Cell Lineage Identification and Stem Cell Culture in a Porcine Model for the Study of Intestinal Epithelial Regeneration

Abstract

Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC) driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM) and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA), Minichromosome Maintenance Complex 2 (MCM2), Bromodeoxyuridine (BrdU) and phosphorylated Histone H3 (pH3) distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/‘reserve’ stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2); enteroendocrine cells by Chromogranin A (CGA), Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII) and sucrase isomaltase (SIM). Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.

Porcine models of digestive disease: the future of large animal translational research

“…For example, the villous projections within the small intestine of both species are finger shaped, whereas in rats they are flat leaf-like structures (30). Additionally, the cell lineages that constitute the intestinal epithelium, their phenotypic appearance, and their expression of unique protein biomarkers used for cellular identification are similar in pigs and humans (68). These include the ultrastructural appearance of stem, goblet, and enteroendocrine cells as well as absorptive enterocytes (68).…”

“…Additionally, the cell lineages that constitute the intestinal epithelium, their phenotypic appearance, and their expression of unique protein biomarkers used for cellular identification are similar in pigs and humans (68). These include the ultrastructural appearance of stem, goblet, and enteroendocrine cells as well as absorptive enterocytes (68). A comparative analysis of sub-cellular features of porcine intestinal epithelial cells located within the crypt base, using transmission electron microscopy, revealed multiple irregularly shaped, small, columnar cells with basally located nuclei and scarce cytoplasm (68).…”

“…These include the ultrastructural appearance of stem, goblet, and enteroendocrine cells as well as absorptive enterocytes (68). A comparative analysis of sub-cellular features of porcine intestinal epithelial cells located within the crypt base, using transmission electron microscopy, revealed multiple irregularly shaped, small, columnar cells with basally located nuclei and scarce cytoplasm (68). This morphologic appearance is consistent with the description of crypt base columnar stem cells in humans (68, 69).…”

“…A comparative analysis of sub-cellular features of porcine intestinal epithelial cells located within the crypt base, using transmission electron microscopy, revealed multiple irregularly shaped, small, columnar cells with basally located nuclei and scarce cytoplasm (68). This morphologic appearance is consistent with the description of crypt base columnar stem cells in humans (68, 69). Taken together, these features contribute to fundamental digestive processes such as mucosal transport and motility that are similar between pigs and humans (19).…”

“…However, based on recent work, a cell sharing ultrastructural qualities attributed to the Paneth cell, including large size, elongated flattened nucleus and located adjacent to the crypt base columnar cells has been identified within porcine small intestine (68). These cells lack certain morphological features historically used to identify Paneth cells such as electron dense apically situated granules.…”