Bone marrow derivation of interstitial cells of cajal in small intestine following intestinal injury

Abstract

Interstitial cells of Cajal (ICCs) in gastrointestinal tract are specialized cells serving as pacemaker cells. The origin of ICCs is currently not fully characterized. In this work, we aimed to study whether bone marrow-derived cells (BMDCs) could contribute to the origin of ICCs in the muscular plexus of small intestine using GFP-C57BL/6 chimeric mice.Engraftment of BMDCs in the intestine was investigated for GFP expression. GFP positive bone marrow mononuclear cells reached a proportion of 95.65% +/- 3.72% at different times in chimerism. Donor-derived cells distributed widely in all the layers of the gastrointestinal tract. There were GFP positive BMDCs in the myenteric plexus, which resembled characteristics of ICCs, including myenteric location, c-Kit positive staining, and ramified morphology. Donor-derived ICCs in the myenteric plexus contributed to a percentage ranging 9.25% +/- 4.9% of all the ICCs in the myenteric plexus. In conclusion, here we described that donor-derived BMDCs might differentiate into gastrointestinal ICCs after radiation injury, which provided an alternative source for the origin of the ICCs in the muscular plexus of adult intestine. These results further identified the plasticity of BMDCs and indicated therapeutic implications of BMDCs for the gastrointestinal dysmotility caused by ICCs disorders.

Figure 1

Recipient's bone marrow was reconstituted by transplantation of donor's GFP positive BMDCs. (a) GFP positive marrow cells took up the majority of chimeric bone marrow two months after transplantation. Bar = 50 μm. (b) the percentage of GFP positive marrow cells in chimeric marrow at two months after transplantation was shown to be 92.05% by flow cytometry.

Figure 2

Detection of donor-derived BMDCs by GFP IHC in the small intestine 3 months after transplantation. (a) GFP positive BMDCs could be integrated into any part of intestine. (b) donor-derived cells could also be observed between longitudinal muscle and circular muscle, and there were some positive processes without ICCs body due to section reason (red arrow). (c) blank control without primary antibody on the serial section. No positive signals could be detected. (d) HE staining of small intestine from transplanted mice.

Figure 3

Whole mount preparation of small intestine from both normal C57 mouse and chimeric mouse. in this figure, donor's BMDCs were detected by GFP antibody. Macrophages were demonstrated by their specific marker F4/80. Upper panel: C57 mouse was used as normal control, in which all the macrophages were negative for GFP antibody. Bar = 50 μm. Lower panel: in the chimerism some macrophages were double positive for GFP and F4/80, suggesting that they were derived from the infused donor's BMDCs. GFP⁺/F4/80⁺ cells were mainly found at the deep myenteric plexus and they were scattered in this layer, which distinguished them from ICCs. Bar = 50 μm.

Figure 4

Confocal image of frozen section demonstrated donor derived ICCs-MY in small intestine of recipient mice 2.5 months after bone marrow transplantation. Upper panel: C57 mouse was used as negative control, in which there were no GFP signals in the intestine. Lower panel: In the chimerism, GFP and c-Kit double positive cells were shown in the merged image. Amplified image of interested cell was shown as inset. Bar = 25 μm.

Figure 5

Representative immunofluorescent image of GFP and c-Kit double positive ICCs on whole mount preparation of small intestine. GFP protein was stained by FITC-labeled goat antirabbit IgG, and c-Kit was demonstrated by Cy3-conjugated goat anti-rat IgG. Upper panel: C57 mouse was used as negative control, in which there were no GFP signals in the intestine. Lower panel: GFP and c-Kit double positive cells were shown in chimeric intestine. Double positive cells could be found on the whole mount preparation, indicating that ICCs could be derived from BMDCs. Yellow arrow was indicated a double positive cell and white arrow directed to its two polar processes. Bar = 50 μm.