Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived

Abstract

In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca(2+) (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3-5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1-3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3-4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic epithelium.

Conflict of interest statement

DISCLOSURE/CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1

Growth of normal human colonic crypts in culture. Markers of growth and differentiation over time in cultured crypts isolated from histologically normal tissue (resected from two patients, representative of 15 total). Light microscopy images at (a) 40 × and (b) 200 × (63-year female). (c) The proliferation marker Ki67 (orange) and differentiation marker E-cadherin (green) (37-year male).

Figure 2

Normal human colonic crypts in culture stained for markers of differentiation, CK20 and MUC2. (a, b) Cytokeratin 20 (green), a marker of mature colon epithelial cells and nuclear DAPI (blue) (64-year male, ascending colon). (c, d) Segregation of CK20 and goblet cell marker MUC2 (red) (41-year female, descending colon). Insets: typical normal colonic mucosa immunoperoxidase-stained for CK20 and MUC2.

Figure 3

Human adenoma in culture. (a) Resected adenoma (preisolation) immunoperoxidase-stained for Ki67 and E-cadherin. (b–d) Cultured adenoma light microscopy images show structural changes over time; budding and tubular formation (white arrow) from the central parent structure (black arrow). (d) At day 15, the adenoma is stained for the proliferation marker Ki67 (orange) and the differentiation marker E-cadherin (green); nuclear DAPI (blue; whitish co-expression with red and green stains). The tissue originated from an 18-year female (donor-1) with a large adenoma and no evidence of familial adenomatous polyposis.

Figure 4

Adenoma-derived structures in culture stained for markers of differentiation, CK20 and MUC2. Resected adenoma (preisolation) immunoperoxidase-stained for (a) CK20 and (b) MUC2. (c) Cultured structures predominately expressed MUC2 at exterior proliferative regions. Inset: CK20 (and MUC2 co-staining) is limited to confocal optical slices interior of the largely MUC2-positive exterior. Donor-1: 18-year female. Donor-2: 69-year female. Donor-3: 51-year male. All no evidence of familial adenomatous polyposis.

Figure 5

Growth of adenoma in culture: segregation of markers for growth and differentiation. (a–f) Proliferating bud regions (white arrows) and parent structure with connecting bud stem regions (black arrows). (a, b) Confocal immunofluorescence at day 24 (donor-2) showing segregation of Ki67 to proliferating outer regions and CK20 to parent structure. (b) Representative high magnification of panel a immunofluorescence marker expression. (c–f) Immunoperoxidase stained cross-section of one structure with multiple budding growths (third passage; day 73). (c) Localization of CK20 to inner epithelium adjoining parent structure. (d) High magnification of buds with proliferating Ki67-positive outer regions. (e) MUC2-positive pre-goblet (overlapping proliferative regions in panel c) and mature goblet cells (overlapping CK20-positive regions in panel c). (f) Cross-sectional cut further into paraffinembedded adenoma showing more parent structure. E-cadherin uniformly expressed throughout epithelium.

Figure 6

Expression of pro-apoptotic marker Bax and apoptotic marker, cleaved Caspase-3 in cultured normal colonic crypts and adenoma. Immunoperoxidase-stained paraffinembedded normal crypts before/after 3 days in culture and adenoma (donor-2) before/after 73 days in culture (main image scale bar 100 mm). Insets: regions enlarged to demonstrate marker localization (scale bars 25 mm). (a–d) Bax as punctate and diffuse cytoplasmic staining, and (e–h) cleaved Caspase-3 as punctate cytoplasmic staining (white arrows) and stained shed bodies (black arrows). (d, top inset) Ki67 expression of a parallel serial section (scale bar 100 µm).

Figure 7

Expression of intestinal stem cell marker Lgr5 in normal colonic mucosa and cultured adenoma. (a, b) Examples of Lgr5 immunoperoxidase-stained normal colonic mucosa from two subjects, demonstrating localized specificity to a few basal cells (inset 2 × image). (c–f) Confocal immunofluorescence with differential interference contrast; Lgr5 (red) and nuclear DAPI (blue). (c) Time-zero isolated normal crypt. (d) Preisolation adenoma. (e) Cultured adenoma (donor-2) at day 42 and (f) day 120.