Study of the colonic epithelial-mesenchymal dialogue through establishment of two activated or not mesenchymal cell lines: Activated and resting ones differentially modulate colonocytes in co-culture

Abstract

Continuous and rapid renewal of the colonic epithelium is crucial to resist the plethora of luminal deleterious agents. Subepithelial fibroblasts contribute to this turnover by regulating epithelial proliferation and differentiation. However, when intestinal homeostasis is disturbed, fibroblasts can acquire an activated phenotype and play a major role in the progression of intestinal pathologies. To evaluate the involvement of fibroblasts in the regulation of colonocytes under homeostatic or pathological conditions, we established resting and activated conditionally immortalized fibroblast cell lines (nF and mF) from mouse colonic mucosa. We then studied the epithelial-mesenchymal interactions between activated or resting fibroblasts and the normal mouse colonocytes (Co) using a co-culture model. Both fibroblastic cell lines were characterized by RT-qPCR, western blot and immunofluorescence assay. Our results showed that nF and mF cells were positive for fibroblastic markers such as vimentin and collagen 1, and negative for cytokeratin 18 and E-cadherin, attesting to their fibroblastic type. They also expressed proteins characteristic of the epithelial stem cell niche such as Grem1, CD90 or Wnt5a. Only rare nF fibroblasts were positive for α-SMA, whereas all mF fibroblasts strongly expressed this marker, supporting that mF cells were activated fibroblasts/myofibroblasts. In coculture, nF fibroblasts and Co cells strongly interacted via paracrine exchanges resulting in BMP4 production in nF fibroblasts, activation of BMP signaling in Co colonocytes, and decreased growth of colonocytes. Activated-type mF fibroblasts did not exert the same effects on Co cells, allowing colonocytes free to proliferate. In conclusion, these two colonic fibroblast lines, associated with Co cells in coculture, should allow to better understand the role of mesenchymal cells in the preservation of homeostasis and the development of intestinal pathologies.

A dual model of normal vs isogenic Nrf2-depleted murine epithelial cells to explore oxidative stress involvement

“…The Co cell line used in these experiments was derived from the Apc +/+ clones described by Forest et al 8 and characterized by Plaisancié et al 20 . To ensure the non-transformed status of our cell line, we performed WGS, focusing specifically on cancer-related genes.…”

Effects of sodium nitrite reduction, removal or replacement on cured and cooked meat for microbiological growth, food safety, colon ecosystem, and colorectal carcinogenesis in Fischer 344 rats

“…Supernatants (fecal waters) were collected and kept at −80 °C until use. Heme was measured with the Heme Assay (Kit MAK316 – Sigma) according to the recommendations of the manufacturer; fecal TBARS were measured in fecal waters according to Ohkawa et al 56 For cytotoxicity and genotoxicity, fecal waters were tested on normal immortalized colon epithelial cells (Co cells), which were established as described previously 57 , 58 . Conditionally immortalized cells were seeded into 96-well plates at 15 × 10 3 cells per well in permissive conditions.…”

Reduction, removal or replacement of sodium nitrite in a model of cured and cooked meat: a joint evaluation of consequences on microbiological issues in food safety, colon ecosystem and colorectal carcinogenesis

“…Heme was measured with the Heme Assay (Kit MAK316 -Sigma) according to the recommendations of the manufacturer; fecal TBARS were measured in fecal waters according to Ohkawa et al [29] For cytotoxicity and genotoxicity, fecal waters were tested on normal immortalized colon epithelial cells (Co cells), which were established as described previously. [30,31] Conditionally immortalized cells were seeded into 96-well plates at 15×10 3 cells per well in permissive conditions. After reaching sub confluence (70%), cells were transferred to 37°C and treated for 24h with filtered fecal waters in serumfree DMEM to avoid any reaction between serum and fecal water.…”