DOI: 10.5061/dryad.tqjq2bw7b
title: Data from: Fibroblasts modulate epithelial cell behavior within the proliferative niche and differentiated cell zone within a human colonic crypt model

description: Colonic epithelium is situated above a layer of fibroblasts that provide
 supportive factors for stem cells at the crypt base and terminally
 differentiated cells in the upper crypt and luminal surface. To study the
 fibroblast-epithelial cell interactions, an in vitro crypt model was
 formed on a shaped collagen scaffold with primary epithelial cells growing
 above a layer of primary colonic fibroblasts. The crypts possessed a basal
 stem cell niche populated with proliferative cells and a differentiated,
 nondividing cell zone at the luminal crypt end. The presence of
 fibroblasts enhanced cell differentiation and accelerated the rate at
 which a high resistance epithelial cell layer formed relative to cultures
 without fibroblasts. The fibroblasts modulated cell proliferation within
 crypts increasing the number of crypts populated with proliferative cells
 but decreasing the total number of proliferative cells in each crypt.
 Bulk-RNA sequencing revealed 41 genes that were significantly upregulated
 and 190 genes that were significantly downregulated in cocultured
 epithelium relative to epithelium cultured without fibroblasts. This
 epithelium-fibroblast crypt model suggests bidirectional communication
 between the two cell types and has the potential to serve as a model to
 investigate fibroblast-epithelial cell interactions in health and disease.
description: Data collection was conducted via transepithelial electrical
 resistance (TEER) measurement with a chopstick electrode, fluorescence
 imaging, and bulk RNA-sequencing. Microsoft Excel, GraphPad Prism, FIJI,
 IMARIS, CellProfiler, STAR v2.7.7a, FastQC 0.11.9, RNA-SeQC 2.3.4, and
 RSeQC 4.0 were used to process these data.
description: # Data from: Fibroblasts modulate epithelial cell behavior within the
 proliferative niche and differentiated cell zone within a human colonic
 crypt model
 [https://doi.org/10.5061/dryad.tqjq2bw7b](https://doi.org/10.5061/dryad.tqjq2bw7b) Data characterizing epithelial cell behavior while cultured within an in vitro model built on collagen hydrogel scaffolds with supportive primary fibroblasts underlying the epithelial monolayer. This work was conducted entirely at the University of Washington in Seattle, WA. The datasets enclosed herein represent the raw data collected for this original research. For any questions, please contact Angelo Massaro (angelom@uw.edu). The corresponding article has been published, on 15 December 2024: Massaro Angelo , Villegas Novoa Cecilia , Wang Yuli , Allbritton Nancy L. Fibroblasts modulate epithelial cell behavior within the proliferative niche and differentiated cell zone within a human colonic crypt model. *Frontiers in Bioengineering and Biotechnology, *Volume 12 - 2024. DOI: 10.3389/fbioe.2024.1506976 Data files include: ### Figure2.xlsx Characterization of epithelial cell phenotype by fluorescence microscopy and characterization of epithelial barrier function by Transepithelial electrical resistance (TEER). In this figure, all cells were exposed to exogenously provided growth factors Wnt, R-spondin, and Noggin (WRN). All data collected by the authors. The area above a certain empirical threshold for fluorescence in images of epithelial monolayers was measured for markers of nuclei, proliferative cells, mucin 2, and cytokeratin-20. The reported values represent the proportion (no units) of the total area imaged that is above the empirical threshold (i.e., 1.0 = full coverage of x marker). Additionally, TEER values are reported for all conditions in Ohms * cm^2. ### Figure3.xlsx Characterization of epithelial cell phenotype by fluorescence microscopy and characterization of epithelial barrier function by Transepithelial electrical resistance (TEER). In this figure, all cells were NOT exposed to exogenously provided growth factors Wnt, R-spondin, and Noggin (WRN). All data collected by the authors. The area above a certain empirical threshold for fluorescence in images of epithelial monolayers was measured for markers of nuclei, proliferative cells, mucin 2, and cytokeratin-20. The reported values represent the proportion (no units) of the total area imaged that is above the empirical threshold (i.e., 1.0 = full coverage of x marker). Additionally, TEER values are reported for all conditions in Ohms * cm^2. ### Figure4.xlsx Characterization of epithelial cells and fibroblasts within colonic crypt structures based on imaging data from confocal microscope pictures and processed via IMARIS.. All data collected by the authors. The area above a certain empirical threshold for fluorescence in 3D reconstructed images of epithelial and cocultured crypts was measured for markers of nuclei and vimentin positive cells (i.e., fibroblasts). The reported values for vimentin are shown as a volume measurement (micrometers^3) and they represent the 3D area occupied by vimentin-expressing fibroblasts. Additionally, the number of nuclei counted for individual crypts is reported as a count (count, cell number) for individual crypts across three treatment groups. ### Figure5.xlsx Characterizing 3D crypts by the amount and location of EdU-positive cells i.e., proliferating cells. First, the percent area (%) of a crypt array structure that was above an empirically set threshold for EdU fluorescent marker is reported. Next, the count for the number of EdU-positive cells (count, cell number) in each individual crypt is reported. Next, the average distance of EdU-positive cells is reported for each individual crypt (micrometers from the crypt base). Finally, the number of EdU-positive cells within each of four quadrants within individual crypts is reported (count, cell number per quadrant). ### Figure6.xlsx Expression data for selected genes from bulk RNA-sequencing data conducted with Illumina NextSeq 2000. The measured expression for three replicates across three treatment conditions is reported for selected genes that are especially relevant to this study. Vimentin, EpCAM, MUC2, KRT20, Goblet-related, Colonocyte-related, Progenitor cell-related, and fibroblast-related genes are reported as transcript counts per million cells (CPM, normalized). ## Description of the data and file structure Data are organized by figure of the referenced manuscript. A separate .xlsx file can be found for Figure 2-6 and within each sub-tab the raw values and relevant statistical analyses are provided for each individual figure panel.