Infant and adult human intestinal enteroids are morphologically and functionally distinct

Abstract

Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex vivo model to advance studies of infant-specific diseases and drug discovery for this population.

Importance: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.

Keywords: development; enteroids; infant gut; intestinal organoids.

Fig 1

Infant HIEs have a different transcriptome than adults. (A) Principal component analysis of infant (J1005, J1006, and J1009) and adult (J2, J3, and J11) HIEs, with the dashed ellipses indicating a 95% confidence interval. (B). Heatmap of genes differentially expressed in infant over adult HIEs (fold change exceeding 1.5, P adj. <0.05). (C) Volcano plot of differential gene expression analysis results. Each gene is represented by a dot; gray dots represent no significant difference between infant and adult HIEs, the blue dots represent significantly downregulated genes in infant HIEs, and red dots represent significantly upregulated genes in infant HIEs. The top 12 genes by statistical significance are annotated. (D) GSEA results using the GO Biological Processes compendium showing the top 10 upregulated and downregulated pathways in infant over adult HIEs. (E) Summary plot for over-representation analysis (ORA) showing 10 significantly enriched pathways selected for further validation. Number and direction of gene changes are shown in the plot on the right.

Fig 2

Cell type composition varies between differentiated infant and adult HIE monolayers on transwells. (A) RNAseq transcript expression for select markers of absorptive (enterocytes) and secretory cells (goblet cells, enteroendocrine cells, Paneth cells, and tuft cells). Data represent mean values and are expressed as a Log2-fold change of gene expression in infant over adult HIEs. Red bars = upregulated in infant HIEs; blue bars = downregulated in infant HIEs. (B) Representative confocal images from four independent experiments, with each experiment including the three infant and three adult HIE lines. Top panel: enterocytes stained for expression of sucrase isomaltase (SI, red), middle panel: goblet cells (Muc2, green), and bottom panel: enteroendocrine cells (ChgA, green). Nuclei are stained with DAPI (blue), Scale bar = 50 μm. (C) Quantification of cell type abundance from two independent experiments. Data represent mean ± SD with each experiment including the three infant (in teal) and three adult (in purple) HIE lines. The P-values were calculated by one-way ANOVA. The asterisks (*** and ****) represent P < 0.001 and P < 0.0001, respectively.

Fig 3

Undifferentiated infant 3D HIEs are more proliferative than adult HIEs. (A). Representative confocal 3D reconstruction images after 24 h 5-ethynyl-2′-deoxyuridine (EdU) incorporation in undifferentiated infant and adult HIEs. (B) Percentage of EdU-positive cells quantified by flow cytometry using data combined from all infant or adult lines. (C) Percentage of EdU-positive cells quantified by flow cytometry in all six lines. Data represent mean ± standard deviation (SD) from two independent experiments, with each experiment including the three infant and three adult HIE lines. The P-values were calculated by student’s t-test, and the asterisk (*) represents P < 0.05.

Fig 4

Infant HIEs have shorter epithelial cell height. (A). Quantitation of cell heights measured in H&E-stained images of 5-day differentiated infant and adult HIEs; P-values were calculated by student’s t-test. (B–D): Three representative images were taken for each HIE line and tissue, and blinded images were provided to three study authors for cell height measurements. A minimum of three single cells per image were measured by each analyst, generating up to 35 data points per sample. Panel D shows paired donor tissue samples for each infant HIE line. (E–G): Each dot indicates the height measurement of a single cell. Data represent mean ± SD with each experiment including the three infant and three adult HIE lines. The P-values were calculated by one-way ANOVA. The asterisks (**, ***, and ****) represent P < 0.01, P < 0.001, and P < 0.0001, respectively. Scale bar = 10 μm.

Fig 5

Infant HIEs have lower barrier integrity than adult HIEs. (A) Log2-fold change of claudin genes expressed in infants over adults, as determined by RNA-Seq. (B) Western blot of expressed CLDN2 in infant and adult HIEs (top) and densitometry-based quantification (bar plots) of CLDN2 normalized to α-Tubulin. (C, D) Transepithelial electrical resistance values of 5-day differentiated HIE transwell monolayers. Data represent mean ± SD of TEER measurements from four independent experiments, with two transwells/line. One measurement was taken for each transwell. The asterisks (** and ***) represent P < 0.01 and P < 0.001, respectively.

Fig 6

Infant HIEs have higher epithelial permeability in response to EGTA than adult HIEs. (A) TEER values of HIEs at baseline and after EGTA treatment. (B) The concentration of basolateral 4kDA FITC-Dextran at baseline and (C) after EGTA treatment. Data represent mean ± SD from four independent experiments, with each experiment including the three infant and three adult HIE lines. The P-values were calculated by student’s t-test, and the asterisks (*, **, and ****) represent P < 0.05, P < 0.01, and P < 0.0001 respectively.

Fig 7

Lactase and lipid metabolism genes are significantly upregulated in infant HIEs. (A) Expression profile of RNA-seq data for lactase and lipid metabolism genes. (B–D) RT-qPCR data for (B) microsomal triglyceride transfer protein (MTTP), (C) apolipoprotein B (APOB), and (D) lactase (LCT) transcripts. Data represent mean ± SD from two independent experiments, with each experiment including the three infant and three adult HIE lines. The P-values were calculated by student’s t-test and the asterisks (*** and ****) represent P < 0.001 and P < 0.0001, respectively.

Fig 8

Infant HIEs have lower epithelial immune responses to oral vaccines than adult HIEs. (A) Monovalent type I poliovirus vaccine (mOPV1) titer in HIEs at 24 hpi. The line at 100 FFU/mL represents OPV-1 titer at 2 hpi (baseline). Quantification of (B) IFNλ2, (C) IFI44L, and (D) IP10 transcripts. (E-H) There are HIE line-specific differences in mOPV1 replication (E) and innate immune response to mOPV1 (F-H). Data represent mean ± SD from two independent experiments, with each experiment including the three infant and three adult HIE lines. The P-values were calculated by student’s t-test and the asterisk (****) represents P < 0.0001.