Isolation and Characterization of Bovine Intestinal Subepithelial Myofibroblasts

Abstract

Abstract.Intestinal subepithelial myofibroblasts (ISMFs) are mesenchymal cells that exist under the epithelium of intestines. Primarily isolated ISMFs from rodents have been applied to experiments. However, due to the size of their intestines, the available cell number is limited. Thus, we attempted to isolate ISMFs from bovine colon as an alternative material. After detachment of smooth muscle and epithelial layers, colonic mucosa was explanted. After 2-week incubation, α -SMA (+) / vimentin (+) / desmin (−) ISMFs were harvested and applied for experiments. First we examined the effect of cell passage on morphology and proliferation activity of bovine ISMFs. Although 3rd and 7th passage bovine ISMFs did not exhibit any changes, 11th passage ISMFs showed rounded enlarged shape and lost proliferation potential. On the contrary, rat ISMFs displayed the above senescent changes at earlier passage (passage 4). In intracellular Ca 2+ concentration measurement, bioactive substances (0.3 -1 µ M ATP, 0.1 -1 µ M serotonin, 10 -100 nM endothelin-1, and 1 -10 nM bradykinin) dose-dependently induced an increase in intracellular Ca 2+ concentration in bovine ISMFs (passage 3 and 7). However, at passage 11, impairment in intracellular Ca 2+ responses was observed. Thus, bovine ISMFs might be a novel useful tool with long life span and good cellular responses to investigate physiological/pathophysiological roles of ISMFs.

Porcine Bladder Urothelial, Myofibroblast, and Detrusor Muscle Cells: Characterization and ATP Release

“…Based solely on the α-SMA results, we cannot exclude the possibility that the myofibroblast cultures contained some smooth muscle cells. In addition, some cells in our muscle cultures, but not myofibroblast cultures, demonstrated positive immunoreactivity to desmin (Paulin and Li, 2004; Iwanaga et al, 2010). Not all cells in the muscle cultures were desmin-immunoreactive, although this is to be expected as expression of desmin is lost with culture (Nair et al, 2011).…”

“…Nevertheless, the branching morphology of myofibroblast cultures was quite different from the narrow, unidirectional appearance of the smooth muscle cultures, which was obvious when comparing their appearance in cultures immunostained with vimentin. Previous authors have used α-SMA as a marker for myofibroblasts in tissue sections (Drake et al, 2006; Sadananda et al, 2008; McCloskey, 2010) and cultured cells (Iwanaga et al, 2010). Although our porcine myofibroblast cultures demonstrated some immunoreactivity for α-SMA, this marker showed stronger immunostaining in detrusor muscle cultures than in myofibroblast cultures.…”

“…AE1/AE3 is a marker for urothelial cells known to react against an antigenic determinant present on the majority of the subfamily A and B cytokeratins (Eichner et al, 1984; Sun et al, 1984), and has previously been employed in studies of the urothelium (Trifillis et al, 1995; Southgate et al, 1999). Smooth muscle actin (α-SMA) is a contractile protein present in both smooth muscle (Roholl et al, 1990) and myofibroblasts (Drake et al, 2006; Sadananda et al, 2008; Iwanaga et al, 2010; McCloskey, 2010). Desmin is an intermediate contractile filament that is a muscle specific protein (Gallanti et al, 1992; Paulin and Li, 2004).…”

Prostaglandin E2Promotes Wound-Induced Migration of Intestinal Subepithelial Myofibroblasts via EP2, EP3, and EP4 Prostanoid Receptor Activation

“…In this study, however, we used bovine ISMFs for reasons described previously (Iwanaga et al, 2010). First, it is easier to isolate a larger number of bovine ISMFs from the animal, whereas the number of ISMFs isolated from one rodent is limited because of their smaller size.…”

Triptolide ameliorates colonic fibrosis in an experimental rat model

“…Established colonies of myofibroblasts possess the physiologic characteristics with immunostaining for α-smooth muscle actin (SMA) and vimentin. α-SMA is a contractile protein present in smooth muscle (19) and myofibroblasts (20). Vimentin is commonly used to stain myofibroblasts and fibroblasts.…”