interstitial cells of cajal ( iccs ) are the specialized cell population of gastrointestinal cells , which perform essential roles in regulating the motility of gastrointestinal tract . according to their location in the intestine , iccs could be divided into four subgroups , including submucosal iccs ( iccs - sm ) , myenteric iccs ( iccs - my ) , intramuscular iccs ( iccs - im ) , and deep muscular plexus iccs ( iccs - dmp ) . iccs express c - kit , which is a kind of tyrosine kinase receptor and at present widely used as the surface marker of iccs . the most obvious morphological characteristics of them are that they usually possess processes and form a network , which distinguish them from mast cells , the other major c - kit positive cells in the myenteric plexus of gi tract . therefore , it could be necessary to define the gastrointestinal iccs as a group of cells that have both c - kit expression and reticular morphological features . based on the current understanding of these cells , iccs in gastrointestinal tract mainly have four different functions . iccs in the myenteric plexus are pacemaker cells , which generate characteristic slow waves spontaneously     [ 2 , 3 ] . iccs in the muscularis propria ( iccs - im ) have been indicated to mediate neurotransmission , including both the excitatory and inhibitory neuronal signals     [ 4 , 5 ] . among all these functions , the most important one is that they are the pacemaker cells that generate characteristic slow waves and regulate gastrointestinal motility . for example , pulsating electromagnetic field ( pemf ) exposure would result in the loss of iccs . it has also been reported that in several motility disorders , such as achalasia , mechanical ileus , intestinal pseudo - obstructions , or malformations , reduced number of iccs or dysfunction of their reticular structure could be observed     [ 9 , 10 ] . recent studies indicate that intestinal surgical operation would cause functional and morphological alterations of iccs in mice , including disrupted electrical rhythmicity , neural responses , and interstitial cell networks .   during the past decades , it has been considered that proliferation of iccs could only be detected in embryonic and neonatal periods . recently , some investigators have shown that there are proliferative iccs in the myenteric plexus after iccs damage . for example , mei et al . reported that iccs could regenerate and restore their normal distribution after disruption by intestinal transection and anastomosis     . these findings excite researchers to explore the origin of iccs .   although the existence of iccs has been identified for several decades , the progenitors of iccs , iccs had been presumed to be the descendants of neural crest cells . using chimeric model and developmental observation , it was shown that iccs came from the mesenchyme of the gut , but not the neural crest . common precursors yielded iccs - my and longitudinal muscular cells     [ 1214 ] . in the stomach of postnatal murine , these cells resembled embryonic iccs precursors . and they could give rise to stomach iccs when they were stimulated by soluble stem cell factor ( scf ) and produce typical slow wave electricity     . there are few reports concerning the origin of iccs in adulthood , and no other cell types have been reported to have the ability to differentiate into iccs . not all adult stem cells are lineage restricted as previously believed . among them , bone marrow - derived cells ( bmdcs ) have the potential to differentiate into several nonhematopoietic parenchymal cells , including neurons , hepatocytes , cardiomyocytes , and epithelial cells . recently , bmdcs have been   shown to successfully accelerate burn wound healing and promote the recovery of ischemic heart failure . therefore , it is promising to exploit the wide differentiation potentiality of bmdcs for the therapeutic treatment of human diseases . so it is necessary to study whether multipotential stem cells in other tissues , such as bmdcs , could be enrolled and reside in gi muscles and participate in the iccs regeneration . in our previous work , we had found that bmdcs could long term repopulate the recipient 's gastrointestinal epithelium . in the present study , we attempted to determine whether bmdcs could differentiate into iccs and contribute to iccs regeneration in gastrointestinal tract using a model of gfp hematopoietic chimera . we found that bmdcs could migrate into the myenteric plexus and differentiate into iccs in vivo after transplantation . gfp - transgenic c57bl/6 mice were used as transplantation donors and purchased from the model animal research center of nanjing university ( aaalac accredited ) . the expression level of egfp in bone marrow mononuclear cells was confirmed by flow cytometry and laser confocal microscope analysis . wild - type c57bl/6 mice were used as transplantation recipients in this study , which were 6~8 weeks old and purchased from experimental animals center of third military medical university . all the animals were acclimated to standard laboratory conditions ( under a 12:12h light - dark cycle ) and kept in specific pathogen free ( spf ) grade environment . all animal experimental operations were conducted in accordance with the animal care guidelines of the third military medical university . briefly , bone marrow was aseptically harvested from femurs and tibias of donor mice by flushing bone cavity with cold dmem / f12 medium containing 10% fetal bovine serum ( fbs ) ( hyclone , beijing , china ) using a syringe containing 100  iu heparin with a 20-gauge needle . marrow samples were sufficiently pipetted , centrifuged , and then filtered through a 20  um nylon mesh to expel dregs . then red blood cells were splited by rbc lysis buffer ( 8.26  g ammonium chloride nh4cl , 1  g potassium bicarbonate khco3 , and 0.037  g edta in 1 liter ddh2o ) . then concentration of bmdcs was determined by beckman coulter z series counter ( beckman coulter , hialeah , fl ) . bmdcs were resuspended in dmem / f12 at a concentration of 1  10 viable nucleated cells per 350  l , a transplantation volume for each recipient . they were housed in autoclaved cages and treated with antibiotics for 10 days before irradiation and 2 weeks after irradiation ( with 280  mg erythromycin and 320  mg gentamicin sulfate per liter of deionized drinking water ) . all the recipient mice were exposed to 10 gy whole body irradiation ( wbi ) using a cobalt-60 source ( theratron-780 model , mds nordion , ottawa , on , canada ) . such an irradiation dose had been used in many other transplantation studies     [ 19 , 20 ] and would cause severe gastrointestinal symptoms , such as diarrhea , bloody stool . within three hours after transplantation , ten female recipient mice received injection of the same volume of pbs via tail vein as transplantation control . the other thirty mice were transplanted via tail vein with 110 gfp bone marrow cells in a volume of 350  l . in order to examine the repopulation effect , bone marrow of chimeric mice after depletion of rbc , mononuclear cells were examined by confocal microscope and flow cytometry . because positive cells expressed gfp intrinsically fresh intestinal samples were immediately immersed into liquid nitrogen or fixed with 4% paraformaldehyde ( pfa ) or 100% acetone . after dewaxing and rehydration , antigen retrieval was carried out in 96c for 10  min using sodium citrate solution ( 0.01  m , ph 6.0 ) . activity of endogenous peroxidase was removed by incubation with 3% h2o2 for 15  minutes at room temperature , and nonspecific binding was blocked by 3% normal goat serum incubation for 30 min at room temperature . donor 's bmdcs were identified by rabbit anti - gfp monoclonal antibody ( 1 : 200 , ag279 , beyotime , hangzhou , china ) . horseradish peroxidase- ( hrp- ) conjugated goat antirabbit secondary antibody was used to bind the gfp antibody . iccs were recognized by c - kit ( ack2 , ebioscience , san diego , ca , usa ) , and macrophages were demonstrated using rat anti - f4/80 monoclonal antibody ( abcam , cambridge , ma , usa ) . fluorescent double staining was employed to detect coexpression of gfp and c - kit . briefly , frozen sections or whole mount preparations were fixed in cold acetone for 15  min at 4c . after this , the samples were rinsed in pbs for 10  min . before adding primary antibody , nonspecific labeling was blocked by incubation in pbs containing 10% normal goat serum and 0.2% triton x-100 at room temperature for 30  min . then they were incubated overnight at 4c with primary antibody against c - kit ( 1 : 100 ) or f4/80 ( 1 : 100 ) . after rinsing in pbs three times for sufficient washing , cy3-conjugated goat after 1  h incubation in the dark and pbs washing , gfp antibody ( 1 : 200 ) and fitc - conjugated goat antirabbit secondary antibody ( 1 : 200 ) were sequentially added and , respectively , incubated overnight at 4c and for 30 min at room temperature . after three times washing , cell nuclei were counterstained with 0.3  m 4,6-diamidino-2-phenylindole , dihydrochloride ( dapi ) ( molecular probes , inc . , eugene , or , usa ) in pbs for 1  min , which was done at room temperature and in the dark . digital images were captured using tcs sp5 confocal laser scanning microscope ( leica , germany ) with an excitation wavelength appropriate for cy3 ( 552  nm ) or fitc ( 488  nm ) . spss version 16.0 ( spss inc . , chicago , usa ) was used to carry out all statistical analyses . differences in the data were evaluated by one - way analysis of variance ( anova ) . mice transplanted with pbs in the control group died of bone marrow failure and intestinal symptoms within 5~7 days after irradiation . on the other hand , all the mice in transplantation group successfully underwent the first week after irradiation with a survival rate more than 93.3% . the survival rate of these two groups had significant difference ( p < .05 ) . transplantation of bmdcs could efficiently prevent recipients dying from bone marrow failure and gastrointestinal symptoms caused by irradiation . mononuclear cells in bone marrow were almost completely replaced by gfp positive cells ( figure 1(a ) ) . by flow cytometry analysis , a representative image of gfp positive cells in all the marrow mononuclear cells at 2.5 months chimeric rates at different timepoints were given in table 1 , and the average level was 95.65%      3.72% . the results indicated that the gfp positive chimeric mice model was successfully established .   using immunohistochemistry ( ihc ) , it was shown that gfp positive bmdcs would migrate into intestine after transplantation . bmdcs appeared in the submucosal lamina propria during the first week , and then the quantity of these cells increased . large amount of gfp positive bmdcs fulfilled the mesenchymal region of intestine ( figure 2(a ) ) . between the longitudinal and circular muscle cells , there were gfp positive cells with the appearance of processes ( figure 2(b ) , red arrow ) . no positive signals could be seen in the same section without gfp antibody ( figure 2(c ) ) . no significant changes could be observed through he staining ( figure 2(d ) ) . in order to examine the definite phenotype of gfp positive bmdcs in the muscular layer , macrophage differentiation was firstly observed . using the antibody against f4/80 , which was a widely - used surface marker for macrophages but the majority of positive macrophages were mainly found in the deep muscular plexus ( dmp ) . for example , at the second month , both gfp positive and f4/80 positive cells could be detected in the dmp layer ( lower panel in figure 3 ) . gfp positive macrophages contributed to a proportion of 50.92    10.98% of all the macrophages in the dmp layer . we then tried to observe the phenotype of bmdcs in the myenteric plexus . in the myenteric plexus c - kit positive iccs - my were well demonstrated using c - kit antibody ( ack2 ) . there were gfp and ack2 double positive cells both in the intestinal sections and in the whole mount preparations . the representative images were demonstrated in figures 4 and 5 , in which the images were taken from recipient intestine 2.5 months after transplantation . double positive cells were indicated by arrow and enlarged in the inset ( merged image in the lower panel of figure 4 ) . donor - derived iccs had bipolar appearance on the whole mount preparations ( white arrow in merged image of the lower panel of figure 5 ) . donor - derived iccs - my reached a proportion of 9.25%    4.9% among all the iccs - my in small intestine . therefore , the transplanted bmdcs were able to engraft in smooth muscle of small intestine and differentiate into iccs after the radiation injury . adult stem cells exhibit more remarkable plasticity than previously believed , and bmdcs could contribute to several nonhematopoietic cell lineages . both experimental and clinic studies have demonstrated that donor 's bmdcs have multipotential differentiation ability in various organs of transplantation recipient , especially the nonhematopoietic differentiation capacity . they could differentiated into neurons , hepatocytes , and kidney tubular epithelial cells     [ 21 , 22 ] . among all these findings , okamato et al reported that bmdcs could also differentiate into epithelial cells to repopulate the damaged intestinal epithelium     [ 23 , 24 ] . in the gastrointestinal tract , bmdcs have also been observed to differentiate into subepithelial myofibroblasts , which are important to regulate intestinal stem cell microenvironment . such exciting reports raised related research concerning the effects of bmdcs in regeneration of various injuries of gastrointestinal tract , such as dextran sulfate sodium ( dss ) or trinitrobenzenesulfonic acid-(tnbs- ) induced ibd model and alcohol - induced stomach epithelial injury     [ 2629 ] . all these studies had shown that bmdcs could participate in the injury restoration process in different damage models .   in our experiments , we established the chimera model using gfp transgenic mice . as confirmed by both flow cytometry and confocal scanning , the bone marrow of transplantation recipients had been repopulated by donor 's bmdcs ( figure 1 and table 1 ) . in the previous study , we found that gfp positive cells could for long term exist in gastrointestinal tract . because of ubiquitous gfp expression , staining of gfp allowed us to detect the engraftment and differentiation of donor - derived bmdcs conveniently . gfp positive bmdcs engrafted into each segment of the gastrointestinal tract without specificity . besides epithelial engraftment , there were certain gfp positive cells in the tunica muscularis . for example , they could migrate into the myenteric plexus and deep muscular plexus as shown in figure 2 . given the location of gfp positive cells , we tried to explore the possibility that whether bmdcs could differentiate into iccs in gastrointestinal tract . macrophages have also been shown to resident in the muscular layer     . therefore , it was necessary for us to exclude whether these cells were macrophages . through gfp and f4/80 double staining , it was demonstrated that gfp positive macrophages could be detected definitely . anyhow , these cells were mainly found in the deep muscular plexus ( figure 3 ) and macrophages were negative for c - kit . using frozen sections and whole mount preparations , we found that there were gfp positive cells with several branches , which resembled the morphological characteristics of iccs . in addition , gfp and c - kit double positive cells could be observed in the myenteric plexus ( figures 4 and 5 ) . the positive cells had at least two processes , which made them distinguishable from mast cells . although mast cells in the myenteric plexus were also positive for c - kit , these cells themselves were morphologically round and without processes . besides this so the double positive cells were iccs - my , but not mast cells in the myenteric plexus . it encouraged us to believe that c - kit positive bmdcs in the myenteric plexus were intestinal iccs - my according to the commonly accepted criteria , including cell location , surface marker , and morphological appearance . therefore , the transplanted bmdcs were able to engraft in smooth muscle of intestine and differentiate into iccs after the radiation injury . at present , it was difficult for us to carry out electrophysiological studies to study whether gfp positive cells could produce the slow wave electricity in this chimeric model , but a recent study had reported that bone marrow transplantation restored gut motility in kit deficient mice , which indicated that these donor - derived iccs played the same function as their wild - type mates . previous studies have demonstrated that iccs originated from mesenchymal cells , but not the neural crest cells     [ 13 , 33 ] . the proliferation of iccs and extension of the processes are only observed in embryonic and neonatal periods in mammals , and even iccs isolated from 6-day neonatal mice could not increase in number in vitro . recently , there were several investigations concerning the plasticity of iccs and loss and recovery of iccs had been reported     [ 35 , 36 ] . another recent study has successfully identified a very small group of c - kitcd44cd34igf - ir cells in murine stomach as the putative progenitors of iccs     . in the adult , it has been identified that bmdcs are able to differentiate into adipocytes , osteocytes , chondrocytes . in the gastrointestinal tract , bmdcs have been reported to have the capacity to differentiate into epithelial cells     [ 17 , 38 ] . in this study we used whole bmdcs as transplantation graft and identified that they could differentiate into iccs . however , bmdcs used here were cell mixtures without purifying specific bone marrow stem cells ( bmscs ) . so we had not demonstrated that these iccs were derived from bone marrow stem cells . it was possible that the gfp positive iccs detected in the gut of recipients could be derived from surviving trapped donor cells . given the current understanding of iccs origin and characteristics of mesenchymal stem cells ( mscs ) , we presumed that mscs , the most widely - investigated bmscs , might be responsible for the donor - derived iccs after transplantation . previous studies had demonstrated that transplanted bmdcs , such as mscs , were preferentially recruited to the injured tissues caused by ischemia , trauma , and disease and could accelerate tissue regeneration . herein , we confirmed the existence of transplanted bone marrow - derived iccs in small intestine several months after irradiation and transplantation . it also meant that bmdcs as a source of iccs under physiological condition could not be completely excluded . because there were still gfp positive iccs several months later after the radiation injury and transplantation . one question was how bmdcs , especially bmscs , such as mscs , could differentiate into iccs . because only about 10% iccs were donor - derived in the recipient 's myenteric plexus , more efforts to study the mechanisms for bmdcs differentiation towards iccs it had been reported that abnormal iccs might be the cause of gastrointestinal stromal tumors ( gists ) . transformation of mscs had also been reported both in vivo and in vitro     [ 39 , 40 ] . therefore , when mscs were used to repopulate iccs damage , the safety was needed to be taken into account . both transdifferentiation and cell fusion have been used to interpret the formation of donor - derived nonhematopoietic cell phenotypes . intestinal epithelial cells derived from donor 's bmdcs were reported to be generated through cell fusion between bmdcs and intestinal stem cells     . we had also performed y - fish and gfp ihc staining to determine whether donor- derived iccs were the products of cell fusion phenomenon , but in our models we did not find occurrence of cell fusion between donor 's bmdcs and host 's iccs ( unpublished data ) , suggesting that there was lack of evidence of cell fusion in the formation process of donor derived iccs . in conclusion , we showed that after irradiation and transplantation donor 's bmdcs repopulated recipient hematopoiesis , migrated into gastrointestinal tract , and had the capacity to differentiate into iccs in the myenteric plexus in the present study . all these results bring us a novel insight into the plasticity of bmdcs and an alternative cell resource for therapy of gastrointestinal dysmotility caused by iccs disorders . in the future the results need to be confirmed using purified bone marrow stem cells to directly demonstrate the origin of donor cells residing in the gut . the specific formation mechanisms of donor - derived iccs , whether cell fusion or transdifferentiation , might be further investigated in the future .
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 .