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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 8, 2003 as doi:10.1096/fj.02-0852fje. |
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The Wistar Institute, Philadelphia, Pennsylvania, USA;
* Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA;
Institute Cancer Research, University of Vienna, Vienna, Austria;
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA;
Department of Internal Medicine I, University of Regensburg, Regensburg, Germany; and
¶ Department of Biostatistics and Epidemiology, Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, Pennsylvania, USA
2Correspondence: The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104, USA. E-mail: herlynm{at}wistar.upenn.edu.
SPECIFIC AIMS
The colonic epithelium undergoes a continuous cycle of proliferation, differentiation, and apoptosis. To characterize factors important for colonic homeostasis and its dysregulation, we developed a novel organotypic culture system of human fetal colonic epithelial cells seeded on a collagen type I matrix with embedded colonic fibroblasts.
PRINCIPAL FINDINGS
1. Development of an organotypic culture model of normal human enteric epithelium
Isolated epithelial were seeded on top of collagen gels with embedded colonic fibroblast. Cells began spreading from clusters 6 h after seeding (Fig. 1
a) and migrated to cover the entire matrix (Fig. 1b-d
, inserts). Constriction of collagen by the fibroblasts started after 24 h and was maximal by day 4, when the matrix had shrunk to 
40% of its original length (Fig. 1d
). As the collagen matrix constricted, epithelial cell morphology changed from flat during the initial migratory phase (Fig. 1a
) to columnar and polarized by day 4 (Fig. 1b
). After 10 days, epithelial cells covered all sides of the collagen matrix (Fig. 1c
). At this time, 2–10% of epithelial cells stained positive for the proliferation marker Ki67 and up to 70% had incorporated BrdU when continuously added to the medium starting at the time of seeding. Total cell numbers began to decrease by day 20, when cells flattened (Fig. 1d
). Mucin-producing goblet cells (Fig. 1b
, arrowheads) and enteroendocrine cells were distributed throughout the epithelial cell layer. Fibroblasts remained dispersed throughout the collagen except for some individual 
-SMA-positive cells migrating close to the epithelial layer (Fig. 1e
), also seen in normal human colon (Fig. 1f
). Cells grown in monolayer or on a collagen matrix without fibroblasts did not proliferate on day 4 as determined by 3H-thymidine incorporation assay and Ki67 immunohistochemistry; they flattened and died by day 8 at the latest.
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2. Carbonic anhydrase isoenzyme II (CA II) is a differentiation marker for fetal colonic enterocytes
CA II is expressed in the cytoplasm of epithelial cells in the upper crypts and villi of the fetal human colon. The percentage of epithelial cells positive for CA II expression was higher than for alkaline phosphatase activity, found at the top of the villi. All epithelial cells were positive for cytokeratin 19.
3. LIF stimulates proliferation but not differentiation of epithelial cells
To identify growth factor effects on epithelial cell, each was added as a single component to the base medium in the absence of serum. When only LIF was added to base medium, the cells flattened and proliferated (day 3; Fig. 2
a); LIF-stimulated cultures formed clusters of polarized epithelial cells by day 7 (Fig. 2b
). Control cultures maintained in the absence of LIF remained round (day 3; Fig. 2d
) and cell numbers remained low (day 7; Fig. 2e
). In the absence of fibroblasts in the collagen matrix, LIF could not sustain cell survival (Fig. 2c
), nor did cells survive in the absence of LIF or fibroblasts (Fig. 2f
).
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Base medium with LIF and 2% FCS allowed migration of the epithelial cells around the entire matrix and formation of a complete monolayer (Fig. 2g
), whereas cells in the absence of LIF covered only a portion of the matrix (Fig. 2h
). Growth of epithelial cells was significantly stimulated by LIF (Fig. 2i-l
). Cells showed significant incorporation of BrdU (Fig. 2i
) with increased cell numbers per area (Fig. 2k
). 3H-thymidine incorporation of epithelial cells was significantly increased on day 3 even in absence of fibroblasts (Fig. 2j
). The proliferation was solely attributable to LIF-mediated stimulation of epithelial cells, not of fibroblasts (Fig. 2l
). LIF did not change collagen constriction by fibroblasts.
Goblet cells represented 4% of all enterocytes on day 10, but differences in relative numbers of LIF-cultured vs. control base medium cultured cells were not significant. There was a trend toward increased numbers of goblet cells per area in LIF-stimulated samples. Cells cultured for 10 days in the presence of BrdU were than double stained for Alcian blue. Approximately one-third of the goblet cells had proliferated during this period. By contrast, relative numbers of enteroendocrine cells were significantly decreased in the presence of LIF. Cultures labeled for 10 days with BrdU were stained, then counterstained with chromogranin A for detection of enteroendocrine cells, revealed no proliferating cells.
CONCLUSIONS AND SIGNIFICANCE
The culture of colonic epithelial cells has proved notoriously difficult. We used an approach that provides a collagen substrate with stromal cells to closely mimic physiological conditions. In 3-dimensional organotypic culture, the isolated human fetal colonic epithelial cells are maintained in their native milieu. LIF was the most significant factor for proliferation of enterocytes but it did not induce differentiation.
In summary, our model provides a novel approach to investigate colon epithelial cell proliferation and differentiation and to dissect the role of individual growth factors in the cross-talk between epithelial and mesenchymal compartments.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0852fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0852fje 
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