Human stool is a heterogeneous mixture of non-digestible food residues, bacteria, cells exfoliated from the gastrointestinal mucosa and other secretory products. We have demonstrated that fresh human stools dispersed in a buffered saline solution can be fractionated over Percoll/BSA gradients to yield 9 discrete bands of cells in the density range of rho 1.033 to 1.139 and which could be further purified over Histopaque 1077. Enzyme-linked immunoassays (ELISA) for colon-specific antigen (CSA) and cytokeratins (CK) were positive. Western blot analysis showed the presence of 3 cytokeratin bands in the 40-kDa to 60-kDa range suggestive of cytokeratins 8, 18, and 19. Fluorescence flow-cytometric analysis of these cells using antibodies against CSA, CK, the blood-group antigens, carcinoembryonic antigen (CEA), non-mucus-secreting columnar-epithelium-specific MAb PR1A3, and to mucus-secreting colonic-epithelium-specific MAb PR5D5 showed varying degrees of reactivity. Expression of the blood-group phenotype suggests that cells from the proximal half of the colon had survived the transit, since in the adult expression of this marker is limited to cells from the proximal region of the colon. In this report we demonstrate the feasibility of studying, non-invasively, cell-specific markers on exfoliated cells isolated from stools. The evidence strongly suggests that almost all the cells are of colonic origin.
Human stools consist of a mixture of undigested food residues, colonic microflora, and cellular components shed from the walls of the gastrointestinal tract. The cellular components are made up mostly of terminally differentiated colonic epithelial cells. Using a combination of Percoll density gradient centrifugation and countercurrent centrifugal elutriation, it is now possible to recover these cells as an enriched fraction from fresh human stools. Cells can be visualized on heat-fixed smears of the enriched fractions stained with modified Wright's stain. The enrichment process is optimized by following the segregation of eukaryotic cells as determined by an ELISA technique using monoclonal antibodies against human double-stranded DNA. This work, demonstrating the feasibility of isolating intact colonic cells from stools, has important applications as a noninvasive approach to the biology of exfoliated cells from the gastrointestinal tract.
Understanding the nature of cell surface markers on exfoliated colonic cells is a crucial step in establishing criteria for a normally functioning mucosa. We have found that colonic cells isolated from stool samples (SCSR-010 Fecal Cell Isolation Kit, NonInvasive Technologies, Elkridge, MD), preserved at room temperature for up to one week, with viability of >85% and low levels of apoptosis (8% -10%) exhibit two distinct cell size subpopulations, in the 2.5 µM -5.0 µM and 5.0 µM -8.0 µM range. In addition to IgA, about 60% of the cells expressed a novel heterodimeric IgA/IgG immunoglobulin that conferred a broad-spectrum cell mediated cytotoxicity against tumor cells. In a cohort of 58 subjects the exclusive absence of this immunoglobulin in two African-Americans was suggestive of a germline deletion. Serial cultures in stem cell medium retained the expression of this heterodimer. Since a majority of the cystic cells expressed the stem cell markers Lgr5 and Musashi-1 we termed these cells as gastrointestinal progenitor stem cells (GIP-C**). CXCR-4, the cytokine co-receptor for HIV was markedly expressed. These cells also expressed CD20, IgA, IgG, CD45, and COX-2. We assume that they originated *This study was supported in part by NIH SBIR grants R44 DK56567 and R44 CA81799. **In this article, "SCSR cells" denote exfoliated cells isolated from stool samples and "GIP-C" are progenitor cells either present in SCSR cell isolates or cultured from SCSR cell isolates.
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