Int J Stem Cells.  2023 Aug;16(3):269-280. 10.15283/ijsc23026.

Peripheral Neuron-Organoid Interaction Induces Colonic Epithelial Differentiation via Non-Synaptic Substance P Secretion

• Che YH
• Choi IY
• Song CE
• Park C
• Lim SK
• Kim JH
• Sung SH
• Park JH
• Lee S
• Kim YJ

• KMID: 2545227
• DOI: http://doi.org/10.15283/ijsc23026

Abstract

Background and Objectives
The colonic epithelial layer is a complex structure consisting of multiple cell types that regulate various aspects of colonic physiology, yet the mechanisms underlying epithelial cell differentiation during development remain unclear. Organoids have emerged as a promising model for investigating organogenesis, but achieving organ-like cell configurations within colonic organoids is challenging. Here, we investigated the biological significance of peripheral neurons in the formation of colonic organoids.
Methods and Results
Colonic organoids were co-cultured with human embryonic stem cell (hESC)-derived peripheralneurons, resulting in the morphological maturation of columnar epithelial cells, as well as the presence of enterochromaffin cells. Substance P released from immature peripheral neurons played a critical role in the development of colonic epithelial cells. These findings highlight the vital role of inter-organ interactions in organoid development and provide insights into colonic epithelial cell differentiation mechanisms.
Conclusions
Our results suggest that the peripheral nervous system may have a significant role in the development of colonic epithelial cells, which could have important implications for future studies of organogenesis and disease modeling.

Keyword

Organogenesis; Colonic organoid; Gut development; Peripheral neuron; Inter-organ crosstalk; Pluripotent stem cell

Figure

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  Fig. 1 Stepwise protocol for generating colonic organoids. (A) Schematic diagram showing the modification of previously reported protocols for colonic organoids, with or without bone morphogenetic protein (BMP) signaling stimulation. (B) Decrease in pluripotency markers OCT4 and NANOG during differentiation process. (C) Expression level of mRNA for SOX17 and FOXA2 on day 3 of differentiation towards definitive endoderm (DE). (D) Immunoblot images of protein expression for SOX17 and FOXA2 on the differentiation stage of DE. GAPDH was used as an internal control. (E) Cells expressing a protein for SOX17 and FOXA2 on the DE stage. (F) Counting results for SOX17 and FOXA2 expressing cells from (E). (G) Expression level of mRNA for CDX1 and KLF5 on day 7 of differentiation towards hindgut (HG) cells. (H) Immunoblot images of protein expression for CDX1 and KLF5 on the differentiation stage of hindgut. GAPDH was used as an internal control. (I) Cells expressing a protein for CDX1 and KLF5 on the hindgut stage. (J) Counting results for CDX1 and KLF5 expressing cells from (I). (K) Schematic diagram showing the embedding process of hindgut cells that formed clusters into an artificial ECM matrix in clump form. (L) BMP4 stimulation during the embedding stage increases expression of hindgut hox genes responsible for posterior development. (M and N) Increase in mRNA and protein expression of colon-restricted chromatin factor SATB2 with BMP4 application. (O) Identification of a cell cluster expressing the SATB2 protein in the inner center of the colonic organoid. Data expressed as mean±SD (n=9, biological repeat). p-values calculated by one-way ANOVA (B, L, M) or unpaired t-test (C, F, G, and J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant. All scale bars=50 μm. DE: definitive endoderm, HG: hindgut, CO: colonic organoid.

• 

  Fig. 2 Characterization of long-term cultured colonic organoids embedded in an ECM matrix. (A) Morphological changes of colonic organoids during long-term culture. (B) Detection of Mucin proteins (MUC) within the organoids at week 6 using MUC2, MUC3, and MUC4 antibodies. (C) Representative FACS plot for EpCAM-expressing cells of colonic organoids. (D) Immunostaining confirmed the formation of an epithelial layer expressing EpCAM, CD133, or Villin on the outer layer of the organoids. (E) Detection of marker proteins representing various cell types within the epithelial layer, including goblet cells (MUC3, MUC4), enterocytes (Villin), Paneth cells (DEFA5), enteroendocrine cells (CHGA), and colonic stem cells (LGR5). (F) Swelling assay confirming the presence of functional epithelial cells in the organoids treated after forskolin. All scale bars=50 μm. CO: colonic organoid.

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  Fig. 3 Reciprocal interaction between colonic organoid and NCSC-derived peripheral neurons. (A) Schematic representation of co-culture of colon organoids with neural crest stem cells (NCSCs). (B) Representative images of NCSCs showing neuronal differentiation after 7 days of co-culture with colon organoids. (C) Representative FACS plot for NCSCs expressing PHOX2B after co-culture with colonic organoids. (D) Increased expression of PHOX2B and NDRG4 in FACS-isolated NCSCs after co-culture with colon organoids. (E) Enrichment of mRNA expression for LMX1A and CHGA in colon organoids after co-culture with NCSCs. (F) Schematic representation of transwell co-culture system. (G) Percentage of PHOX2B-expressing NCSCs induced by co-culture with colon organoids using direct contact or transwells. (H) Verification of PHOX2B-expressing cells after co-culture with colon organoids. (I) Expression of NDRG4 and LMX1A in colon organoids after co-culture with NCSCs using transwells. (J) Differentiated neuronal cells expressing TUJ1 containing serotonin (lower panel) and epithelial cells of colon organoids labeled with eGFP. Columnar morphology of epithelial cells are indicated by dotted red line in the right panel. (K) Proportion of EpCAM-expressing epithelial cells in colon organoids after co-culture with NCSCs. (L) Representative immunostaining image of Ki-67-expressing proliferating cells within colon organoids after co-culture with NCSCs, and the counting result. (M) Representative FACS plot for the isolation of EpCAM-expressing cells of colonic organoid, and representative images for attached cells after re-plating. (N) SOX9 expression in proliferating cells from EpCAM-expressing cells after 10 days of re-cultivation. Data expressed as mean±SD (n=9, 12 for G, biological repeat). p-values calculated by unpaired t-test (D, E, H, I, K, L) or one-way ANOVA (G). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant. All scale bars=50 μm. CO: colonic organoid.

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  Fig. 4 Developing neurons influence colonic organoids. (A) Tracking the expression changes of PHOX2B and NDRG4 in NCSCs and LMX1A in colonic organoids according to the co-culture period of colonic organoids and NCSCs. (B) Immunostaining images of NCSC-derived neurons expressing TUJ1 as a key difference between co-culture time points of 4 and 6 days. (C) Significant increase in substance P in the co-culture medium on day 6 compared day 4, as measured by ELISA. (D) Expression of colonic epithelial cell marker proteins of colonic organoid after substance P administration were validated by immunoblotting. (E) Scattering distribution of cells expressing CHGA in the colonic organoid was confirmed using immunofluorescence staining. (F) Presence of enteroendocrine cells detectable by 5-HT within the colonic organoid, and columnar epithelial cell morphology (red dotted line). (G) The expression of NK1R and NK3R mRNA in colonic organoids was validated using qRT-PCR, with or without treatment with substance P. (H) Protein expression levels of CHGA in colonic organoids were measured using ELISA after inhibition of NK1R with specific antagonists. Data expressed as mean±SD (n=s, biological repeat). p-values calculated by two-way ANOVA (A and C) or one-way ANOVA (G and H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant. Scale bars=25 μm (B and E) or 10 μm (F). CO: colonic organoid, SP: substance P.

Reference

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