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[Preprint]. 2023 Jan 13:2023.01.13.523825. [Version 1] doi: 10.1101/2023.01.13.523825

Figure 1. Crypt regeneration mainly starts 3 days after irradiation.

Figure 1.

(A) Demonstration of intestinal regeneration following 12 Gy of irradiation of mice. Crypt cells are lost by 2 days post-irradiation (12 Gy) but restoration begins at Day 3 after irradiation, when highly proliferative regenerative clusters of cells expand, as evidenced by H&E staining and immunohistochemistry staining of stem/proliferative markers (brown color) including Ki67, OLFM4 and CD44 (representative of 3 biological replicates). (B) Immunostaining of BrdU (proliferative marker, brown color; representative of 3 biological replicates). Mice were injected with 1 mg of BrdU at Day 2 post-irradiation. Intestinal tissues were harvested after 2, 4, 6, 8, 10 and 24 hours of BrdU injection. (C) Heatmap depicts transcript levels of fetal/regenerative marker genes and regenerative stem cell-associated genes that are highly expressed at Day 3 post-irradiation (GSE165157 (Qu et al., 2021), RNA-seq, n=2 biological replicates per time-point). (D) GSEA confirms that gene signatures of regenerative epithelium, fetal spheroid and revival stem cells (Ayyaz et al., 2019; Wang et al., 2019; Yui et al., 2018) are elevated at Day 3 post-irradiation (GSE165157 (Qu et al., 2021), crypt cells, n=2 biological replicates per time-course, Kolmogorov-Smirnov test, P < 0.001). See expanded panel in Figure S1E. (E) scRNA-seq reveals that fetal/regenerative transcripts are elevated in irradiated crypt cells after 3 days of irradiation. Cell numbers per condition (GSE117783 (Ayyaz et al., 2019)) for irradiated crypt cells: n=4252 and normal crypt cells: n=4328. (F) Immunostaining of Ki67 after 56 hours of irradiation vs. non-irradiation (proliferative marker, brown color; representative of 3 biological replicates). (G) scRNA-seq reveals that fetal/regenerative and reserve stem cell transcripts are elevated in sorted Ki67-RFP positive cells after 56 hours of irradiation. Number of cells in each condition was Non-IR Ki67-RFP positive cells: n=1739; IR 56h Ki67-RFP positive cells replicate 1: n=677; IR 56h Ki67-RFP positive cells replicate 2: n= 669. Ki67-RFP positive cells were isolated and sorted from crypt cells of Mki67tm1.1Cle/J mice after 56 hours of IR vs. non-IR control. IR: irradiation; Non-IR: non-irradiation (normal control). Also see Figure S1F.