Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration

• Published in: PloS one 2013-06, Vol.8 (6), p.e66465-e66465
• Main Authors: Gonzalez, Liara M, Williamson, Ian, Piedrahita, Jorge A, Blikslager, Anthony T, Magness, Scott T
• Format: Article
• Language: English
• Subjects: Absorptivity; Animal models; Animals; Antibodies; Apoptosis; Bioindicators; Biology; Biomarkers; Biomarkers - metabolism; Biomedical engineering; Bromodeoxyuridine; Candidate species; Carbonic anhydrase; Carbonic anhydrase II; Cell culture; Cell Culture Techniques; Cell Cycle; Cell Differentiation; Cell division; Cell Lineage; Cell Proliferation; Cells (biology); Cells, Cultured; Crypts; Disease; Electron microscopy; Enterocytes; Epithelial cells; Epithelial Cells - physiology; Epithelial Cells - ultrastructure; Epithelium; Gastrin; Gene expression; Goblet cells; Goblet Cells - physiology; Goblet Cells - ultrastructure; Histone H3; Hogs; Intestinal Mucosa - physiology; Intestinal Mucosa - ultrastructure; Intestine; Ischemia; Medicine; Mice; Mortality; Mucin; Permeability; Physical characteristics; Physiology; Proliferating cell nuclear antigen; Reagents; Regeneration; Rodents; Small intestine; Somatostatin; Sox9 protein; Stem cells; Stem Cells - physiology; Studies; Suidae; Sus scrofa; Translation; Transmission electron microscopy
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0066465

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.