Generation of an enteric smooth muscle cell line from the pig ileum

Abstract

Smooth muscle cells (SMCs) play an important role in physiology and production in farm animals such as pigs. Here, we report the generation of a pig SMC line. Our original objective was to establish an enteroendocrine cell line from the pig ileum epithelium through lentiviral transduction of the Simian Virus (SV) 40 large T antigen. However, an initial expression analysis of marker genes in nine cell clones revealed that none of them were enteroendocrine cells or absorptive enterocytes, goblet cells, or Paneth cells, some of the major cell types existing in the ileum epithelium. A more detailed characterization of one clone named PIC7 by RNA-seq showed that these cells expressed many of the known smooth muscle-specific or -enriched genes, including smooth muscle actin alpha 2, calponin 1, calponin 3, myosin heavy chain 11, myosin light chain kinase, smoothelin, tenascin C, transgelin, tropomyosin 1, and tropomyosin 2. Both quantitative PCR and RNA-seq analyses showed that the PIC7 cells had a high expression of mRNA for smooth muscle actin gamma 2, also known as enteric smooth muscle actin. A Western blot analysis confirmed the expression of SV40 T antigen in the PIC7 cells. An immunohistochemical analysis demonstrated the expression of smooth muscle actin alpha 2 filaments in the PIC7 cells. A collagen gel contraction assay showed that the PIC7 cells were capable of both spontaneous contraction and contraction in response to serotonin stimulation. We conclude that the PIC7 cells are derived from an enteric SMC from the pig ileum. These cells may be a useful model for studying the cellular and molecular physiology of pig enteric SMCs. Because pigs are similar to humans in anatomy and physiology, the PIC7 cells may be also used as a model for human intestinal SMCs.

Keywords: epithelium; ileum; pigs; small intestine; smooth muscle.

Figure 1.

Western blot analysis of SV40 T antigen protein in nine cell clones. Cell clones are indicated as 1 to 9. M, molecular weight ladder. NC (negative control) = pig ileum epithelial cells prior to SV40 T antigen transduction. β-tubulin was detected as a loading control.

Figure 2.

Correlation of gene expression levels estimated by two duplicated RNA-seq analyses of PIC7 cells. The x-axis represents gene expression levels as log₁₀(FPKM+1) from one RNA-seq analysis; the y-axis represents gene expression levels from the other RNA-seq analysis. R², the square of Pearson correlation coefficient.

Figure 3.

Representative micrographs of PIC7 cells. (A, B) Images of cells before reaching confluency; (C, D) Images of cells at confluency; (A, C) 100× magnification; (B, D) 200× magnification.

Figure 4.

Immunocytochemical detection of ACTA2 in PIC7 cells. ACTA2 staining is shown in red; nuclear staining by 4′,6-diamidino-2-phenylindole is shown in blue. (A, B, C) 200× magnification; (D, E, F) 400× magnification.

Figure 5.

Contractility analysis of PIC7 cells. (A) Representative photographs of collagen gel lattices embedded with PIC7 cells treated with 100 μM 5-HT or an equal volume of PBS (control) at different times of treatment. (B) Quantification of changes in gel surface areas from four independent experiments. Data are expressed as Mean ± SEM. *denotes P < 0.05 compared with control within time.