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.
Highlights
Complete physiologic renewal of the intestinal epithelium occurs in approximately one week and is driven by a pool of intestinal epithelial stem cell (IESC) at the crypt base [1]
This study focuses on eliminating many of the obstacles that limit the pig as a translational model to study IESC-driven regeneration of the intestinal epithelium
The utility of the pig as a large animal model has been well-documented for many body systems [15,16,17,18,19,20,21,82,83,84,85,86,87,88]; and the similarities between the pig and human gastrointestinal system position the pig as a promising species for animal models of gastrointestinal disease
Summary
Complete physiologic renewal of the intestinal epithelium occurs in approximately one week and is driven by a pool of IESCs at the crypt base [1]. Logistical and ethical issues minimize the use of humans or human- derived tissues for research and discovery pertaining to conditions of the intestinal epithelium These obstacles highlight the need for a research model that closely mimics human intestinal anatomy, physiology, disease and injury processes. Rats and mice in particular represent an important, cost effective animal model for basic genetic, cellular and molecular biology of IESC-driven regeneration of the intestinal epithelium. Despite these advantages, significant differences between rodents and humans confound or prohibit translational studies [7]
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