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| Status |
Public on Dec 02, 2022 |
| Title |
Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids [scRNA-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The intestinal epithelium is an immunologically active barrier adapted for a low oxygen environment. Its role is implicated in the pathophysiology of various diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Patient-derived intestinal epithelial organoids (IEOs) mimic the architecture and cell type composition of the intestine and can be used for disease modeling and personalized drug screening. IEOs are usually cultured in atmospheric oxygen concentrations (20% oxygen) without accounting for the physiological hypoxia in the colonic epithelium, where the oxygen concentration range from 3% to <1%. Previously, we showed that human colon derived IEOs (colonoids) are viable and respond to the pro-inflammatory cytokines TNF and IL17 after short-term (40 hours) cultivation in low (2%) oxygen. We suggested that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of intestinal organoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia for the entire in vitro culture period and compare growth, differentiation, and immunological responses in colonoids at 2% and 20% oxygen. Growth from single cells to differentiated organoids was monitored by brightfield images captured throughout cultivation and evaluated with a linear mixed model. Cell composition in undifferentiated vs. differentiated colonoids was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing. Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. There were significantly more cells with proliferation potential (KI67 positive) in undifferentiated than in differentiated colonoids; and significantly higher expression of cell markers for goblet cells (MUC2) and absorptive cells (MUC2 negative, CK20 positive) in differentiated colonoids. Enteroendocrine cells (CGA positive) were only present in differentiated colonoids. No differences in expression of any of the cell marker proteins were found between colonoids cultured in 2% and 20% oxygen, but the single-cell RNA-sequencing analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CX3CL1, CXCL2, CXCL5, CXCL6, CXCL10, CXCL12, CCL20, CCL25, and NGAL upon TNF + poly(I:C) treatment. However, there appeared to be a lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.
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| Overall design |
Single cell RNA-sequencing data of undifferentiated and differentiated intestinal epithelial organoids (untreated) cultured in 2% oxygen and 20% oxygen throughout cultivation.
***Please note that raw data is not provided as Norwegian law does not allow public access to human sequences raw data***
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| Web link |
https://pubmed.ncbi.nlm.nih.gov/36793710/
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| Contributor(s) |
Walaas GA, Gopalakrishnan S, Bakke I, Skovdahl HK, Flatberg A, Østvik AE, Sandvik AK, Bruland T |
| Citation(s) |
36793710 |
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| Submission date |
Nov 23, 2022 |
| Last update date |
Mar 13, 2023 |
| Contact name |
Gunnar Andreas Walaas |
| E-mail(s) |
gunnar.a.walaas@ntnu.no
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| Organization name |
Norwegian University of Science and Technology
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| Department |
Department of Clinical and Molecular Medicine
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| Street address |
Prinsesse Kristinas gate 1
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| City |
Trondheim |
| ZIP/Postal code |
7030 |
| Country |
Norway |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA904706 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE218623_RAW.tar |
113.2 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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