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Cited by 138Wnt/β-catenin signaling in development and cancerDigestive system and related healthCancer Cells and Metastasis

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Establishment of intestinal organoid cultures modeling injury-associated epithelial regeneration

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Qu, Molong, Xiong, Liang, Lyu, Yulin, Zhang, Xiannian, Shen, Jie, Guan, Jingyang, Chai, Peiyuan, Lin, Zhongqing, Nie, Boyao, Li, Cheng, Xu, Jun, Deng, Hongkui·2021·Cell Research
DOI 10.1038/s41422-020-00453-xPMC PMC8027647
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Abstract

The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing stem cells and their derivatives. Here, we established a novel intestinal organoid culture system composed of 8 components, mainly including VPA, EPZ6438, LDN193189, and R-Spondin 1 conditioned medium, which mimics the gut epithelium regeneration that produces hyperplastic crypts following injury; therefore, these organoids were designated hyperplastic intestinal organoids (Hyper-organoids). Single-cell RNA sequencing identified different regenerative stem cell populations in our Hyper-organoids that shared molecular features with in vivo injury-responsive Lgr5 + stem cells or Clu + revival stem cells. Further analysis revealed that VPA and EPZ6438 were indispensable for epigenome reprogramming and regeneration in Hyper-organoids, which functioned through epigenetically regulating YAP signaling. Furthermore, VPA and EPZ6438 synergistically promoted regenerative response in gut upon damage in vivo. In summary, our results demonstrated a new in vitro organoid model to study epithelial regeneration, highlighting the importance of epigenetic reprogramming that pioneers tissue repair.

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Fig. 1 Establishment of intestinal organoid cultures enriched for an injury-associated regenerative signature. a qPCR analyses of Clu and regenerative gene expression in organoids cultured under the indicated conditions (n = 2 wells). P values were determined by two-sided unpaired t-test. b Typical morphology of intestinal organoids cultured under the indicated conditions. c Immunofluorescence staining of CLU and regenerative markers in organoids cultured under the indicated conditions. d FACS analysis of SCA1+ cells in organoids cultured under the indicated conditions (n = 3 mice). P values were determined by two-sided unpaired t-test. e Quantification of CLU+ cells in each organoid cultured under the indicated conditions (n = 15 organoids from three mice). P values were determined by two-sided unpaired t-test. f Heatmap displaying the expression of an injury-associated regenerative signature in different organoids and primary crypts from different intestinal injury models (n = 3 mice). The gene expression profile of organoids cultured in the ENR and 8C conditions was plotted. Representative genes are shown on the left. Gran and Non-gran indicate crypts overlying and adjacent to granulomas, respectively. DSS and Non-DSS indicate crypts from repairing epithelium in DSS-associated colitis, and normal epithelium without DSS treatment, respectively. **P < 0.01; ***P < 0.001. Scale bars, 100 μm. The experiments in a–c were independently repeated at least three times with similar results.
Fig. 1 Establishment of intestinal organoid cultures enriched for an injury-associated regenerative signature. a qPCR analyses of Clu and regenerative gene expression in organoids cultured under the indicated conditions (n = 2 wells). P values were determined by two-sided unpaired t-test. b Typical morphology of intestinal organoids cultured under the indicated conditions. c Immunofluorescence staining of CLU and regenerative markers in organoids cultured under the indicated conditions. d FACS analysis of SCA1+ cells in organoids cultured under the indicated conditions (n = 3 mice). P values were determined by two-sided unpaired t-test. e Quantification of CLU+ cells in each organoid cultured under the indicated conditions (n = 15 organoids from three mice). P values were determined by two-sided unpaired t-test. f Heatmap displaying the expression of an injury-associated regenerative signature in different organoids and primary crypts from different intestinal injury models (n = 3 mice). The gene expression profile of organoids cultured in the ENR and 8C conditions was plotted. Representative genes are shown on the left. Gran and Non-gran indicate crypts overlying and adjacent to granulomas, respectively. DSS and Non-DSS indicate crypts from repairing epithelium in DSS-associated colitis, and normal epithelium without DSS treatment, respectively. **P < 0.01; ***P < 0.001. Scale bars, 100 μm. The experiments in a–c were independently repeated at least three times with similar results.CC BY · PubMed Central
Fig. 2 The lineage composition of hyperplastic intestinal organoids resembles that of primary crypts upon damage in vivo. a UMAP visualizations of scRNA-seq data from Hyper- and ENR-organoids. Left, colors indicate the unsupervised clusters. Right, colors indicate different organoids: ENR-organoids (orange dots) and Hyper-organoids (purple dots). b Expression of cell type-specific markers that identify distinct cell types. EE enteroendocrine, PC Paneth cell, GC goblet cell, EC enterocyte, EP enterocyte progenitor, TA transit amplifying. c Stacked histograms showing the proportions of the included cell types in Hyper-organoids derived from either ENR-organoids (up) or primary crypts (down). Hyper-organoids derived from ENR-organoids had a lineage composition similar to those derived from primary crypts. d Schematic diagram of the genetic lineage tracing strategy used to trace endogenous Lgr5 activation. e Representative images of ENR- and Hyper-organoids (with or without 4-OHT induction) using the lineage tracing system. Scale bars, 100 μm. f FACS analysis of tdTomato+ cells in ENR- and Hyper-organoids (with or without 4-OHT induction) (n = 3 wells). P values were determined using one-way ANOVA. ***P < 0.001. Experiments in e and f were independently repeated at least twice with similar results.
Fig. 2 The lineage composition of hyperplastic intestinal organoids resembles that of primary crypts upon damage in vivo. a UMAP visualizations of scRNA-seq data from Hyper- and ENR-organoids. Left, colors indicate the unsupervised clusters. Right, colors indicate different organoids: ENR-organoids (orange dots) and Hyper-organoids (purple dots). b Expression of cell type-specific markers that identify distinct cell types. EE enteroendocrine, PC Paneth cell, GC goblet cell, EC enterocyte, EP enterocyte progenitor, TA transit amplifying. c Stacked histograms showing the proportions of the included cell types in Hyper-organoids derived from either ENR-organoids (up) or primary crypts (down). Hyper-organoids derived from ENR-organoids had a lineage composition similar to those derived from primary crypts. d Schematic diagram of the genetic lineage tracing strategy used to trace endogenous Lgr5 activation. e Representative images of ENR- and Hyper-organoids (with or without 4-OHT induction) using the lineage tracing system. Scale bars, 100 μm. f FACS analysis of tdTomato+ cells in ENR- and Hyper-organoids (with or without 4-OHT induction) (n = 3 wells). P values were determined using one-way ANOVA. ***P < 0.001. Experiments in e and f were independently repeated at least twice with similar results.CC BY · PubMed Central
Fig. 3 VPA and EPZ6438 are critical for establishing a hyperplastic phenotype in intestinal organoids. a The effects of individual components of the 8C medium on the expression of an injury-associated regenerative signature in Hyper-organoids (n = 3 mice). Representative genes are shown on the left. b UMAP visualizations of scRNA-seq data across different organoids. The stem cell clusters and cluster 17 annotated in Fig. 2a are plotted. Colors indicate different organoids: ENR-organoids (orange dots), Hyper-organoids (purple dots), and organoids cultured in 8C minus VPA/EPZ6438 condition (–VE) (green dots). c Expression of the fetal gene signature was overlaid on the UMAP shown in b. Hyper- and –VE/ENR-organoids are separated by the purple and green dotted line. d Integrated analysis of stem cell clusters from different organoids in vitro and the irradiation model in vivo. e Expression of the revival stem cell signature was overlaid on the UMAP shown in d. Hyper- and –VE/ENR-organoids are separated by the orange and blue dotted line. f Trajectory reconstruction of single cells from Hyper-organoids with and without VPA/EPZ6438 treatment. Left panel: pre-branch (blue dots, before bifurcation), successful branch (red dots), and failed branch (green dots). Middle panel: cells from the Hyper-organoids (red dots) and –VE-organoids (green dots) were overlaid on the regenerative trajectory, respectively. Right panel: distribution of homeostatic (green dots), injury-responsive (red dots) Lgr5+ cells, and other cells (gray dots) were overlaid on the regenerative trajectory.
Fig. 3 VPA and EPZ6438 are critical for establishing a hyperplastic phenotype in intestinal organoids. a The effects of individual components of the 8C medium on the expression of an injury-associated regenerative signature in Hyper-organoids (n = 3 mice). Representative genes are shown on the left. b UMAP visualizations of scRNA-seq data across different organoids. The stem cell clusters and cluster 17 annotated in Fig. 2a are plotted. Colors indicate different organoids: ENR-organoids (orange dots), Hyper-organoids (purple dots), and organoids cultured in 8C minus VPA/EPZ6438 condition (–VE) (green dots). c Expression of the fetal gene signature was overlaid on the UMAP shown in b. Hyper- and –VE/ENR-organoids are separated by the purple and green dotted line. d Integrated analysis of stem cell clusters from different organoids in vitro and the irradiation model in vivo. e Expression of the revival stem cell signature was overlaid on the UMAP shown in d. Hyper- and –VE/ENR-organoids are separated by the orange and blue dotted line. f Trajectory reconstruction of single cells from Hyper-organoids with and without VPA/EPZ6438 treatment. Left panel: pre-branch (blue dots, before bifurcation), successful branch (red dots), and failed branch (green dots). Middle panel: cells from the Hyper-organoids (red dots) and –VE-organoids (green dots) were overlaid on the regenerative trajectory, respectively. Right panel: distribution of homeostatic (green dots), injury-responsive (red dots) Lgr5+ cells, and other cells (gray dots) were overlaid on the regenerative trajectory.CC BY · PubMed Central
Fig. 4 Epigenetic regulation of YAP is involved in the VPA-and-EPZ6438-driven regenerative response in vitro. a Expression of the YAP gene signature was overlaid on the regenerative trajectory from Hyper-organoids with and without VPA/EPZ6438 treatment shown in Fig. 3f. b The expression dynamics of YAP signature was cataloged in a pseudotime manner shown as a red line (successful reprogramming), a green line (failed reprogramming) and a blue line (pre-branch before bifurcation). Thick lines indicate the average gene expression patterns in each branch. c Volcano plot displaying the results of differential gene expression analysis performed between successful branch and failed branch. The dots representing YAP target genes are indicated as red. d Expression of the YAP gene signature was overlaid on the UMAPs shown in Fig. 3b and d, respectively. Hyper- and –VE/ENR-organoids are separated by the orange and blue dotted line. e Violin plots showing the entire range of metagene expression levels per single cell per cluster for the transcriptional programs of all clusters (left) and stem cell clusters (right) in different organoids. f Heatmap displaying H3K27me3 signals at promoter regions of YAP target genes in different organoids. g ChIP-seq (H3K27me3) and RNA-seq tracks of YAP target genes in different organoids.
Fig. 4 Epigenetic regulation of YAP is involved in the VPA-and-EPZ6438-driven regenerative response in vitro. a Expression of the YAP gene signature was overlaid on the regenerative trajectory from Hyper-organoids with and without VPA/EPZ6438 treatment shown in Fig. 3f. b The expression dynamics of YAP signature was cataloged in a pseudotime manner shown as a red line (successful reprogramming), a green line (failed reprogramming) and a blue line (pre-branch before bifurcation). Thick lines indicate the average gene expression patterns in each branch. c Volcano plot displaying the results of differential gene expression analysis performed between successful branch and failed branch. The dots representing YAP target genes are indicated as red. d Expression of the YAP gene signature was overlaid on the UMAPs shown in Fig. 3b and d, respectively. Hyper- and –VE/ENR-organoids are separated by the orange and blue dotted line. e Violin plots showing the entire range of metagene expression levels per single cell per cluster for the transcriptional programs of all clusters (left) and stem cell clusters (right) in different organoids. f Heatmap displaying H3K27me3 signals at promoter regions of YAP target genes in different organoids. g ChIP-seq (H3K27me3) and RNA-seq tracks of YAP target genes in different organoids.CC BY · PubMed Central
Fig. 5 Combined treatment with VPA and EPZ6438 promotes regenerative responses in the intestinal epithelium after irradiation. a Schematic diagram of the administration of VPA/EPZ6438 to C57BL/6 mice in the irradiation-induced injury model. b H&E staining of small intestinal crypts at 3 and 5 days post-irradiation (dpi). The green arrows between the two dashed lines show the length of the crypts. Scale bars, 50 μm. c Quantification of crypt number (10 images were analyzed per mouse) and crypt length (10 crypt–villus axes were counted per mouse) at 3 and 5 dpi (n = 6 mice). P values were determined by two-tailed Mann–Whitney test. d Heatmap displaying the expression of the YAP signature in small intestinal crypts with or without VPA/EPZ6438 treatment at 3 dpi. Representative genes are shown on the right. (n = 2 mice). e Heatmap displaying H3K27me3 signals at promoter regions of YAP target genes in small intestinal crypts at 3 dpi. **P < 0.01; ns, not significant. The experiments in b, c, and e were independently repeated at least twice with similar results.
Fig. 5 Combined treatment with VPA and EPZ6438 promotes regenerative responses in the intestinal epithelium after irradiation. a Schematic diagram of the administration of VPA/EPZ6438 to C57BL/6 mice in the irradiation-induced injury model. b H&E staining of small intestinal crypts at 3 and 5 days post-irradiation (dpi). The green arrows between the two dashed lines show the length of the crypts. Scale bars, 50 μm. c Quantification of crypt number (10 images were analyzed per mouse) and crypt length (10 crypt–villus axes were counted per mouse) at 3 and 5 dpi (n = 6 mice). P values were determined by two-tailed Mann–Whitney test. d Heatmap displaying the expression of the YAP signature in small intestinal crypts with or without VPA/EPZ6438 treatment at 3 dpi. Representative genes are shown on the right. (n = 2 mice). e Heatmap displaying H3K27me3 signals at promoter regions of YAP target genes in small intestinal crypts at 3 dpi. **P < 0.01; ns, not significant. The experiments in b, c, and e were independently repeated at least twice with similar results.CC BY · PubMed Central

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Authors
Qu, M., Xiong, L., Lyu, Y., Zhang, X., Shen, J., Guan, J., Chai, P., Lin, Z., Nie, B., Li, C., Xu, J., & Deng, H.
Affiliations
  • Peking University (CN)
  • Capital Medical University (CN)
Corresponding author
  • Li, Cheng
  • Xu, Jun
  • Deng, Hongkui
ORCID
Venue
Cell Research
Publisher
Springer Science and Business Media LLC
Published
2021-01-08
Volume
31
Issue
3
Pages
259-271
ISSN
  • 1001-0602 (ISSN-L)
  • 1748-7838
DOI
10.1038/s41422-020-00453-x
PMC
PMC8027647
OpenAlex
W3119003316
Cited by
138
References
51
Citations by year
2026: 21 · 2025: 31 · 2024: 41 · 2023: 18 · 2022: 21 · 2021: 6
FWCI
6.83
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Research (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Greater South Information System
Funders
  • National Natural Science Foundation of China (31521004, 31871266, 2017YFA0103000, 31730059)
  • Peking University
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
  • Health Sciences › Medicine › Gastroenterology › Digestive system and related health
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
Keywords
intestinal organoids, epithelial regeneration, intestinal injury, valproic acid, LDN-193189, RSPO1, Lgr5+ stem cells, epigenetic reprogramming, YAP signaling, tissue repair, single-cell RNA sequencing
MeSH terms
Animals, Benzamides, Biphenyl Compounds, Cells, Cultured, Colitis, Disease Models, Animal, Female, Intestinal Mucosa, Intestines, Male, Mice, Inbred C57BL, Mice, Transgenic, Morpholines, Organoids, Pyridones, Radiation Injuries, Regeneration, Stem Cells, Valproic Acid, Signal Transduction, Dextran Sulfate, Treatment Outcome, Culture Media, Conditioned, Tissue Culture Techniques, Mice

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