Development of Colonic Organoids Containing Enteric Nerves or Blood Vessels from Human Embryonic Stem Cells

Abstract

The increased interest in organoid research in recent years has contributed to an improved understanding of diseases that are currently untreatable. Various organoids, including kidney, brain, retina, liver, and spinal cord, have been successfully developed and serve as potential sources for regenerative medicine studies. However, the application of organoids has been limited by their lack of tissue components such as nerve and blood vessels that are essential to organ physiology. In this study, we used three-dimensional co-culture methods to develop colonic organoids that contained enteric nerves and blood vessels. The development of enteric nerves and blood vessels was confirmed phenotypically and genetically by the use of immunofluorescent staining and Western blotting. Colonic organoids that contain essential tissue components could serve as a useful model for the study of colon diseases and help to overcome current bottlenecks in colon disease research.

Keywords: blood vessel; colon; enteric nervous system; human embryonic stem cell; organoid.

Figure 1

Timeline for the development of enteric nervous system (ENS) and blood vessel-containing human colonic organoids (HCOs) from human embryonic stem cells (hESCs). The dashed line indicates the 3D co-culture.

Figure 2

Development and characterization of HCOs. (A) HCO differentiation protocol. (B) Expression of pluripotency markers (OCT4 and NANOG) and endoderm differentiation markers (SOX17 and FOXA2) in the definitive endoderm (DE) of HCOs and untreated hESCs as detected via qRT-PCR. SOX17 and FOXA2 expression was detected by Western blotting, with GAPDH used as a loading control. (C) Hindgut (HG) morphology on bright-field (BF) microscopy and H&E staining. Expression of hindgut markers (CDX2 and KLF5) as detected via qRT-PCR. (D) Development of morphologic changes in HCOs over time. Crypt-like structures had formed by day 28. Expression of colon-specific marker (SATB2); posterior HOX markers (HOXA13, HOXB13, HOXD12, and HOXD13); adult stem cell marker (LGR5); goblet cell markers (MUC2, MUC3, and MUC4); and markers for paneth cells (DEFA5), enteroendocrine cells (CHGA), and enterocytes (villin) by HCOs as detected via qRT-PCR. Detection of SATB2, LGR5, MUC4, DEFA5, CHGA, and villin by Western blotting, with GAPDH used as the loading control. The scale bars in panels (a–e) indicate 200 µm; the scale bar in panel (f) indicates 100 µm. (E): H&E and immunofluorescent staining of HCOs. The scale bar in panel (a) indicates 200 µm; the scale bar in panel (b) indicates 50 µm. * p ≤ 0.1, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. Scale bar: 200 µm.

Figure 2

Development and characterization of HCOs. (A) HCO differentiation protocol. (B) Expression of pluripotency markers (OCT4 and NANOG) and endoderm differentiation markers (SOX17 and FOXA2) in the definitive endoderm (DE) of HCOs and untreated hESCs as detected via qRT-PCR. SOX17 and FOXA2 expression was detected by Western blotting, with GAPDH used as a loading control. (C) Hindgut (HG) morphology on bright-field (BF) microscopy and H&E staining. Expression of hindgut markers (CDX2 and KLF5) as detected via qRT-PCR. (D) Development of morphologic changes in HCOs over time. Crypt-like structures had formed by day 28. Expression of colon-specific marker (SATB2); posterior HOX markers (HOXA13, HOXB13, HOXD12, and HOXD13); adult stem cell marker (LGR5); goblet cell markers (MUC2, MUC3, and MUC4); and markers for paneth cells (DEFA5), enteroendocrine cells (CHGA), and enterocytes (villin) by HCOs as detected via qRT-PCR. Detection of SATB2, LGR5, MUC4, DEFA5, CHGA, and villin by Western blotting, with GAPDH used as the loading control. The scale bars in panels (a–e) indicate 200 µm; the scale bar in panel (f) indicates 100 µm. (E): H&E and immunofluorescent staining of HCOs. The scale bar in panel (a) indicates 200 µm; the scale bar in panel (b) indicates 50 µm. * p ≤ 0.1, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. Scale bar: 200 µm.

Figure 3

Development of HCOs containing ENS. (A) Ectoderm differentiation protocol and the expression of pluripotency markers (OCT4 and NANOG) and ectoderm differentiation markers (NESTIN and OTX2) as detected via qRT-PCR. (B) Neural crest differentiation protocol and the expression of neural crest differentiation markers (ZIC1, SOX10, and FOXD3) as detected via qRT-PCR. (C) Development of HCOs with ENS development protocol. Morphology of HCO containing ENS at different time points. Detection of SATB2 and TUJ1 by Western blotting, with GAPDH used as the loading control. H&E staining of HCO containing ENS, and immunofluorescent staining of of TUJ1. Expression of neuron differentiation markers (TUJ1 and NDRG4), and colon-specific marker (SATB2) as detected via qRT-PCR. ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. n.s. indicates non significance. The scale bars indicate 200 µm.

Figure 4

Development of HCOs containing blood vessels. (A) Timeline for development of blood vessel-containing HCOs. (B) Expression of pluripotency markers (OCT4 and NANOG) and mesoderm differentiation markers (EOMES and MIXL1) as detected via qRT-PCR. (C) The morphology of blood vessel structures at days 8, 16, 20, 24, and 28. Immunofluorescent staining of blood vessel endothelial cell marker (CD31). Detection of SATB2 and CD31 by Western blotting, with GAPDH used as the loading control. H&E staining of HCO containing blood vessels, and immunofluorescent staining of CD31. Expression of colon-specific marker (SATB2) and blood vessel endothelial cell marker (CD31) as detected via qRT-PCR. n.s. indicates non significance. * p ≤ 0.1, ** p ≤ 0.01, and *** p ≤ 0.001. All the scale bars indicate 200 µm.

Figure 4

Development of HCOs containing blood vessels. (A) Timeline for development of blood vessel-containing HCOs. (B) Expression of pluripotency markers (OCT4 and NANOG) and mesoderm differentiation markers (EOMES and MIXL1) as detected via qRT-PCR. (C) The morphology of blood vessel structures at days 8, 16, 20, 24, and 28. Immunofluorescent staining of blood vessel endothelial cell marker (CD31). Detection of SATB2 and CD31 by Western blotting, with GAPDH used as the loading control. H&E staining of HCO containing blood vessels, and immunofluorescent staining of CD31. Expression of colon-specific marker (SATB2) and blood vessel endothelial cell marker (CD31) as detected via qRT-PCR. n.s. indicates non significance. * p ≤ 0.1, ** p ≤ 0.01, and *** p ≤ 0.001. All the scale bars indicate 200 µm.