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. 2022 May 10;17(5):1138-1153.
doi: 10.1016/j.stemcr.2022.03.007. Epub 2022 Apr 7.

Acquisition of NOTCH dependence is a hallmark of human intestinal stem cell maturation

Yu-Hwai Tsai  1 , Angeline Wu  1 , Joshua H Wu  1 , Meghan M Capeling  2 , Emily M Holloway  3 , Sha Huang  1 , Michael Czerwinkski  1 , Ian Glass  4 , Peter D R Higgins  1 , Jason R Spence  5
Affiliations

Acquisition of NOTCH dependence is a hallmark of human intestinal stem cell maturation

Yu-Hwai Tsai et al. Stem Cell Reports. .

Abstract

NOTCH signaling is a key regulator involved in maintaining intestinal stem cell (ISC) homeostasis and for balancing differentiation. Using single-cell transcriptomics, we observed that OLFM4, a NOTCH target gene present in ISCs, is first expressed at 13 weeks post-conception in the developing human intestine and increases over time. This led us to hypothesize that the requirement for NOTCH signaling is acquired across human development. To test this, we established a series of epithelium-only organoids (enteroids) from different developmental stages and used γ-secretase inhibitors (dibenzazepine [DBZ] or DAPT) to functionally block NOTCH signaling. Using quantitative enteroid-forming assays, we observed a decrease in enteroid forming efficiency in response to γ-secretase inhibition as development progress. When DBZ was added to cultures and maintained during routine passaging, enteroids isolated from tissue before 20 weeks had higher recovery rates following single-cell serial passaging. Finally, bulk RNA sequencing (RNA-seq) analysis 1 day and 3 days after DBZ treatment showed major differences in the transcriptional changes between developing or adult enteroids. Collectively, these data suggest that ISC dependence on NOTCH signaling increases as the human intestine matures.

Keywords: NOTCH; enteroid; intestinal stem cell; intestine; organoid.

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Figures

Figure 1
Figure 1
OLFM4 gene expression increases concurrently with age (A) UMAP visualizations of ages and clusters from EPCAM+ cells by scRNA-seq analysis from fetal tissue 47–132 days post-conception, corresponding to Figure S1. (B) Feature plot and dot plot of OLFM4+ cells from extracted epithelium across time points corresponding to (A). (C) Feature plot and dot plot of LGR5+ cells from extracted epithelium across time points corresponding to (A). (D) H&E staining of fetal duodenum at 59, 91, 103, and 132 days post-conception and 65-year-old adult duodenum biopsy. (E) Immunofluorescence (IF) protein staining for OLFM4 (pink) and ECAD (blue) with DAPI (gray) on fetal duodenum. (F) Fluorescence in situ hybridization (FISH) staining for OLFM4 (green), LGR5 (red), and IF staining for ECAD (blue) with DAPI (gray) on fetal duodenum (n = 10 [n = 2 biological replicates per time point shown]). Scale bars represent 100 μm.
Figure 2
Figure 2
NOTCH2 and JAG1 are the major NOTCH functional components in human intestinal epithelial stem cells (A) Dot plots of scRNA-seq analysis of Notch components from fetal and adult duodenum. (B) FISH staining for OLFM4 (green); NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL3, DLL4, JAG1, and JAG2 (red); and IF staining for ECAD (blue) with DAPI (gray) on fetal duodenum aged 59 and 132 days post-conception and adult duodenum (n = 6 [n = 2 biological replicates per time point shown]). (C) UMAP plots of cells computationally extracted from cluster 2 (C2), cluster 0 (C0), and cluster 8 (C8) from the analysis of EPCAM+ cells in Figure 1A. All cells within each cluster were computationally extracted and included in subsequent cluster-specific analyses. (D) Dot plots of Notch components from C2, C0, and C8. Scale bars represent 100 μm.
Figure 3
Figure 3
Short-term γ-secretase inhibition increases stem cell survival in fetal enteroids compared with adult enteroids (A) Experimental schematic for data presented in (B)–(D). (B) Stereomicroscope images of enteroids derived from respective fetal ages and adult after LWRN and LWRN + DBZ treatments for 7, 10, and 13 days. (C) Quantification of enteroid survival after 10 days of treatments corresponding to (B) for all three age groups (n = 3–9 experimental replicates [indicated by individual data points carried out on n = 1 biological replicate]). (D) Measurements of surface area of individual enteroid size using ImageJ after 10 days of treatments corresponding to (B) for all three age groups. (E) Experimental schematic for data presented in (F)–(I). (F) Stereomicroscope images for the passages of enteroids derived from fetal tissue (59 days post-conception) and adult (33 years old) on LWRN and LWRN + DAPT treatments (n = 4 [two biological replicates per each time point]). (G) Real-time PCR of KI67 of CYST and DENSE enteroids corresponding to (F) (n = 3 experimental replicates on n = 1 biological replicate). (H) Quantification of the percentage of cystic enteroids to total enteroids over five passages corresponding to (F) (n = 3 experimental replicates on n = 1 biological replicate). (I) Quantification for percentage of DENSE enteroids to total enteroids over five passages corresponding to (F) (n = 3 experimental replicates on n = 1 biological replicate). Scale bars represent 2 mm. All statistics were analyzed with unpaired t tests by GraphPad Prism 7.0, and data were presented as the mean ± SEM. In all figures, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
Figure 4
Figure 4
Fetal and adult human enteroids show major transcriptional differences in response to γ-secretase inhibition (A) Experimental schematic for data presented in (B)–(D). (B) Principal-component analysis (PCA) with PC1 showing maturity of the enteroids and PC2 showing treatments of the enteroids. (C) Gene expression differentiation was analyzed by Venny 2.1.0 between control and DBZ treatments. (D) TMM normalized counts of OLFM4 on LWRN-control and LWRN-DBZ for fetal (59 days post-conception) and adult (33 years old) enteroids (n = 3 experimental replicates on n = 1 biological replicate per time point). (E) FISH staining for OLFM4 (green), LGR5 (pink), and IF staining for ECAD (blue) with DAPI (gray) on fetal (59 days post-conception) and adult (33 years old) enteroids cultured in LWRN (n = 4 [n = 2 biological replicates per time point]). Scale bars represent 100 μm. All statistics were analyzed with unpaired t tests by GraphPad Prism 7.0, and data are presented as the mean ± SEM. In all figures, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
Figure 5
Figure 5
Reduced self-renewal of epithelium cultured under γ-secretase inhibition coincides with the onset of OLFM4 expression (A) Experimental schematic for data presented in (B)–(F). (B) Stereomicroscope images of P1 enteroids derived from fresh epithelium of fetal tissues aged 58, 70, 91, 125, and 140 days post-conception after LWRN and LWRN + DBZ treatments for 10 days. Scale bar represents 2 mm. (C) FISH staining for OLFM4 (green) and LGR5 (red) with DAPI (gray) on respective time points corresponding to (B). Scale bar represents 100 μm. (D) FISH staining for OLFM4 (green) with DAPI (gray) on respective time points corresponding to (B) and (C). Scale bar represents 200 μm. (E) IF staining for KI67 (red) with DAPI (gray) on respective time points corresponding to (B)–(D) (n = 5 [n = 1 biological replicate per time point]). Scale bar represents 200 μm. (F) Manual quantification of the percent changes of OLFM4HIGH and KI67HIGH enteroids compared with total enteroids with respective time points corresponding to (D) and (E) (n = 5 [n = 1 biological replicate per time point]; the other time points [n = 6] are present in Figure S4).
Figure 6
Figure 6
Chronic γ-secretase inhibition promotes higher stem-cell survival in fetal enteroids younger than 20 weeks post-conception (A) Experimental schematic for data presented in (B)–(D). (B) FISH staining for OLFM4 (green) and LGR5 (red) with DAPI (gray) on fetal duodenum aged 58, 59, 70, 78, 91, 100, 110, 132, and 140 days post-conception, as well as 42- and 65-year-old adult duodenum (n = 11 [n = 1 biological replicates per time point]). Scale bar represents 50 μm. (C) Stereomicroscope images of different passages for respective enteroids time points after LWRN and LWRN + DBZ treatments (n = 3 experimental replicates per time point from n = 1 biological replicate per time point). Scale bars represent 2 mm. (D) Manual counting of enteroid number from respective time points and passages corresponding to (C) (n = 3 experimental replicates per time point from n = 1 biological replicate per time point). All statistics were analyzed with unpaired t tests by GraphPad Prism 7.0, and data were presented as the mean ± SEM. In all figures, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
Figure 7
Figure 7
Primary fetal explant cultures respond to γ-secretase inhibition differently across fetal development (A) Experimental schematic for data presented in (B)–(E). (B) FISH staining for OLFM4 (red) and IF staining for ECAD (blue) with DAPI (gray) on fetal duodenum 59 days post-conception after LWRN (a) and LWRN + DBZ (b) treatments. Scale bars represent 200 μm. (C) IF staining for KI67 (red) and ECAD (blue) with DAPI (gray) on fetal duodenum at 59 days post-conception after LWRN (a) and LWRN + DBZ (b) treatments. Scale bars represent 200 μm. (aʹ) represents high magnification of (a), and (bʹ) represents high magnification of (b) (n = 2 biological replicates). Scale bars represent 100 μm. (D) FISH staining for OLFM4 (red) and IF staining for ECAD (blue) with DAPI (gray) on fetal duodenum at 125 days post-conception after LWRN (a) and LWRN + DBZ (b) treatments. Scale bars represent 200 μm. (E) IF staining for KI67 (red) and ECAD (blue) with DAPI (gray) on fetal duodenum at 125 days post-conception after LWRN (a) and LWRN + DBZ (b) treatments. Scale bars represent 200 μm. (aʹ) represents high magnification of (a), and (bʹ) represents high magnification of (b) (n = 1 biological replicate). Scale bars represent 100 μm.
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References

    1. Barker N., Clevers H. Tracking down the stem cells of the intestine: strategies to identify adult stem cells. Gastroenterology. 2007;133:1755–1760. - PubMed
    1. Barker N., van Es J.H., Kuipers J., Kujala P., van den Born M., Cozijnsen M., Haegebarth A., Korving J., Begthel H., Peters P.J., et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007;449:1003–1007. - PubMed
    1. Beatus P., Lundkvist J., Öberg C., Lendahl U. The Notch 3 intracellular domain represses Notch 1-mediated activation through Hairy/Enhancer of split (HES) promoters. Development. 1999;126:3925–3935. - PubMed
    1. Blondel V.D., Guillaume J.-L., Lambiotte R., Lefebvre E. Fast unfolding of communities in large networks. J. Stat. Mech. Theor. Exp. 2008;2008:1–12.
    1. Bray N.L., Pimentel H., Melsted P., Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 2016;34:525–527. - PubMed

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