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Primary Myofibroblasts Maintain Short-Term Viability following Submucosal Injection in Syngeneic, Immune-Competent Mice Utilizing Murine Colonoscopy
- Source :
- PLoS ONE, PloS one, vol 10, iss 5, PLoS ONE, Vol 10, Iss 5, p e0127258 (2015)
- Publication Year :
- 2014
-
Abstract
- The myofibroblast is an important stromal cell of the gastrointestinal tract. Current in vitro and in vivo models either do not accurately recreate stromal-epithelial interactions or are not specific to myofibroblasts. We sought to create an animal model that would allow the study of myofibroblast-epithelial interactions. We isolated and cultured colonic myofibroblasts from FVB mice. Cells were α-SMA and vimentin positive but desmin negative on immunoblot analysis. We injected the myofibroblasts into the colonic submucosa of syngeneic adult mice (n = 8) via a miniendoscopic system. We then isolated green fluorescent protein (GFP) positive colonic myofibroblasts from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and injected them into the colonic lamina propria of C57BL/6J mice at 1x10(5) (n = 14), 1x10(6) (n = 9), or 5x10(6) cells/mL (n = 4). A subset of mice were injected with serum-free media and ink without cells (n = 3). Mice underwent repeat endoscopy and euthanasia one or 7 days after injection. Colons were isolated and either fixed in 10% formalin or the inked sites were individually excised and lysed for DNA. We assessed the injection sites via histology and immunohistochemical stains for α-SMA and GFP. We used qPCR to quantify GFP DNA transcripts at the lamina propria injection sites. Submucosal injection of myofibroblasts resulted in the formation of a subepithelial wheal on endoscopy, which persisted to day 7. Myofibroblasts injected either into the submucosa or lamina propria maintained viability on post-injection day 7 as evidenced by positive α-SMA staining. qPCR of lamina propria injections showed a dose-dependent increase in GFP DNA transcripts on post-injection day 1, whereas the number of transcripts on day 7 was equivalent for the concentrations injected. We demonstrate short-term survival of primary cultured colonic myofibroblasts in syngeneic mice. This may prove to be a valuable model for studying the role of myofibroblasts in states of health and disease.
- Subjects :
- Male
Pathology
medicine.medical_specialty
Stromal cell
Time Factors
General Science & Technology
Cell Survival
Colon
1.1 Normal biological development and functioning
lcsh:Medicine
Vimentin
Inbred C57BL
Oral and gastrointestinal
Green fluorescent protein
Injections
03 medical and health sciences
Mice
0302 clinical medicine
Intestinal mucosa
Underpinning research
Submucosa
medicine
Animals
Intestinal Mucosa
lcsh:Science
Myofibroblasts
030304 developmental biology
0303 health sciences
Lamina propria
Multidisciplinary
biology
lcsh:R
Histology
Colonoscopy
3. Good health
Mice, Inbred C57BL
medicine.anatomical_structure
030220 oncology & carcinogenesis
biology.protein
lcsh:Q
Female
Myofibroblast
Immunocompetence
Research Article
Subjects
Details
- ISSN :
- 19326203
- Volume :
- 10
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- PloS one
- Accession number :
- edsair.doi.dedup.....e748ead50dcc8692f3abacdfb2530f84