title: 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.
authors.affiliation: Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Liara M
authors.initials: LM
authors.lastname: Gonzalez
authors.forename: Ian
authors.initials: I
authors.lastname: Williamson
authors.forename: Jorge A
authors.initials: JA
authors.lastname: Piedrahita
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Blikslager
authors.forename: Scott T
authors.initials: ST
authors.lastname: Magness
mesh_terms: D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002453:Cell Cycle; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000502:physiology / Q000648:ultrastructure; D020397:Goblet Cells / Q000502:physiology* / Q000648:ultrastructure; D007413:Intestinal Mucosa / Q000502:physiology / Q000648:ultrastructure; D012038:Regeneration; D013234:Stem Cells / Q000502:physiology*; D034421:Sus scrofa
chemical_list: D015415:Biomarkers