Journal: bioRxiv
Article Title: Preservation of Human Colonic Stem Cells Requires an ERK Dynamics Checkpoint Mediated by AKT
doi: 10.64898/2026.04.02.715982
Figure Lengend Snippet: (A) Model depicting organoid monolayer preparation. Patient colonic tissue is digested, single celled, and plated in a bubble of Matrigel. Organoids are supplemented with media containing surplus of Wnt, EGF, R-spondin, and Noggin. Once organoids are fully grown in 3D conditions, cells are disassociated and plated on a thin layer of Matrigel. Within 5-7 days, cells grow into a monolayer with distinct stem cell (5-10% of cells), TA cell (5-10% of cells) and differentiated cell (80-90% of cells) niches. Stem cell niches, called nodes, are dense, regularly spaced niches made up of OLFM4/Sox9/LGR5/MYC positive stem cells. (B) Representative image of stem cell niche (node) characterized by OLFM4+ cells with quantification. OLFM4 measured through immunofluorescent staining, population makes up around 10% of monolayers, although almost all cells in node are OLFM4+. Cells were portioned into node or non-node and percent of OLFM4+ cells out of total node or non-node cells was used for quantification. (C) Representative image of transit-Amplifying (TA) cells characterized by Ki-67 immunofluorescent staining. TA cells surround the OLFM4+ stem cells and make up around 10% of the monolayer. (D) Differentiated cells shown by CK20 immunostaining makes up the surrounding monolayer, around 80% of total cells. (E) Total EGFR representative image and quantification of almost all cells in monolayer expressing EGFR uniformly. (F) P-EGFR-Y1068 representative image and quantification preferentially activated in nodes. (G) Representative image and quantification of P-AKT-S473 exclusively activated within nodes and low in differentiated compartment. (H) P-ERK1/2 representative image and quantification. Activation excluded from nodes and on in a small percentage of differentiated cells at any given time (10-20%). (I) Co-stain representative image of P-AKT and P-ERK mutual exclusivity. (J) Quantification of AKT+, ERK+, Double Positive and Double Negative cells within monolayer. Red asterisks marking around 5% total cells that are double positive, uninsulated cells that coactivate AKT and ERK. Three to four biological replicates were performed. Data shown is from analysis of 4 technical replicates with at least 150 total cells quantified per replicate. Data are represented as mean ± SEM. All scale bars are 100uM, significance calculated with Welch’s t-test, ** P ≤ 0.01, *** P ≤ 0.001. See also Figure S1 and S2.
Article Snippet: Antibodies and dyes Rabbit monoclonal OLFM4 (CST, 14369S), Rat monoclonal anti-Ki67 (Invitrogen, 14-5698-82), Mouse monoclonal P-ERK1/2 (Invitrogen, 14-9109-82), Rabbit Monoclonal P-AKT-Ser473 (CST, 4058), Rabbit monoclonal P-RAF1-S259 (Invitrogen, 44-502), Rabbit monoclonal P-EGFR-Y1068 (abcam, ab40815), Mouse monoclonal EGFR (Invitrogen, MA5-13070), Click-iT EdU Cell Proliferation Kit for Imaging, DAPI (Thermofisher, D21490), Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Invitrogen, A32731), Goat anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A21247), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (Invitrogen, A32728), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 546 (Invitrogen, A11030).
Techniques: Staining, Immunostaining, Expressing, Activation Assay
