Dragos Cretoiu

Interstitial cells of Cajal in pancreas

We show here (presumably for the first time) a special type of cell in the human and rat exocrine pancreas. These cells have phenotypic characteristics of the enteric interstitial cells of Cajal (ICC). To identify pancreatic interstitial cells of Cajal (pICC) we used routine light microscopy, non‐conventional light microscopy (less than 1 μm semi‐thin sections of Epon‐embedded specimens cut by ultramicrotomy and stained with Toluidine blue), transmission electron microscopy (TEM), and immunocytochemistry. The results showed that pICC can be recognized easily by light microscopy, particularly on semi‐thin sections, as well as by TEM. Two‐dimensional reconstructions from serial photos suggest a network‐like spatial distribution of pICC. pICC represent 3.3±0.5% of all pancreatic cells, and seem to establish close spatial relationships with: capillaries (43%), acini (40%), stellate cells (14%), nerve fibres (3%). Most of pICC (88%) have 2 or 3 long processes (tens of μm) emerging from the cell body. TEM data show that pICC meet the criteria for positive diagnosis as ICC (e.g. numerous mitochondria, 8.7±0.8% of cytoplasm). Immunocytochemistry revealed that pICC are CD117/c‐kit and CD34 positive. We found pICC positive (40–50%) for smooth muscle α‐actin or S‐100, and, occasionally, for CD68, NK1 neurokinin receptor and vimentin. The reactions for desmin and chromogranin A were negative in pICC. At present, only hypotheses and speculations can be formulated on the possible role of the pICC (e.g., juxtacrine and/or paracrine roles).

Novel type of interstitial cell (Cajal‐like) in human fallopian tube

We describe here ‐ presumably for the first time‐a Cajal‐like type of tubal interstitial cells (t‐ICC), resembling the archetypal enteric ICC. t‐ICC were demonstrated in situ and in vitro on fresh preparations (tissue cryosections and primary cell cultures) using methylene‐blue, crystal‐violet, Janus‐Green B or Mito Tracker‐Green FM Probe vital stainings. Also, t‐ICC were identified in fixed specimens by light microscopy (methylene‐blue, Giemsa, trichrome stainings, Gomori silver‐impregnation) or transmission electron microscopy (TEM). The positive diagnosis of t‐ICC was strengthened by immunohistochemistry (IHC; CD117/c‐kit+ and other 14 antigens) and immunofluorescence (IF; CD117/c‐kit+ and other 7 antigens). The spatial density of t‐ICC (ampullar‐segment cryosections) was 100–150 cells/mm2. Non‐conventional light microscopy (NCLM) of Epon semithin‐sections revealed a network‐like distribution of t‐ICC in lammina propria and smooth muscle meshwork. t‐ICC appeared located beneath of epithelium, in a 10–15μ thick ‘belt’, where 18±2% of cells were t‐ICC. In the whole lamina propria, t‐ICC were about 9%, and in muscularis ∼7%. In toto, t‐ICC represent ∼8% of subepithelial cells, as counted by NCLM. In vitro, t‐ICC were 9.9±0.9% of total cell population.

Interstitial Cajal-like cells in human uterus and fallopian tube.

Abstract:  Recently, parallels have been drawn between enteric interstitial cells of Cajal (ICC) and similar cells outside the gut—interstitial Cajal‐like cells (ICLC). This article reviews our laboratory findings on ICLC in the female reproductive tract. Since the morphology and function of ICLC are still a subject of debate, our purpose was to investigate whether ICLC are present in the fallopian tube and/or uterus, and if they share ultrastructural and immunohistochemical (IHC) features and/or functional roles. We studied ICLC presence in the human fallopian tube and myometrium primarily by light microscopy, and then by transmission electron microscopy (TEM), in tissue samples and at a single cell level. Taking advantage of our ICLC studies of several organs (pancreas, mammary gland, myocardium), we assembled a set of criteria, derived from ultrastructural features of ICLC, called “platinum standard.” Besides the putative pacemaker function, ICLC might have other physiological roles, depending on tissue type (e.g., intercellular signaling, immune surveillance, steroid sensors). Consequently, there is a great urge for a conceptual framework that could allow a better understanding, from a functional point of view, and more so, as the ICLC processes are the longest cellular prolongations (except neurons).

C-kit immunopositive interstitial cells (Cajal-type) in human myometrium.

Previous reports describing Cajal‐like interstitial cells in human uterus are contradictory in terms of c‐kit immunoreactivity: either negative (but vimentin‐positive) in pregnant myometrium, or positive, presumably in the endometrium. The aim of this study was to verify the existence of human myometrial Cajal‐like interstitial cells (m‐CLIC). Six different, complementary approaches were used: 1) methylene‐blue supravital staining of tissue samples (cryosections), 2) methylene blue and Janus green B vital staining (m‐CLIC and mitochondrial markers, respectively), and 3) extracellular single‐unit electrophysiological recordings in cell cultures, 4) non‐conventional light microscopy on glutaraldehyde/osmium fixed, Epon‐embedded semi‐thin sections (less than 1μm) stained with toluidine blue (TSM), 5) transmission electron microscopy (TEM), and 6) immunofluorescence (IF). We found m‐CLIC in myometrial cryosections and in cell cultures. In vitro, m‐CLIC represented ∼7% of the total cell number. m‐CLIC had 2–3 characteristic processes which were very long (∼ 60 μm), very thin (±0.5μm) and moniliform. The dilated portions of processes usually accomodated mitochondria. In vitro, m‐CLIC exhibited spontaneous electrical activity (62.4 ± 7.22 mV field potentials, short duration: 1.197 ± 0.04ms). Moreover, m‐CLIC fulfilled the usual TEM criteria, the so‐called ‘gold’ or ‘platinum’ standards (e.g. the presence of discontinuos basal lamina, caveolae, endoplasmic reticulum, and close contacts between each other, with myocytes, nerve fibers and/or capillaries etc.). IF showed that m‐CLIC express CD117/c‐kit, sometimes associated with CD34 and with vimentin along their processes.

The connective connection: interstitial cells of Cajal (ICC) and ICC‐like cells establish synapses with immunoreactive cells.: Electron microscope study in sity.

We present transmission electron microscope (TEM) evidence that ICC and ICC‐like cells frequently establish close contacts (synapses) with several types of immunoreactive cells (IRC): lymphocytes, plasma cells, eosinophils, basophils, macrophages and mast cells. Such synapses were found in various organs: human mammary gland and myometrium, as well as rat stomach, gut, bladder and uterus. Specimens were observed by conventional TEM on ultrathin sections. Based on morphometric analyses and computer‐aided 3‐D reconstructions from serial sections, we propose an operational definition of ICC‐IRC synapses: cell‐to‐cell close contacts where the two cells are separated by only ∼15nm, equivalent to twice the plasmalemmal thickness. Two types of such synapses were found: (i) uniform (‘plain’) synapses (PS) ‐ close contact extending for >200 nm, and (ii) multicontact (‘kiss and run’) synapses (MS) ‐ with multiple, focal, close‐contact points alternating with regions of wider intermembrane distance. For instance, a typical PS between a rat bladder ICC‐like cell and an eosinophil was 2.48 μm long and 11±4nm wide. By contrast, a MS synapse in rat myometrium (between an ICC‐like cell and an eosinophil) was 8.64 μm long and had 13 contact points. The synaptic cleft measured 15±8nm at contact points and ∼100nm or more in wider areas. These synapses are different from gap junctions usually seen between ICC and between ICC and smooth muscle cells.

Insights into the interstitium of ventricular myocardium: interstitial Cajal-like cells (ICLC).

We have previously described interstitial Cajal‐like cells (ICLC) in human atrial myocardium. Several complementary approaches were used to verify the existence of ICLC in the interstitium of rat or human ventricular myocardium: primary cell cultures, vital stainings (e.g.: methylene blue), traditional stainings (including silver impregnation), phase contrast and non‐conventional light microscopy (Epon‐embedded semithin sections), transmission electron microscopy (TEM) (serial ultrathin sections), stereology, immunohistochemistry (IHC) and immunofluorescence (IF) with molecular probes. Cardiomyocytes occupy about 75% of rat ventricular myocardium volume. ICLC represent ∼32% of the number of interstitial cells and the ratio cardiomyocytes/ICLC is about 70/1. In the interstitium, ICLC establish close contacts with nerve fibers, myocytes, blood capillaries and with immunoreactive cells (stromal synapses). ICLC show characteristic cytoplasmic processes, frequently two or three, which are very long (tens up to hundreds of μm), very thin (0.1‐0.5μm thick), with uneven caliber, having dilations, resulting in a moniliform aspect. Gap junctions between such processes can be found. Usually, the dilations are occupied by mitochondria (as revealed by Janus green B and Mito Tracker Green FM) and elements of endoplasmic reticulum. Characteristically, some prolongations are flat, with a veil‐like appearance, forming a labyrinthic system. ICLC display caveolae (about 1 caveola/1μm cell membrane length, or 2‐4% of the relative cytoplasmic volume, Mitochondria and endoplasmic reticulum (rough and smooth) occupy 5‐10% and 1‐2% of cytoplasmic volume, respectively. IHC revealed positive staining for CD34, EGFR and vimentin and, only in a few cases for CD117. IHC was negative for: desmin, CD57, tau, chymase, tryptase and CD13. IF showed that ventricular ICLC expressed connexin 43. We may speculate that possible ICLC roles might be: intercellular signaling (neurons, myocytes, capillaries etc.) and/or chemomechanical sensors. For pathology, it seems attractive to think that ICLC might participate in the process of cardiac repair/remodeling, arrhythmogenesis and, eventually, sudden death.

Comparative proteomic analysis of human lung telocytes with fibroblasts

Telocytes (TCs) were recently described as interstitial cells with very long prolongations named telopodes (Tps; www.telocytes.com). Establishing the TC proteome is a priority to show that TCs are a distinct type of cells. Therefore, we examined the molecular aspects of lung TCs by comparison with fibroblasts (FBs). Proteins extracted from primary cultures of these cells were analysed by automated 2‐dimensional nano‐electrospray ionization liquid chromatography tandem mass spectrometry (2D Nano‐ESI LC‐MS/MS). Differentially expressed proteins were screened by two‐sample t‐test (P < 0.05) and fold change (>2), based on the bioinformatics analysis. We identified hundreds of proteins up‐ or down‐regulated, respectively, in TCs as compared with FBs. TC proteins with known identities are localized in the cytoskeleton (87%) and plasma membrane (13%), while FB up‐regulated proteins are in the cytoskeleton (75%) and destined to extracellular matrix (25%). These identified proteins were classified into different categories based on their molecular functions and biological processes. While the proteins identified in TCs are mainly involved in catalytic activity (43%) and as structural molecular activity (25%), the proteins in FBs are involved in catalytic activity (24%) and in structural molecular activity, particularly synthesis of collagen and other extracellular matrix components (25%). Anyway, our data show that TCs are completely different from FBs. In conclusion, we report here the first extensive identification of proteins from TCs using a quantitative proteomics approach. Protein expression profile shows many up‐regulated proteins e.g. myosin‐14, periplakin, suggesting that TCs might play specific roles in mechanical sensing and mechanochemical conversion task, tissue homoeostasis and remodelling/renewal. Furthermore, up‐regulated proteins matching those found in extracellular vesicles emphasize TCs roles in intercellular signalling and stem cell niche modulation. The novel proteins identified in TCs will be an important resource for further proteomic research and it will possibly allow biomarker identification for TCs. It also creates the premises for understanding the pathogenesis of some lung diseases involving TCs.

Interstitial Cajal-like cells of human Fallopian tube express estrogen and progesterone receptors

Cells of the female reproductive tract are subject to hormonal control via sex steroid genomic receptors expressed at nuclear level. We previously showed that interstitial Cajal-like cells (ICLC) of human myometrium expressed estrogen and progesterone receptors (ER/PR). Our aim, based on these results, was to see if ER and/or PR could be found also in tubal ICLC. Indeed, we present here immunohistochemical evidence that ICLC of human Fallopian tube (isthmic region) have such receptors. Stromal ICLC, as well as ICLC among smooth muscle layers, were identified in tissue sections by their morphological features (e.g. several very long, moniliform, prolongations of cell body) as well as by c-kit positivity, vital staining with methylene blue or silver impregnation. Additional evidence was provided by sequential staining for c-kit and for PR on the same cell, by ‘sandwich method’. In vitro, the 4th passage cell cultures from Fallopian tube muscularis exhibiting ICLC morphology showed the presence of ER-alpha and/or PR-A by immunofluorescence. In conclusion, our data suggest that ICLC could function as steroid sensors, and might be implicated in Fallopian tube motility (via gap junctions or juxta- and/or paracrine mechanisms).

Myometrial interstitial cells and the coordination of myometrial contractility.

•  Introduction •  Nomenclature •  Uterine contractions and the ‘functional syncytium theory’ •  Interstitial cells of Cajal in the gastrointestinal and urinary tracts •  Are there m‐ICLC in myometrium? •  The effect of c‐kit inhibition on myometrial contractility •  Electrophysiology of m‐ICLC •  Imaging of tissue level signals in myometrium •  Emerging concepts – the possible role of extracellular ATP in myometrial contractility •  Where do m‐ICLC fit in with a model for the generation of myometrial contractions? •  Future research directions •  Conclusion

Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells and lymphocytes.

Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC‐specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8+ T cells from bronchial lymph nodes (T‐BL) and CD8+ T cells from lungs (T‐LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up‐regulated and 70% down‐regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over‐expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types.