate number
FindRaftTime: time to locate target microraft in s
Count: number of needle actuations required to release microraft from array
NeedleTimes_{actuation number}: time for single needle actuation in s
CollectionTimes: time to collect and deposit microraft in collection plate in s
SingleRaftTimes: total time to locate, release, and collect each microraft in s
Automated Microraft Isolation Times_Means.csv
Variable: FindRaftTimes, NeedleTimes, CollectionTimes, SingleRaftTimes (see above)
Replicate/Summary: replicate number or summary [mean or standard deviation (stdev)] across replicates
Value: value of variable
Bead Distribution
Area and distance to microraft edge of superparamagnetic beads within individual microrafts.
Bead Distribution.csv
Replicate: replicate number
ArrayLoc: array location (middle or peripheral)
Raft: microraft number
RaftArea: area of microraft in px^2
RaftCen: pixel coordinates (x, y) of microraft centroid within image
BeadArea: area of beads in px^2
NormBeads: area of beads normalized by area of microraft
FromEdge_Mean: mean distance of bead pixels to microraft edge in px
FromEdge_Stdev: standard deviation of distance of bead pixels to microraft edge in px
Bead Distribution_Means.csv
Replicate: replicate number or across all replicates (all)
ArrayLoc: array location (middle or peripheral)
NormBead_Mean: mean normalized area of beads
NormBead_Stdev: standard deviation for normalized area of beads
FromEdge_Mean: mean distance of bead pixels to microraft edge in px
FromEdge_Stdev: standard deviation for distance of bead pixels to microraft edge in px
Fixed Gastruloid Fluor Analysis
Features extracted from high-quality fixed gastruloids.
Fixed Gastruloid Fluor Analysis.csv
Replicate: replicate number
Raft: microraft number
Variable: ThreshVal_{channel} [intensity threshold value used in segmentation]; FArea_{channel} [fluorescence area of segmented region in px^2]; NormFArea_{channel} [fluorescence area of segmented region normalized to area of gastruloid]; Eccen_{channel} [eccentricity of segmented region]; Radius_{channel} [radius of segmented region in px]
Value: value of variable
Fixed Gastruloid Fluor Analysis_Means.csv
Replicate: replicate number or across all replicates (all)
Count: sample size (number of microrafts for replicate-level means or number of replicates)
Variable: ThreshVal_{channel} [intensity threshold value used in segmentation]; FArea_{channel} [fluorescence area of segmented region in px^2]; NormFArea_{channel} [fluorescence area of segmented region normalized to area of gastruloid]; Eccen_{channel} [eccentricity of segmented region]; Radius_{channel} [radius of segmented region in px]
Mean: replicate-level mean or mean across replicates
Stdev: replicate-level standard deviation or standard deviation across replicates
Living Gastruloid Fluor Analysis
Features extracted from high-quality living gastruloids.
Living Gastruloid Fluor Analysis.csv
Replicate: replicate number
Condition: euploid or aneuploid
Raft: microraft number
Variable: Eccen [eccentricity of calcein stain]; Farea_Hull [fluorescence area of calcein stain in px^2]; NormDNA [fluorescence area of Hoechst 33342 stain normalized by fluorescence area of calcein stain]; Radius [radius of calcein stain in px]
Value: value of variable
Living Gastruloid Fluor Analysis_Means.csv
Replicate: replicate number or across all replicates (all)
Condition: euploid or aneuploid
Count: sample size (number of microrafts for replicate-level means or number of replicates)
Variable: Eccen [eccentricity of calcein stain]; FArea_Hull [fluorescence area of calcein stain in px^2]; NormDNA [fluorescence area of Hoechst 33342 stain normalized by fluorescence area of calcein stain]; Radius [radius of calcein stain in px]
Mean: replicate-level mean or mean across replicates
Stdev: replicate-level standard deviation or standard deviation across replicates
SEM: standard error across replicates (only calculated across replicates, NaN otherwise)
Living Gastruloid NOG & KRT7 qPCR
Measurements and calculations of noggin (NOG) and keratin 7 (KRT7) gene expression via qPCR. “Living Gastruloid NOG & KRT7 qPCR_Filtered.csv” contains manually filtered data (outlier GAPDH CT values and CT values exceeding 38 were excluded from furth analysis) from “Living Gastruloid NOG & KRT7 qPCR.csv”.
Living Gastruloid NOG & KRT7 qPCR.csv
Plate: individual plates for NOG and KRT7 assays (both plates contained GAPDH assays)
Well Position: well positions for each sample
Sample Name: {replicate number}_{condition: euploid (Eu) or aneuploid (An)}_{sample number}
Target Name: gene expression assay target (GAPDH, NOG, KRT7)
CT: cycle threshold
Living Gastruloid NOG & KRT7 qPCR_Filtered.csv
Plate: individual plates for NOG and KRT7 assays (both plates contained GAPDH assays)
Well Position: well positions for each sample
Sample Name: {replicate number}_{condition: euploid (Eu) or aneuploid (An)}_{sample number}
Target Name: gene expression assay target (GAPDH, NOG, KRT7)
CT: cycle threshold
Living Gastruloid NOG & KRT7 qPCR_RelativeExpression.csv
Samp_Name: {replicate number}_{condition: euploid (Eu) or aneuploid (An)}_{sample number}
Replicate: replicate number
Condition: euploid (Eu) or aneuploid (An)
Samp_Num: sample number
Target: gene expression assay target (NOG or KRT7)
dCT: delta cycle threshold (CT) calculated by subtracting CT by average CT value for GAPDH
Average dCT: average dCT for euploid condition (control) across sample numbers for same replicate
ddCT: delta dCT calculated by subtracting each dCT from “Average dCT”
2^-ddCT: calculated for each ddCT value to determine relative expression level (each cycle is two-fold difference)
NormDNA: normalized DNA content (fluorescence area of Hoechst stain divided by total area of gastruloid)
Living Gastruloid NOG & KRT7 qPCR_RelativeExpression_Means.csv
Replicate: replicate number or across all replicates (all)
Condition: euploid (Eu) or aneuploid (An)
Target: gene expression assay target (NOG or KRT7)
Mean: replicate-level mean or mean across replicates for relative expression level (2^-ddCT)
Stdev: replicate-level standard deviation or standard deviation across replicates for relative expression level (2^-ddCT)
Microraft Dimensions
Measurements of microraft dimensions (edge length, flatness, thickness).
Microraft Dimensions_Edge.csv
Replicate: replicate number
ArrayLoc: array location (middle or peripheral)
Raft: microraft number
Edge: edge number
EdgeLen: edge length in µm
Microraft Dimensions_Flatness.csv
Replicate: replicate number
ArrayLoc: array location (middle or peripheral)
Raft: microraft number
RaftLen: microraft edge length from cross-section view in µm
FlatLen: length of flat portion of microraft from cross-section view in µm
FlatProp: flat proportion calculated by dividing “FlatLen” by “RaftLen”
Microraft Dimensions_Thickness.csv
Replicate: replicate number
ArrayLoc: array location (middle or peripheral)
Raft: microraft number
Orthogonal: cross-sectional view [XZ (1) or YZ (2)]
RaftLoc: microraft location (center or edge)
Thickness: thickness in µm
Microraft Dimensions_Means.csv
Variable: Edge [edge length in µm]; Flatness [length of flat portion of microraft divided by total microraft edge length]; Thickness [thickness of microraft in µm]
Replicate: replicate number or across all replicates (all)
ArrayLoc: array location (middle, peripheral, Total)
Raft: microraft number or across all microrafts (all)
RaftLoc: microraft location (center or edge) only for thickness, NaN otherwise
Mean: replicate-level mean or mean across replicates
Stdev: replicate-level standard deviation or standard deviation across replicates
Microraft Grading Count
Numbers of microrafts graded as centered [centered gastruloid on microraft], missing [missing microraft], empty [empty microraft], or border [gastruloid contacting border of microraft].
Microraft Grading Count.csv
Replicate: replicate number
ArrType: array type (fixed or living)
Grade: microraft grade (see above)
ArrLoc: array location (middle, peripheral, total)
Count: number of microrafts for each grade
PercRaft: percentage of microrafts for each grade (out of 529 microrafts per replicate)
Microraft Grading Count_Means.csv
Grade: microraft grade (see above)
ArrLoc: array location (middle, peripheral, total)
Mean: mean percentage of microrafts for each grade (out of 529 microrafts per replicate)
Stdev: standard deviation for percentage of microrafts for each grade
Patterning Metrics
Metrics describing AF647-labeled Matrigel micropatterns on microrafts.
Patterning Metrics_Diameter.csv
Replicate: replicate number
ArrayLoc: array location (middle or peripheral)
Pattern: micropattern number
Diameter: diameter of micropattern in µm
Patterning Metrics_Diameter_Means.csv
Replicate: replicate number or across all replicates (all)
ArrayLoc: array location (middle, peripheral, total)
Mean Diameter: replicate-level mean or mean across replicates for micropattern diameter in µm
Stdev Diameter: replicate-level standard deviation or standard deviation across replicates for micropattern diameter in µm
SEM Diameter: replicate-level standard error or standard error across replicates for micropattern diameter in µm (only calculated for across replicates, NaN otherwise)
Mean Radius: replicate-level mean or mean across replicates for micropattern radius in µm (only calculated for across replicates, NaN otherwise)
Stdev Radius: replicate-level standard deviation or standard deviation across replicates for micropattern radius in µm (only calculated for across replicates, NaN otherwise)
SEM Radius: replicate-level standard error or standard error across replicates for micropattern radius in µm (only calculated for across replicates, NaN otherwise)
Patterning Metrics_FromCenter.csv
Replicate: replicate number
ArrayLoc: array location (middle or peripheral)
Pattern: micropattern number
Distance: distance along line drawn across widest part of micropattern in µm
Intensity: intensity value in arbitrary units along line drawn across widest part of micropattern
Patterning Metrics_FromCenter_Smooth.csv
Empty cells because of rolling average calculation (50 sliding window size).
Replicate: replicate number
ArrayLoc: array location (middle or peripheral)
Pattern: micropattern number
Distance: distance along line drawn across widest part of micropattern in µm
Intensity: intensity value in arbitrary units along line drawn across widest part of micropattern
CenDist: half-length of line drawn across widest part of micropattern in µm
FromCenDist: distance from the center of line drawn across widest part of micropattern in µm
CenInt: average intensity within 10 µm of micropattern center
NormInt: normalized intensity by dividing intensity value by “CenInt” for each micropattern
NormInt_Smooth: “NormInt” smoothed via rolling average (50 sliding window size)
Patterning Metrics_Grading.csv
Replicate: replicate number
Grade: micropattern grade [Centered (centered micropattern within microraft), Missing (missing micropattern), Border (micropattern contacting microraft border)]
ArrayLoc: array location (middle, peripheral, total)
Count: number of micropatterns for each grade
PerRaft: percentage of microrafts for each grade (out of 529 microrafts per replicate)
Patterning Metrics_Grading_Means.csv
Grade: micropattern grade [Centered (centered micropattern within microraft), Missing (missing micropattern), Border (micropattern contacting microraft border)]
ArrayLoc: array location (middle, peripheral, total)
Mean: mean percentage of microrafts for each grade (out of 529 microrafts per replicate)
Stdev: standard deviation for percentage of microrafts for each grade
Plate vs Microraft Gastruloid Grading Count
Numbers of microrafts with fully developed gastruloids on multi-well plates and microraft arrays.
Plate vs Microraft Gastruloid Grading Count.csv
Replicate: replicate number
Substrate: substrate for gastruloid formation (plate or microraft)
ArrayLoc: array location (middle or peripheral)
Grade: gastruloid grade (good or malformed)
Count: number of microrafts for each grade
PercRaft: percentage of microrafts for each grade (out of 16 per array location per replicate)
Template Dimensions
Measurements of master template dimensions.
Template Dimensions_Gap.csv
Template: template number
Gap: gap number
Start: starting coordinate along x-axis of template cross-section in mm
End: ending coordinate along x-axis of template cross-section in mm
Gap Size: gap size calculated by subtracting “End” by “Start” in µm
Template Dimensions_Height.csv
Template: template number
Feature: feature number
Height: height of feature in µm
Template Dimensions_Width.csv
Template: template number
Feature: feature number
Start: starting coordinate along x-axis of template cross-section in mm
End: ending coordinate along x-axis of template cross-section in mm
Width: Width of feature calculated by subtracting “End” by “Start” in µm
Template Dimensions_Means.csv
Variable: Gap [gap width in µm], Height [feature height in µm], Width [feature width in µm]
Template: template number or across all templates (all)
Mean: replicate-level mean or mean across templates
Stdev: replicate-level standard deviation or standard deviation across templates
File: EGOMRA Images.zip
Individual microraft (raft) images [see “Individual Raft Images” subdirectory] and stitched composite images [see “Stitched Images” subdirectory] of array for both fixed and living gastruloid analysis. For each replicate of the living gastruloids “Array 1” and “Array 2” refer to the euploid and aneuploid arrays, respectively.
Fixed channels:
BF = brightfield (transmitted light)
Blue = Hoechst 33342
Green = SOX2
Red = Brachyury (BRA)
FarRed = CDX2
Living channels:
BF = brightfield (transmitted light)
Blue = Hoechst 33342
Green = Calcein AM
Code/software
For code used, please refer to https://github.com/ijan780/EGOMRA.