Characterization of Interstitial Cajal Progenitors Cells and Their Changes in Hirschsprung’s Disease

<div><p>Interstitial cells of Cajal (ICC) are critical to gastrointestinal motility. The phenotypes of ICC progenitors have been observed in the mouse gut, but whether they exist in the human colon and what abnormal changes in their quantity and ultrastructure are present in Hirschsprung’s disease (HSCR) colon remains uncertain. In this study, we collected the surgical resection of colons, both proximal and narrow segments, from HSCR patients and normal controls. First, we identified the progenitor of ICC in normal adult colon using immunofluorescent localization techniques with laser confocal microscopy. Next, the progenitors were sorted to observe their morphology. We further applied flow cytometry to examine the content of ICC progenitors in these fresh samples. The ultrastructural changes in the narrow and proximal parts of the HSCR colon were observed using transmission electron microscopy (TEM) and were compared with the normal adult colon. The presumed early progenitor (c-Kit^lowCD34⁺Igf1r⁺) and committed progenitor (c-Kit⁺CD34⁺Igf1r⁺) of ICC exist in adult normal colon as well as in the narrow and proximal parts of the HSCR colon. However, the proportions of mature, early and committed progenitors of ICC were dramatically reduced in the narrow segment of the HSCR colon. The proportions of mature and committed progenitors of ICC in the proximal segment of the HSCR colon were lower than in the adult normal colon. Ultrastructurally, ICC, enteric nerves, and smooth muscle in the narrow segment of the HSCR colon showed severe injury, including swollen vacuola or ted mitochondria, disappearance of mitochondrial cristae, dilated rough endoplasmic reticulum, vesiculation and degranulation, and disappearance of the <i>caveolae</i> on the ICC membrane surface. The contents of ICC and its progenitors in the narrow part of the HSCR colon were significantly decreased than those of adult colon, which may be associated with HSCR pathogenesis.</p></div

Morphology of single normal human ICC progenitor before and after sorting in suspension.

<p>Upper lane: before sorting, laser confocal microscope displayed phenotypes of ICC progenitor in total cell population from adult normal colon. ICC progenitors showed fluorescence of c-Kit (pink), CD34 (red) and Igf1r (green), merged with blue nuclear stained DAPI. At the same time, other cells only show DAPI staining as negative control, without other fluorescence. Lower lane: after sorting, laser confocal microscopy detected the single intact ICC progenitor with three-color fluorescence. But after immunofluorescence-activated sorting, the fluorescence of ICC progenitors recessed.</p

Mature and progenitor ICC located in human adult normal colon by laser confocal.

<p>(<b>A–D</b>) In AP of normal adult colon, the c-Kit positive mature ICCs connected to each other formed extending chords and around AP. ICC progenitors were co-localization of c-Kit⁺CD34⁺Igf1r⁺ found by overlapping fluorescence images. (<b>E–H</b>) In the proximal segment of HSCR, c-Kit positive cells and some of c-Kit⁺/CD34⁺/Igf1r⁺ cells were found occasionally. (<b>I–L</b>) In the narrow segment of the HSCR colon, it was very difficult to find positive ICC and their network structure was damaged, the c-Kit⁺/CD34⁺/Igf1r⁺ cells could not be located by overlapping fluorescence images. Green, red and pink fluorescence represent c-Kit, CD34 and Igf1r, respectively.</p

Statistical analysis of different proportions of ICC and progenitors in adult normal colon and narrow and proximal segments of HSCR colon.

<p>Numbers indicate total colon muscularis cell frequencies (%). (<b>A</b>) The proportions of mature, early and committed progenitors of ICC in the narrow segment of HSCR colon are all remarkably less than in the proximal segment. This is self-control by paired-sample <i>t</i> tests, (<i>n = </i>11). (<b>B</b>) In the proximal segment of the HSCR colon, the proportions of mature and committed progenitor of ICC differ from those of the normal adult colon (<i>n = </i>11); but the proportion of early ICC progenitor is similar to that of the normal adult colon (<i>n = </i>11). This is a comparison of different age groups by independent-sample <i>t</i> tests. (<b>C</b>) In adult normal colon, between the two genders, the proportions of mature, early and committed progenitors of ICC do not differ, as compared by self-controlled paired-sample <i>t</i> tests (female = 5, male = 6). (<b>D</b>) The narrow segment of the HSCR colon, which is the site of the lesion, shows no difference in the proportions of mature, early and committed progenitors of ICC between females (<i>n = </i>5) and males (<i>n = </i>6). They are compared by paired-sample <i>t</i> tests. (<b>E</b>) In the proximal segment of the HSCR colon, the mature ICC proportion in female colons is lower than in male colons, (female = 5, male<i> = </i>6). But the early and committed ICC progenitors show no difference. They are compared by paired-sample <i>t</i> tests. The detailed <i>P</i>-values were marked in the columns for each group. <i>P</i><0.05 was used as cut-off for statistical significance.</p

Immunofluorescent illustrations and 3D reconstruction in narrow and proximal segments of HSCR colon, compared with normal adult colon.

<p>(<b>A, B</b>) The 3D network structures of c-Kit⁺ and c-Kit⁺/CD34⁺/Igf1r⁺ cells were clearly visible in AP of normal adult colon. (<b>C, D</b>) The 3D structures in proximal segment of HSCR were not well formed. (<b>E, F</b>) The 3D structures in narrow segment of HSCR were completely destroyed.</p

Flow cytometry sorting pathway to purify the mature, early and committed progenitors of ICC.

<p>Analysis following gating: (<b>A</b>) Selection of living mononuclear cells on the histogram with Side Scatter (SSC)/Forward Scatter (FSC). R1 gate was used to select the cells with light scatter properties characteristic of live cells. (<b>B</b>) R2 gate was used to select the cells not expressing macrophage markers (F4/80, CD11b) and the general hematopoietic marker CD45 (FITC- cells): gating on histogram SSC/CD45. (<b>C</b>) Detection of ICC phenotypes: the c-Kit⁺ cells population was gated in R4 and further analyzed in step <b>E</b>; the <b>c-</b>Kit^low cells population was gated in R3 and further analyzed in next step <b>D</b>. (<b>D</b>) The cells in R5 were <b>c-</b>Kit^lowCD34⁺Igf1r⁺. (<b>E</b>) The cells in R6 were <b>c-</b>Kit⁺CD34⁺Igf1r⁺, and the cells in R7 were c-Kit⁺CD34⁻Igf1r⁻. (<b>F</b>) To confirm the presence of the c-Kit^low population (R3) reliably between c-Kit⁻ and c-Kit⁺ populations (R4). Pink discontinuous straight line was used as the dividing line between c-Kit⁻ and c-Kit^low populations.</p

Ultrastructural changes in the narrow and proximal segment of the HSCR colon compared with normal adult colon.

<p>In normal human adult colon, typical ultrastructural features were observed by TEM (<b>A–D</b>). (<b>A</b>) Normal ICC had an oval nucleus with condensed heterochromatin distributed in the periphery, abundant mitochondria (m) and rough endoplasmic reticulum (rER). (<b>B</b>) <i>Caveolae</i> was located along the cell membrane (white arrow). (<b>C</b>) ICCs had long processes. Occasionally they were engaged in multicontact synapses with other ICC (pink dotted line). (<b>D</b>) In AP of human normal colon, there were normal mitochondria (m) inside of normal nerve bundles. (<b>E–G</b>) In the narrow segment of the HSCR colon, ICC showed evidence of severe injury. In almost all of the ICCs, the main features included swollen or vacuolated mitochondria, lack of mitochondrial cristae, dilatated and vesiculated rough endoplasmic reticulum (rER), and degranulated ribosomes. The <i>caveolae</i> on the ICCs plasma membrane were disappeared. Orange arrows in (<b>E</b>) and (<b>F</b>) indicate vesiculated rER. Yellow arrows in (<b>E</b>) indicate swollen or vacuolated mitochondria. (<b>E, F</b>) Injured ICCs were surrounded by large amounts of collagen fibrils (coll), ICC processes were hindered by these collagen fibrils from extending to form connections between cells. (<b>G</b>) High magnification displayed swollen or vacuolated mitochondria and vesiculated rER inside of ICC, which appeared as high density clumps inside of mitochondria (black small arrow). (<b>H</b>) Injured nerve bundles were surrounded by many collagen fibrils (coll). Inside of nerve bundles, swollen mitochondria and lysosomes were identified (black arrows). (<b>I</b>) Swollen or vacuolated mitochondria and dilatated rER inside of smooth muscle cells (SMC) in the narrow segment of the HSCR colon were identified as high density clumps present inside of some SMC mitochondria, which were similar to ICC. (<b>J</b>) Smooth muscle cells in the proximal segment of the HSCR colon were relatively normal. (<b>K, L</b>) In the proximal segment of the HSCR colon, there were long processes of ICC surrounding nerve bundles, not hindered by collagen fibrils (<b>K</b>). There were some swollen mitochondria and dilatated rER inside of ICC (orange font), at the same time some ICC presented with normal rER (black font). A few collagen fibrils (coll) presented beside ICC, but the amount was not greater than in the narrow segment of the HSCR colon.</p