Human Milk Supports Robust Intestinal Organoid Growth, Differentiation, and Homeostatic Cytokine Production

Abstract

Background and aims: Necrotizing enterocolitis is a severe gastrointestinal complication of prematurity. Using small intestinal organoids derived from fetal tissue of a gestational age similar to an extremely preterm infant, this study aims to assess the effect of diet on intestinal epithelial growth and differentiation to elucidate the role nutrition type plays in intestinal development and modifies the risk for necrotizing enterocolitis.

Methods: Organoids were cultured for 5 days in growth media and 5 days in differentiation media supplemented 1:40 with 4 different diets: parental milk, donor human milk, standard formula, or extensively hydrolyzed formula. Images were captured daily and organoids were quantified. Organoids were preserved for RNA sequencing and immunofluorescence staining with Ki67, cleaved caspase 3, and chromogranin-A. Media was saved for cytokine/chemokine and growth factor analysis.

Results: Human milk supplementation improved growth and differentiation of intestinal organoids generating larger organoids during the growth phase and organoids with longer and wider buds during differentiation compared to formula. Ki67 staining confirmed the proliferative nature of milk-supplemented organoids and chromogranin A staining proved that MM-supplemented organoids induced highest enteroendocrine differentiation. Human milk supplementation also upregulated genes involved in Wnt signaling and fatty acid metabolism pathways and promoted a homeostatic immune landscape, including via increased secretion of tumor necrosis factor-related apoptosis-inducing ligand among other cytokines. Conversely, organoids supplemented with formula had a downregulation of cell-cycle-promoting genes and a more inflammatory immune signature, including a reduced level of leukemia inhibitory factor.

Conclusion: Our results demonstrate that parental milk, and to a lesser extent donor human milk, support robust intestinal epithelial proliferation, differentiation, and homeostatic cytokine production, suggesting a critical role for factors enriched in human milk in intestinal epithelial health.

Keywords: Breast Milk; Intestinal Development; Necrotizing Enterocolitis.

Figure 1

Human Milk Increases Organoid Size. (A) Day 5 organoid survival, case 3. 3. (B) Day 5 organoid survival, all cases. (C) Representative images from day 5, case 3. (D) Organoid area on day 1 and 5, case 3. (E) Transformed organoid area on day 5, all cases. Graphs display mean and standard error of the mean (SEM). One-way ANOVA with Tukey’s correction for multiple comparisons in individual case analysis; for aggregate data analysis, data was transformed by dividing each case by the average of its respective control group and analyzed with main effects-only two-way ANOVA with Tukey’s correction for multiple comparisons. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 2

Human Milk Increases Organoid Proliferation. (A) Mean fluorescence intensity (MFI) of Ki67 staining in growth phase organoids, case 3 and case 4. (B) Transformed MFI of Ki67 staining in growth phase organoids, all cases. (C) Representative growth phase images from case 3 when available; control from case 4. (D) MFI of cleaved caspase-3 (CC3) staining in growth phase organoids, representative case 3. (E) Transformed MFI of CC3 staining in growth phase organoids, all cases. (F) Representative growth phase images from case 3 when available; control from case 4. Graphs display mean and standard error of the mean (SEM). One-way ANOVA with Tukey’s correction for multiple comparisons in individual case analysis; for aggregate data analysis, data was transformed by dividing each case by the average of its respective control group and analyzed with main effects-only two-way ANOVA with Tukey’s correction for multiple comparisons. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 3

Human Milk Improves Organoid Differentiation. (A) Percentage of differentiating organoids per hpf, day 8, case 3 and transformed percentage of differentiating organoids per hpf, day 8, all cases. (B) Number of buds/organoid, day 8, case 3 and transformed number of buds/organoid, day 8, all cases. (C) Bud length, day 8, case 3 and transformed bud length, day 8, all cases. (D) Bud diameter, day 8, case 3, and transformed bud diameter, day 8, all cases. (E) Example bud measurement and representative day 8 images from case 3. Graphs display mean and standard error of the mean (SEM). One-way ANOVA with Tukey’s correction for multiple comparisons in individual case analysis; for aggregate data analysis, data was transformed by dividing each case by the average of its respective control group and analyzed with main effects-only two-way ANOVA with Tukey’s correction for multiple comparisons. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 4

Parental Milk Induces Enteroendocrine Cell Differentiation. (A) MFI of chromogranin-A (CHGA) staining in growth phase organoids, case 3. (B) Transformed MFI of CHGA staining in growth phase organoids, all cases. (C) Representative growth phase images from case 3. (D) MFI of CHGA staining in differentiation phase organoids, case 3. (E) Transformed MFI of CHGA staining in differentiation phase organoids, all cases. (F) Representative differentiation phase images from case 3 when available; HF from case 4. Graphs display mean and standard error of the mean (SEM). One-way ANOVA with Tukey’s correction for multiple comparisons in individual case analysis; for aggregate data analysis, data was transformed by dividing each case by the average of its respective control group and analyzed with main effects-only two-way ANOVA with Tukey’s correction for multiple comparisons. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 5

Bulk Sequencing Demonstrates Differential Wnt Signaling. (A) Heatmap showing differential gene expression (DGE) between HM and formula supplemented organoids. (B) Volcano plot showing DGE of HM supplemented organoids. (C) Volcano plot showing DGE of formula supplemented organoids. (D) Wnt signaling pathway schematic derived from DAVID Functional Annotation Analysis and created with BioRender.com with genes downregulated in both organoids supplemented with human milk and formula (red) and those supplemented with formula (blue) highlighted.

Figure 6

Human Milk Increases Immunomodulatory Cytokines. (A) Concentration of EGF, out of range (OOR) > values set to 7,000,000. (B) Concentration of FGF-2. (C) PCA plot of cytokine concentration levels in media. (D) Volcano plot showing DGE of HM supplemented organoids. (E) Concentration of TRAIL. (F) Concentration of LIF. Media pooled from day 5 of growth and day 3 of differentiation (day 8). OOR < set to 0. Graphs display mean and standard error of the mean (SEM). One-way ANOVA with Tukey’s correction for multiple comparisons in individual cytokine analysis. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. PCA, principal component analysis.

Figure 7

DEG and Cytokines Upregulated by Human Milk are Reduced in NEC. (A) Volcano plot showing DGE of neonatal samples compared to NEC samples in RNA bulk sequencing data from Egozi et al, 2023. Genes and cytokines up/downregulated in human milk organoids are highlighted. (B) Volcano plot showing DGE of neonatal samples compared to NEC samples in single cell sequencing data from Egozi et al, 2023. Genes and cytokines up/downregulated in human milk organoids are highlighted. P < .05 considered significant.