GASTROENTEROLOGY 1996;111:901–910
Interstitial Cells of Cajal in Human Colon and in
Hirschsprung’s Disease
JEAN–MARIE VANDERWINDEN,* JÜRI J. RUMESSEN,‡ HAO LIU,§ DOMINIQUE DESCAMPS,*
MARC–HENRI DE LAET,§ and JEAN–JACQUES VANDERHAEGHEN*
*Laboratoire de Neurophysiologie et Physiopathologie du Système Nerveux, Faculté de Médecine, Université Libre de Bruxelles, Brussels,
Belgium; §Département de Chirurgie Pédiatrique, Hôpital Universitaire des Enfants Reine Fabiola, Brussels, Belgium; and ‡Department of
Gastroenterology 261 and Institute of Pathology, Hvidovre Hospital, Hvidovre, Denmark
Background & Aims: Subpopulations of interstitial cells
of Cajal are regarded as the source of spontaneous
slow waves of the gut musculature (pacemaker cells).
Their ontogeny remains unclear, but a role of the tyro-
sine kinase receptor c-kit in their development has re-
cently been recognized. This study examined the inter-
stitial cells in the human colon and in Hirschsprung’s
disease (aganglionosis). Methods: The distribution of
the c-kit receptor was studied using specific antibodies
in 5 normal patients, 10 patients with Hirschsprung’s
disease, and 3 patients with diversion loop enterosto-
mies. c-kit immunohistochemistry was also combined
with reduced nicotinamide adenine dinucleotide phos-
phate diaphorase histochemistry or with c-kit ligand
(stem cell factor) immunohistochemistry. Transmission
electron microscopy was performed in 1 patient with
Hirschsprung’s disease. Results: c-kit immunoreactiv-
ity labeled a network of interstitial cells at the outer
edge of the submucosa, in the muscular layers, and
around the myenteric plexus. In aganglionic segments,
interstitial cells were scarce and its network appeared
disrupted. Interstitial cells of Cajal were identified in
aganglionic regions by electron microscopy. Interstitial
cells of Cajal are identifiable in newborns and exhibit
similar distribution in diversion loops independent of
contact with luminal nutrients. Conclusions: Our mor-
phological data may explain the abnormal spontaneous
electrical activity in aganglionic segments of Hirsch-
sprung’s disease and may give new insight into the
ontogeny of interstitial cells.
H irschsprung’s disease (HD) is a human disease char-
acterized by aganglionosis, i.e., lack of the intrinsic
enteric nervous system (ENS). The affected segment ex-
tends cranially from the anus and encompasses a variable
portion of the gut. In the classical form of HD, only the
rectosigmoid is affected; however, in some cases a longer
segment (long-segment HD) or even the entire colon
may be involved (total colonic aganglionosis). Function-
ally, the lack of propulsive movements may lead either
to an early obstructive syndrome in infancy or to a severe
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constipation with dilatation of the proximal ganglionic
segment presenting in childhood or even later. Numerous
abnormalities of innervation have been described in HD,
including the lack of the potent inhibitory neurotrans-
mitter nitric oxide,1 supporting the view of a relaxation
defect of the aganglionic segment.2 However, the func-
tional significance of these abnormalities has not been
fully clarified because the obstructive symptoms are not
directly related to the length of the aganglionic seg-
ment.3
Interstitial cells of Cajal (ICC) at distinct locations are
regarded as the pacemaker cells of the gut because they
generate the spontaneous slow waves of the smooth mus-
cle layers.4,5 Their distribution has been difficult to study
with light microscopy because of the lack of specificity
and reliability of the procedures used, and unequivocal
identification of ICC requires transmission electron-mi-
croscopic criteria.6 – 9 Previous ultrastructural studies have
addressed the distribution of ICC in the normal human
colon.10 – 12 A potential role of ICC in human gastrointes-
tinal diseases has not yet been identified.
The ontogeny of the ICC is currently unknown. Ultra-
structural studies in the mouse have suggested that ICC
develop postnatally under the influence of the ENS, and
a role of alimentation has been implied.13 Recent reports
indicate that the transmembrane tyrosine-kinase receptor
c-kit is essential for the development and function of the
ICC.14 – 17 The ligand for the c-kit receptor is a cytokine
known as stem cell factor (SCF), mast cell growth factor,
steel factor, or c-kit ligand.18 c-kit immunoreactivity is
present in various cell types,19 but in the gut, c-kit is
expressed only in ICC and mast cells.14 – 17
In the present study, we studied the distribution of
c-kit and SCF immunoreactivity in the normal human
Abbreviations used in this paper: ABC, avidin-biotin complex; DAB,
3,3*-diaminobenzidine; ENS, enteric nervous system; HD, Hirsch-
sprung’s disease; ICC, interstitial cells of Cajal; NADPH, reduced
nicotinamide adenine dinucleotide phosphate; SCF, stem cell factor.
q 1996 by the American Gastroenterological Association
0016-5085/96/$3.00
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Table 1. Antibodies Used

Clone/
Antibody catalog no. Characteristics Source Working dilution

c-kit 0/#SC39 Affinity-purified polyclonal rabbit antiserum raised to a synthetic Santa Cruz 1:250
peptide corresponding to residues 961–976 within the C- Biotechnology
terminal domain of the human c-kit receptor (100 mg
immunoglobulin G/mL).
1D9 3D6 Mouse monoclonal immunoglobulin G1 raised to human c-kit. Boehringer 10 mg/mL
Recognizes an epitope on the extracellular domain.43
SCF MGF-M1/0 Rat monoclonal immunoglobulin G2a raised to recombinant Immunex Corp. 2 mg/mL
murine SCF/MGF.27
MGF-M3/0 4 mg/mL

colon and HD. Electron microscopy was used to confirm
the presence of ICC in the aganglionic colon.
Materials and Methods
Tissues were obtained from 10 patients with histologi-
cally proven HD (age range, 3 weeks to 2 years; 8 boys and
2 girls) at the time of corrective surgery (Soave pull-through).
The level of adequate resection was determined during the
procedure by identification of myenteric ganglia on routine
frozen sections. The aganglionic segment involved the rectosig-
moid in 7 cases (classical form of HD), and the aganglionic
zone extended to the transverse colon or to the hepatic flexure
in 3 cases (long-segment HD). The proximal part of each
specimen, which always contained enteric ganglia, served as
an internal positive control. Specimens from normal rectosig-
moid and colon were also obtained from 5 patients (two autop-
sies within 6 hours of death and three organ donors; age, 1
day, 4 months, and 13, 17, and 30 years, respectively; 4 males
and 1 female). Specimens from diversion loop enterostomies
(two ileostomies and one left colostomy) performed shortly
after birth in neonates with imperforated anus were also ob-
tained when digestive continuity was restored (age, 6 and 10
weeks and 7 months, respectively; 2 boys and 1 girl).
The study was approved by the Institutional Ethical Com-
mittee of the Hôpital Universitaire des Enfants Reine Fabiola
(Brussels, Belgium).
For immunohistochemistry, specimens were harvested and
processed as described previously.1 Briefly, the surgical speci-
mens were opened along their antimesenteric edge, pinned
flat, fixed overnight in fresh 4% paraformaldehyde solution in
0.1 mol/L phosphate buffered saline (PBS) at 47C, cryopre-
served in graded solutions of sucrose (10%, 20%, 30%; over-
night each), embedded in Tissue-Tek OCT compound (Miles,
Elkhart, IN), snap-frozen in 2-methyl butane that had been
cooled on dry ice, and stored at 0807C. Longitudinal sections
(15 mm thick) were cut on a cryostat, mounted on slides coated
with 0.1% poly-L-lysine (Sigma Chemical Co., St. Louis, MO),
and stored at 0207C until use. In addition, fresh samples taken
at various levels from one case of HD were embedded in OCT
compound, snap-frozen on dry ice, cut on a cryostat, air-dried
for 20 minutes, stored at 0207C, and fixed for 10 minutes in
acetone at 47C immediately before use.
Immunohistochemistry was performed with commercially
available kits using the avidin-biotin complex (ABC) system
(Vectastain ABC), according to the instructions of the supplier
(Vector Laboratories, Burlingame, CA). Briefly, sections were
first incubated in blocking normal serum for 20 minutes, incu-
bated with the primary antibody diluted in normal serum for
3 hours, rinsed in PBS for 10 minutes, incubated with the
biotinylated secondary antibody for 30 minutes, rinsed in PBS,
and incubated with the ABC complex for 1 hour. Different
kits were used according to the species of the primary antibod-
ies (Table 1). ABC conjugated with horseradish peroxidase
was routinely used. Horseradish peroxidase was revealed by
incubation for 10–15 minutes at room temperature with the
Immunopure metal-enhanced 3,3*-diaminobenzidine (DAB)
substrate kit (Pierce, Rockford, IL) (referred to below as DAB-
Metal), which produces a brown deposit. Some selected sections
were subsequently counterstained with Mayer’s hematoxylin.
Histochemical staining for reduced nicotinamide adenine
dinucleotide phosphate (NADPH) diaphorase, as a marker for
the neuronal isoform of constitutive NO synthase, was per-
formed as previously described.1 Briefly, the slides were incu-
bated in the dark in a solution of 0.1 mol/L Tris-HCl buffer
containing 1 mmol/L NADPH (Sigma Chemical Co.), 0.2
mmol/L nitro blue tetrazolium (Sigma Chemical Co.), and
10% dimethyl sulfoxide at 377C for 75–90 minutes. Omission
of NADPH was used as negative control and resulted in the
absence of staining.
Double-labeling procedures were performed on three se-
lected specimens of normal colon.
NADPH-diaphorase histochemistry and c-kit immuno-
histochemistry. The two techniques were performed sequen-
tially as described above with NADPH-diaphorase histochem-
istry performed first.
Double immunohistochemistry. c-kit immunohisto-
chemistry with the polyclonal antiserum was performed first
and revealed with DAB-Metal. Subsequently, immunohisto-
chemistry for SCF was performed in the same way but ABC
conjugated with alkaline phosphatase was used. The phos-
phatase activity was revealed with the 1-Step NBT/BCIP
substrate kit (Pierce), giving a blue-purple precipitate
clearly distinct from the brown product of the DAB-Metal
reaction.

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October 1996 INTERSTITIAL CELLS IN HIRSCHSPRUNG’S DISEASE 903

Figure 1. c-kit immunoreactivity in the normal colon. (A ) c-kit immunoreactivity (in brown) in ICC and nerve fibers expressing NO synthase
stained by NADPH-diaphorase histochemistry (in dark blue). ICC form a submucosal plexus at the edge of the circular musculature and the
submucosa (scale bar Å 50 mm). (B ) c-kit immunoreactivity in ICC surrounding a submucosal ganglion. Hematoxylin counterstain. Arrow indicates
submucosal ganglion (scale bar Å 50 mm). (C ) c-kit immunoreactivity (in brown) in the submucosal plexus. Hematoxylin counterstain. Apposition
of the soma of two adjacent ICC (scale bar Å 50 mm). (D ) c-kit immunoreactivity (in brown) in ICC around two myenteric plexus ganglia. c-kit
immunoreactivity is present around, but not within, the ganglia. Neurons expressing NO synthase are stained by NADPH-diaphorase histochemis-
try (in dark blue) (scale bar Å 100 mm). CM, Circular musculature; LM, longitudinal musculature; SM, submucosa.

Controls of the immunohistochemical procedures.
No staining was observed when the primary antibodies were
omitted. The optimal working dilution was empirically deter-
mined for each antibody by serial dilutions. Liquid phase pre-
absorption was performed for the c-kit antiserum according to
the instructions of the supplier (Santa Cruz Biotechnology,
Santa Cruz, CA), i.e., overnight incubation at 47C with 4 mg/
mL (a 10-fold by weight excess of peptide antigen for a work-
ing concentration of the c-kit antiserum of 0.4 mg immuno-
globulin G/mL) of peptide antigen (Santa Cruz Biotechnology;
catalog no. SC39-P). This totally abolished the signal. For all
the double-staining studies, separate procedures were identi-
cally performed at the same time as controls.
For electron microscopy, a full-thickness specimen from the
most distal part of the aganglionic portion from 1 of the
patients included in the histological study (a 1-year-old girl
with familial classical rectosigmoid form of HD) was used.
After resection, the tissue was immediately chopped with a
razor blade into small pieces (approximately 3 1 2 mm), im-
mersed in a fixative containing 4% glutaraldehyde in 0.1 mol/
L PBS, pH 7.4, for 90 minutes at 47C, rinsed, and stored
overnight in PBS. The pieces were postfixed in 0.2% OsO4 in
PBS for 30 minutes, dehydrated in alcohol, and embedded in
Epon 832 R (Merck, Darmstadt, Germany). Semithin sections
were stained with toluidine blue to select areas suitable for
transmission electron microscopy. Ultrathin sections (50–70
nm) were cut, mounted on copper grids, contrasted with uranyl
acetate and lead citrate, and examined in a Jeol 1010 electron
microscope at 80 kV.
Results
c-kit Immunoreactivity in Normal Colon
The polyclonal c-kit antiserum labeled numerous
elongated cells forming a network at the border between
the circular muscle layer and the submucosa (Figure 1A).
These cells had the appearance of ICC. Ganglia from the
deep submucosal plexus were also surrounded by ICC
(Figure 1B). Close apposition between two adjacent ICC
(Figure 1C) and between an ICC and a cell unlabeled

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904 VANDERWINDEN ET AL. GASTROENTEROLOGY Vol. 111, No. 4

Figure 2. c-kit immunoreactivity in the normal colon. (A ) Distribution of ICC across the colonic musculature. *Myenteric plexus (scale bar Å
200 mm). (B ) c-kit immunoreactivity in the proximal part (where nutrient passed) of a left colostomy performed during the neonatal period.
*Myenteric ganglia (scale bar Å 50 mm). (C ) c-kit immunoreactivity in the distal part (where no contact with nutrient occurred) of the colostomy
of the same patient. The orientation of the specimen differed slightly from B. Although the intensity of the staining appears weaker, the pattern
of distribution of ICC appears similar to the proximal part. *Myenteric ganglia (scale bar Å 50 mm). CM, Circular musculature; LM, longitudinal
musculature.

with the c-kit antiserum (not shown) were observed. ICC
were fusiform with two long main processes along the
cephalocaudal (longitudinal) axis. The nucleus usually
had dispersed chromatin and one nucleole.
c-kit immunoreactivity also labeled ICC around the myen-
teric ganglia (Figures 1D and 2A). They appeared similar to
the ICC at the submucosal border with a fusiform shape,
but they often had several short primary processes in various
directions. In the muscular layers (Figure 2A), ICC had one
or two long processes running parallel to the muscle bundles.
c-kit–positive cells did not display NADPH-diapho-
rase activity but were often close to nerve fibers (Figure
1A) or myenteric neurons (Figure 1D) expressing
NADPH-diaphorase/neuronal NO synthase.

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October 1996 INTERSTITIAL CELLS IN HIRSCHSPRUNG’S DISEASE 905

The ICC network was prominent throughout the

thickness of the musculature (Figure 2A). The distribu-
tion of ICC was similar in the different parts of the colon
and at the different ages, including the newborn.
The distal side of the neonatal diversion ileostomies
(n Å 2) and of the colostomy (n Å 1), deprived since birth
of contact with luminal nutrients, after a few months
displayed a pattern of ICC similar to the proximal side,
which had been bathed by the chyme (Figure 2B and C,
respectively). In the ileal specimen, ICC were observed
in the myenteric plexus layer and in the muscular layers.
ICC at the level of the plexus muscularis profundus20
were not observed (data not shown).
In the inner part of the colonic submucosa, the lamina
propria, and the mucosa, no elongated c-kit immunoreac-
tive cell was encountered. In contrast, numerous round
cells were labeled. They were identified as mast cells by
their metachromasia after toluidine blue staining. Occa-
sionally, similar c-kit–positive round cells were observed
in the muscular layers and in the serosal connective tissue,
especially in specimens from enterostomies where inflam-
matory infiltrates were more significant (data not shown).
Satisfactory labeling with the c-kit monoclonal anti-
body 3D6 was achieved only in unfixed tissues. The pat-
tern of labeling was similar to that observed in fixed
tissues with the polyclonal c-kit antiserum, but nonspe-
cific background staining was somewhat higher.

SCF Immunoreactivity
SCF-positive cells were isolated, round cells
mainly scattered in the submucosa. Some SCF-positive
cells were observed in the circular muscle layer near the
submucosal edge (Figure 3A), but very few were found
in the outer part of the circular layer and around the
myenteric plexus. Some SCF-positive cells were also ob-
served in the mucosa and in the serosal connective tissue.
In the submucosa, SCF-positive cells were often sur-
rounded by granules of immunoreactivity apparently ex-
tending into the extracellular matrix (Figure 3B). Both
SCF-specific antibodies used produced that same pattern.
In double-staining studies, ICC labeled with the c-kit
antiserum and SCF-positive cells were never close, and
contacts between them were not apparent (Figure 3C).
No difference in the distribution of SCF immunoreac-
tivity was apparent between the normal colon (n Å 5) Figure 3. SCF immunoreactvity in the colon. (A ) SCF-positive cells in
and specimen of HD (n Å 3). the submucosa and inner part of the circular musculature (arrows)
(scale bar Å 100 mm). (B ) SCF-positive granules (arrows) surrounding
c-kit Immunoreactivity in HD an SCF-positive cell (the cell body is slightly out of the focal plane) in
the submucosa (scale bar Å 20 mm). (C ) Double staining with c-kit
The distribution of ICC in the proximal, gangli- and SCF immunohistochemistry (black and white print of a color slide,
see text for details of the double-labeling technique). c-kit–labeled
onic portion of HD appeared similar to that observed in
ICC at the submucosal border (arrowheads). SCF-positive cells are
the normal colon (data not shown). located with distances in the submucosa (arrows) (scale bar Å 50
In the aganglionic segment, the density of c-kit immu- mm). CM, Circular musculature; SM, submucosa.

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October 1996 INTERSTITIAL CELLS IN HIRSCHSPRUNG’S DISEASE 907

Figure 4. c-kit immunoreactivity in the aganglionic colon. (A ) Distribution of ICC in the external muscle layers. The number of ICC labeled is
clearly reduced. *Myenteric plexus (scale bar Å 200 mm). (B ) c-kit immunoreactivity (monoclonal antibody 3D6 on unfixed tissue). Few ICC
(arrows) are observed at the edge of the circular musculature and the submucosa, and they do not form the network present at that level in
the ganglionic colon (see Figure 1A for comparison) (scale bar Å 100 mm). (C ) c-kit immunoreactivity in the aganglionic myenteric plexus layer
(*). There were no apparent close associations between ICC and the hypertrophic nerve bundles (arrow) (scale bar Å 100 mm). (D ) c-kit
immunoreactivity (monoclonal antibody 1D9 3D6 on unfixed tissue) in an isolated ICC (arrow) at the edge of the submucosa and the circular
musculature. The shape of the cell is similar to that of its counterparts in the ganglionic colon (scale bar Å 50 mm). (E ) c-kit immunoreactivity
(monoclonal antibody 1D9 3D6 on unfixed tissue) in the myenteric plexus layer of an aganglionic segment. ICC are present between the
muscular layers. *Aganglionic myenteric plexus layer (scale bar Å 100 mm). CM, Circular musculature; LM, longitudinal musculature; SM,
submucosa.
b

noreactivity was markedly reduced (Figure 4A, compare tion. ICC were clearly distinguished from fibroblast-like
with Figure 2A). However, ICC were readily observed in cells, which were characterized by abundant granular endo-
the most distal musculature of the resected specimen. The plasmic reticulum, frequent Golgi areas, and coated vesicles.
distribution appeared similar along the entire aganglionic They had few caveolae and short membrane-associated dense
zone, even in the cases with long-segment HD (n Å 3). bands but no basal lamina. Macrophage-like cells were infre-
ICC were scarce in the musculature and at the border of quent. The morphology of fibroblast-like cells and macro-
the circular muscle layer (Figure 4B). ICC were rather phage-like cells corresponded to previous descriptions in
frequent in the aganglionic space between the muscle lay- normal adult human colon.12
ers, but the cellular density was reduced in comparison
with normal tissue or ganglionic HD tissue (Table 2). No
specific relationships with the hypertrophic extrinsic nerve
bundles were observed (Figure 4C). Individual ICC in the
submucosal region (Figure 4D) and between the two mus-
cular layers (Figure 4E) appeared similar to ICC in the
normal colon but, due to the reduced cellular density of
ICC, their network appeared disrupted.
Ultrastructural Identification of ICC in the
Aganglionic Distal Colon
ICC were identified by electron microscopy in the
submucosal region associated with the circular muscle layer
(Figure 5). ICC had a complete basal lamina. Caveolae and
dense bodies were present but fewer in number as compared
with smooth muscle cells. Intermediate (10 nm) and thin
(5 nm) filaments were abundant in ICC processes. Thick
filaments (15 nm) were not observed. The main axis orienta-
tion of ICC was in the cephalocaudal (longitudinal) direc-

Table 2. Distribution of c-kit Immunoreactivity in HD

HD

Normal Ganglionic Aganglionic

colon (proximal) (distal)

Longitudinal musculature // // {
Myenteric plexus layer //a //a /b
Circular muscle // // 0/{ Figure 5. Ultrastructure of ICC in the aganglionic colon. Small ICC
Submucosal border // // { bundle (N, nucleus; *processes) in the submucosa (SU) adjacent to
the innermost circular muscle in the affected segment of the colon
NOTE. The cellular density of c-kit immunoreactive cells was assessed in a 1-year-old girl with HD. Intermediate filaments are characteristic
semiquantitatively as follows: 0, no labeling, {, occasional; /, re- of the ICC processes (*), whereas thick filaments are not found. ICC
duced; //, abundant. have a complete basal lamina (arrowheads) and occasional caveolae
a
Around the myenteric ganglia. (thin arrows). A basal body from a cilium is seen in this ICC (Ci).
b
No apparent association with the hypertrophic nerve bundles present Fibroblast-like cells (FLC) have no basal lamina but occasional caveo-
at that level. lae (broad arrow) (original magnification 32,0001; bar Å 1 mm).

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908 VANDERWINDEN ET AL.
Discussion
The elongated cells that display c-kit immunore-
activity in the musculature of the human colon were
similar to those observed in the human pylorus.21 These
cells match the general description of ICC.8,9,12 Their
distribution at the submucosal border of the circular
muscle layer corresponded to previous descriptions of
ICC in adult human colon by osmic acid/iodide tech-
niques and electron microscopy.12 ICC labeled with c-kit
immunoreactivity in the human gut appeared strikingly
similar to the ICC identified with light microscopy in
the mouse.14,16 Recent reports indicate that c-kit is re-
quired for the development and function of the ICC
associated with the myenteric plexus in the mouse.14 – 17
The c-kit rabbit antiserum used in the present study
recognizes a specific peptide of the intracellular domain
of the c-kit receptor (Table 1). The c-kit monoclonal
antibody used in this study, and the several other mono-
clonal antibodies that we tested (unpublished data,
March 1995), recognize epitopes of the extracellular do-
main of the c-kit receptor. In our hands, these mono-
clonal antibodies were suitable for immunohistochemis-
try only on unfixed tissue. Although the pattern of
staining was identical, they usually compared unfavor-
ably with the polyclonal antiserum in terms of sensitivity
and nonspecific background staining. This might partly
explain the absence of c-kit immunoreactivity in the
gut reported in a study using yet another monoclonal
antibody.22 In addition, the delay of harvesting in that
study was 1–2 hours, contrasting with our material har-
vested without delay. ICC might be sensitive to hyp-
oxia,23 but the effect of delayed retrieval on c-kit immu-
noreactivity has not been investigated in humans. A
study using several c-kit–specific rabbit antisera in fresh
frozen human tissues mentioned the presence of c-kit
immunoreactivity in so-called glial cells in the enteric
plexuses of the gut.19 Our data indicate that under ade-
quate circumstances, immunohistochemistry with anti-
bodies for the human c-kit receptor appears valuable for
the study of the distribution of ICC with light micros-
copy in the human gut. This may present significant
advantages in combination with identification of ICC
with electron microscopy.
c-kit immunoreactivity did not stain elements within
the myenteric and submucosal ganglia. ICC did not dis-
play NADPH-diaphorase activity but were frequently
close to nerve fibers expressing NADPH-diaphorase/neu-
ronal NO synthase, and ICC might play a physiological
role of amplification of the NO signal produced by neu-
rons.24
SCF-positive cells were not identified; these cells ap-
peared similar to mesenchymal cells in the submucosa.
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GASTROENTEROLOGY Vol. 111, No. 4
SCF has been found in fibroblasts.25 However, only a
minority of cells in the submucosa were SCF positive,
and the morphology of these cells do not correspond
to the morphology of colonic submucosal fibroblasts as
revealed by osmic acid/iodide methods.12 Mast cells and
nerves were apparently unlabeled by SCF antibodies.
Therefore, by exclusion, it is possible that SCF immuno-
reactivity is present in macrophages. Two forms of SCF
have been described: a membrane-bound molecule and
a soluble, circulating cytokine produced by proteolytic
cleavage of the membrane-bound SCF.26 A granular pat-
tern of staining, apparently extending into the extracellu-
lar matrix, was observed with both SCF antibodies in the
human colon, similar to observations made previously in
the human pylorus.21 This pattern was also reported in
a study of cutaneous mast cells using the same antibody,27
strongly suggesting the labeling of high concentration
of extracellular soluble SCF in the matrix around the
SCF-positive cells.27 Direct interactions between SCF im-
munoreactive cells and ICC seem unlikely because these
cells were apparently not in close contact. However, the
immunohistochemical technique might not be sensitive
enough to detect low levels of SCF expressed by other cell
types. The source of SCF responsible for the physiological
activation of the c-kit receptors expressed by the ICC
and the identification of the SCF immunoreactive cells
deserve further studies.
Our observations give new insight into the ontogeny
of the human ICC. In the normal mouse colon, neither
ICC nor their precursors could be identified by electron
microscopy in term fetuses. ICC were suggested to differ-
entiate only after birth from the gut mesenchyme under
the influences of the ENS and alimentation.7 However
c-kit messenger RNA is already expressed in the colonic
mesenchyme of 1212-day-old mouse embryo.28 In human
fetuses, we observed c-kit positive cells very similar to
ICC in the foregut at 14 weeks of gestation and in the
hindgut at 23 weeks of gestation (Vanderwinden, unpub-
lished observations, February 1994). In contrast to the
mouse, our data indicate that c-kit–positive ICC are
present in the distal colon of human newborns. In 3
neonates with diversion enterostomies, no difference in
ICC distribution was observed between the proximal
part, where nutrients passed, and the distal part, which
was deprived of contact with the chyme. These data sug-
gest that, at least in humans, ICC already develop in the
fetus and that the contact with luminal nutrients is not
essential for their development.
HD is a multigenic disease, and three genes have been
identified thus far to confer susceptibility to agangli-
onosis of the colon by their involvement in neural crest
migration: the RET protooncogene,29,30 the endothelin
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October 1996
3 gene, and the endothelin B receptor gene.31 Another
still unidentified gene located on chromosome 21q22
might also be involved.31 c-kit has been considered in
the past as a possible candidate in the search for genes
involved in hereditary forms of HD,32 but recent develop-
ments in genetics made that hypothesis obsolete. The
human c-kit gene is located on chromosome 4q12-4q13,
and in a large Mennonite kindred, no linkage was found
between HD and the region of the c-kit gene.33
We identified ICC by c-kit immunohistochemistry
in HD, and their presence was confirmed by electron
microscopy. However, the cellular density of ICC ap-
peared markedly reduced in the aganglionic segment.
This may suggest either that the presence of the ENS is
required for full differentiation of the ICC network or
that an underlying mechanism in the mesenchyme affects
both the migration of the neural crest derivatives and
the differentiation of ICC. The embryological origin of
ICC has not been settled,6 but the present data do not
support a neural crest origin. The mesenchymal origin
of ICC has been recently shown in the chicken embryo.34
The distribution of ICC was similar in all the cases in
this study. We did not, however, identify the genetic
defects leading to aganglionosis in our patients, and sub-
tle differences in the distribution of ICC may have been
overlooked. Therefore, a link between a specific genetic
defect leading to aganglionosis and the differentiation of
ICC in patients with HD cannot be ruled out.
In the normal gut, ICC exhibit autorhythmicity, and
electrical coupling between ICC and between ICC and
smooth muscle cells has been documented.4,5,35,36 Electri-
cal coupling is essential for the electrophysiological be-
havior of the entire gut musculature.36 – 38 The number of
ICC associated with the myenteric plexus is dramatically
reduced in BALB/c mice by neonatal passive immuniza-
tion with c-kit antibodies.14 Spontaneous mutations at
the dominant-white spotting/c-kit (W) locus in mouse
cause the absence of ICC in the pacemaking zone of the
small intestine around the myenteric ganglia leading to
the absence of spontaneous slow waves in the muscula-
ture,15 – 17 incoordination of the peristalsis,16 and signs of
pseudo-obstruction.14 There is accumulating evidence,
especially from the canine colon, that ICC at the submu-
cosal border are primary pacemaker cells of the co-
lon.37,39 – 41 Colonic ICC at other locations have not been
extensively studied. Previous pathological studies of the
human colon have not included ICC. Our data indicate
that the network of ICC is disrupted in the aganglionic
colon as compared with the ganglionic colon. This sug-
gests a loss of electrical coupling and gives morphological
support to the lack of spontaneous slow waves in the
aganglionic segment in HD studied in vitro by intracel-
/ 5e12$$0018 09-04-96 13:08:22 gasa
INTERSTITIAL CELLS IN HIRSCHSPRUNG’S DISEASE 909
lular microelectrode recordings.2 A recent preliminary
report on patients with HD seems to confirm the absence
of rhythmic electrical waves in vivo, assessed using elec-
trorectograms.42 The physiology of the residual ICC in
the aganglionic bowel is currently unknown, but ICC
might retain some of their electrical properties. This
might partly explain the poor correlation between the
length of the aganglionic segment and the clinical onset
of obstructive symptoms in HD.3
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Received February 1, 1996. Accepted June 17, 1996.
Address requests for reprints to: Jean-Marie Vanderwinden, M.D.,
Laboratoire de Neurophysiologie et Physiopathologie du Système
Nerveux, Faculté de Médecine, Université Libre de Bruxelles, Cam-
pus Erasme, CP 601, 808 route de Lennik, B-1070 Brussels, Bel-
gium. Fax: (32) 2-555-41-21. e-mail: jmvdwin@ulb.ac.be.
Supported by Belgian grants from Fonds de la Recherche Scienti-
fique Médicale (3.4582.94-97), Fondation Médicale Reine Elisabeth
(1992–1995), Ministère de la Politique Scientifique (Pôle d’Attrac-
tion Interuniversitaire 22, 1990–1995), and National Lottery (1992
and 1994). Dr. Vanderwinden is a Senior Research Assistant at the
National Fund for Scientific Research (Belgium; 1995–1997). Dr.
Liu is a visiting scientist from the Department of Pediatric Surgery,
Xian Children Hospital (Xian, People’s Republic of China).
Preliminary results of this work were presented at the VIIth Belgian
Week of Gastroenterology, March 1995, in Knokke, Belgium.
The authors thank Roberte Menu, Michèle Authelet, Jean-Louis
Conreur, Susan Peters, Niels Johansen, and Susanne Østergård for
skillfull technical assistance. The MGF antibodies were kindly pro-
vided by Michael B. Widmer (Immunex Corp., Seattle, WA).
WBS-Gastro