Human Colonoid-Myofibroblast Coculture for Study of Apical Na+/H+ Exchangers of the Lower Cryptal Neck Region

Abstract

Cation and anion transport in the colonocyte apical membrane is highly spatially organized along the cryptal axis. Because of lack of experimental accessibility, information about the functionality of ion transporters in the colonocyte apical membrane in the lower part of the crypt is scarce. The aim of this study was to establish an in vitro model of the colonic lower crypt compartment, which expresses the transit amplifying/progenitor (TA/PE) cells, with accessibility of the apical membrane for functional study of lower crypt-expressed Na⁺/H⁺ exchangers (NHEs). Colonic crypts and myofibroblasts were isolated from human transverse colonic biopsies, expanded as three-dimensional (3D) colonoids and myofibroblast monolayers, and characterized. Filter-grown colonic myofibroblast-colonic epithelial cell (CM-CE) cocultures (myofibroblasts on the bottom of the transwell and colonocytes on the filter) were established. The expression pattern for ion transport/junctional/stem cell markers of the CM-CE monolayers was compared with that of nondifferentiated (EM) and differentiated (DM) colonoid monolayers. Fluorometric pH_i measurements were performed to characterize apical NHEs. CM-CE cocultures displayed a rapid increase in transepithelial electrical resistance (TEER), paralleled by downregulation of claudin-2. They maintained proliferative activity and an expression pattern resembling TA/PE cells. The CM-CE monolayers displayed high apical Na⁺/H⁺ exchange activity, mediated to >80% by NHE2. Human colonoid-myofibroblast cocultures allow the study of ion transporters that are expressed in the apical membrane of the nondifferentiated colonocytes of the cryptal neck region. The NHE2 isoform is the predominant apical Na⁺/H⁺ exchanger in this epithelial compartment.

Keywords: SLC9 family; claudins; colonic electrolyte transport; human intestinal organoids; intestinal barrier; pHi regulation; sodium–hydrogen exchange.

Figure 1

Colonoids generated from crypts isolated from human transverse colon and cultured in nondifferentiating (expansion) medium and in differentiating medium lacking Wnt signaling stimuli. (A) The morphological features of 3D colonoids at day 4 in the respective expansion or differentiation medium. (B) IHC staining of whole mount organoids with many proliferative (KI67-positive) cells when cultured in expansion medium (upper panels), and with differentiation markers MUC2, NHE3 and SLC26A3 when cultured in differentiation medium (lower panels). Scale bar 50 µm. The dotted squares indicate the areas whose magnification is displayed in the upper right and left corners of the respective micrographs.

Figure 2

Expression of ion transporters in human colonoid cultures and colon tissue. (A) Log₁₀ fold change in mRNA expression levels of a panel of genes (mostly coding for ion transport proteins) between the nondifferentiated state (3D in expansion medium for several days after the removal of Y-27632 and CHIR-99021), representing cryptal base and lower neck cells, and the differentiated state, representing the cells in the cryptal mouth/surface. n ≥ 5 separate cultures. Genes that assemble close to zero in this graph likely have a homogenous expression pattern along the cryptal axis. (B) Western blot analysis of the absorptive enterocyte markers SLC26A3 and NHE3 in nondifferentiated and differentiated 3D colonoids. (C) Formalin-fixed, paraffin-embedded (FFPE) section of human transverse colon stained for SLC26A3 (green) and nuclei (blue). SLC26A3 is strongly expressed on the luminal side of mucosal cells from the surface and crypt neck regions, where colonic epithelium is terminally differentiated. (D) Expression of SLC26A3 in human colonoid monolayer after four days of differentiation. 3D reconstruction of a region of monolayer (left panel) shows that SLC26A3 is expressed at different levels in different cells. Right panel shows top view and cross-section of the selected area in higher magnification where SLC26A3 is apically localized. Green: SLC26A3, red: F-actin, blue: nuclei (excluded in XYZ).

Figure 3

Morphological features and mRNA expression panel of myofibroblast cultured from colonic lamina propria. (A) Immunohistochemical staining for smooth-muscle actin (SMA), CD90, phalloidin (F-actin) and DAPI (nuclei). (B) Myofibroblast (Myofibr.) mRNA expression of a panel of genes encoding for proliferative and differentiation factors in the myofibroblasts, in comparison to HEK293T cells. n = 5–7 different myofibroblast cultures, t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Colonic myofibroblast (CM)–colonic epithelial cells (CE) coculture system. (A) Schematic diagram of the CM-CE transwell monolayer cultures. (B) Development of the transepithelial electrical resistance (TEER) during the days of the monolayers in culture. In the subconfluent phase, the colonoid cells were cultured in “expansion” medium, the myofibroblasts were plated onto the bottom of transwell plates as described in material and methods. After confluency, the transwell cups were placed in wells containing myofibroblast monolayers at the bottom (coculture) or in wells containing expansion medium without myofibroblasts. TEER increased much faster in the CM-CE cocultures. (C) IHC for KI67 (surface rendering in 3D reconstructions for facilitated counting of KI67-positive nuclei (yellow) and MUC2 positive mucin granules (green) in 2D organoid cultures, EM: cultured for 4 days after confluency in expansion medium, CM-CE: 4 days coculture, DM: 4 days in differentiation medium. Ratio of KI67 positive nuclei versus total nuclei was quantified and illustrated in (D). t-test, ** p < 0.01.

Figure 5

Different stages of colonoid cultures represent different regions of the colonic crypts in gene expression profile. Relative mRNA expression of a panel of proliferation/differentiation markers in addition to ion transporters with differential spatial expression along cryptal axis is shown in stem cell enriched 3D colonoids (SE) and non-differentiated (EM), CM-CE and differentiated (DM) colonoid monolayers; t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Gene expression for a panel of tight junction proteins in different stages of colonoid culture. (A) Colonoids grown as stem cell enriched 3D organoids (SE), as monolayers in expansion medium (EM), as CM-CE cocultures, and as differentiated monolayers (DM) for four days. The conspicuous difference between EM and CM-CE monolayers is the downregulation of claudin-2. t-test, * p < 0.05, *** p < 0.001. (B) IHC of SE, EM, and CM-CE monolayers, displaying the dramatic downregulation of claudin-2 (Green: CLDN2, blue: nucleus, yellow: ZO-1, red: F-actin).

Figure 7

NHE expression and activity in CM-CE monolayers. (A) The mRNA expression levels of NHE isoforms in SE 3D organoids and EM, CM-CE and DM monolayers, characterized in Figure 4, Figure 5 and Figure 6. (B,C) Basolateral NHE activity analyzed fluorometrically in the CM-CE monolayers by removal and re-addition of Na⁺ to the basolateral perfusate. (D–I) Apical NHE activity in the CM-CE monolayers measured by removal and re-addition of Na⁺ to the apical perfusate in the absence or presence of NHE-specific inhibition. For apical NHE activity measurements, the basolateral NHE1 was inhibited by removal of Na⁺ and addition of 3 µM HOE642 in the basolateral perfusate. Representative pH_i traces for apical NHE activity in control (D), with 3 μM HOE642 to inhibit NHE8 (E), with 3 μM HOE642 plus 1 μM tenapanor to inhibit NHE3 and NHE8 (F), and with 60 μM HOE642 to inhibit NHE8 and NHE2 (G) conditions are illustrated. (H) The pH_i recovery rates shown as ΔpH/min without and with the different inhibitor concentrations. (I) Method of estimating the respective NHE isoform activities: Na⁺-dependent pH_i recovery rate in the absence of inhibitors was set to 100%, and by subtracting the respective percentage rates in the presence of the respective inhibitor concentrations from the total apical activity (100%), the relative NHE-specific activities were calculated. The calculated exact values in percentage of total apical NHE activity are as follows: NHE2 82 ± 10.7; NHE8 21.7 ± 17.2; NHE3 1.21 ± 12.9 (each dot represents one individual CM-CE monolayer, n = 5, mean ± SEM, t-test, * p < 0.05, *** p < 0.001).