Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization

We have previously reported (Hinescu & Popescu, 2005) the existence of interstitial Cajal-like cells (ICLC), by transmission electron microscopy, in human atrial myocardium. In the present study, ICLC were identified with non-conventional light microscopy (NCLM) on semi-thin sections stained with toluidine blue and immunohistochemistry (IHC) for CD117/c-kit, CD34, vimentin and other additional antigens for differential diagnosis. Quantitatively, on semi-thin sections, ICLC represent about 1-1.5% of the atrial myocardial volume (vs. approximately 45% working myocytes, approximately 2% endothelial cells, 3-4% for other interstitial cells, and the remaining percentage: extracellular matrix). Roughly, there is one ICLC for 8-10 working atrial myocytes in the intercellular space, beneath the epicardium, with a characteristic (pyriform, spindle or triangular) shape. These ICLC usually have 2-3 definitory processes, emerging from cell body, which usually embrace atrial myocytes (260 nm average distance plasmalemma/sarcolemma) or establish close contact with nerve fibers or capillaries (approximately 420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.15+/-0.1 microm), very long for a non-nervous cell (several tens of microm) and moniliform (uneven caliber). Stromal synapses between ICLC and other interstitial cells (macrophages) were found (e.g. in a multicontact type synapse, the average synaptic cleft was approximately 65 nm). Naturally, the usual cell organelles (mitochondria, smooth and rough endoplasmic reticulum, intermediate filaments) are relatively well developed. Caveolae were also visible on cell prolongations. No thick filaments were detected. IHC showed that ICLC were slightly and inconsistently positive for CD117/c-kit, variously co-expressed CD34 and EGF receptor, but appeared strongly positive for vimentin, along their prolongations. Some ICLC seemed positive for a-smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S-100. In conclusion, we provide further evidence of the existence of ICLC in human atrial myocardium, supporting the possible ICLC role in pacemaking, secretion (juxta- and/or paracrine), intercellular signaling (neurons and myocytes). For pathology, ICLC might as well be 'players' in arrhythmogenesis and atrial remodeling.     

Interstitial Cajal-like cells in rat mesentery: an ultrastructural and immunohistochemical approach

Interstitial Cajal-like Cells (ICLC) were recently recognized in a plethora of non-digestive organs. Here, we describe a cell type of rat mesentery sharing ultrastructural and immunohistochemical features with ICLC. Mesenteric ICLC were demonstrated by transmission electron microscopy (TEM) and further tested by light microscope immunohistochemistry. The cell described here fulfils the TEM diagnostic criteria accepted for ICLC: location in the connective interstitium; close vicinity to nerves, capillaries and other interstitial cells; characteristic long, moniliform cell processes; specialized cell-to-cell junctions; caveolae; mitochondria at 5-10% of cytoplasmic volume; rough endoplasmic reticulum at about 1-2%; intermediate and thin filaments, microtubules; undetectable thick filaments. The processes of this mesenteric ICLC were particularly long, with a mean length of 24.91 microm (10.27-50.83 micorm), and a convolution index of 2.32 (1.37-3.63) was calculated in order to measure their potential length. Mean distances versus main target cells of ICLC-nerve bundles, vessels, adipocytes and macrophages-were 110.69, 115.80, 205.07 and 34.65 nm, respectively. We also tested the expression of CD117/c-kit, CD34, vimentin, alpha-smooth muscle actin, nestin, NK-1, tryptase and chymase and the antigenic profile of the mesenteric ICLC was comparable if not identical with that recently observed in ICLC from other extra-digestive tissues. Due to the peculiar aspect of the mesenteric ICLC processes it can be hypothesized that these cells form a three-dimensional network within the mesentery that is at the same time resistant and deformable following stretches consequent to intestine movements, mainly avoiding blood vessels closure or controlling blood vessels rheology. It remains, however, to be established if and how such cells are connected with the archetypal enteric ICC.     

Immunolocalization and expression of small‐conductance calcium‐activated potassium channels in human myometrium

Small‐conductance calcium‐activated potassium (SK3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro. The aims of this study were to identify the precise localization of SK3 channels and to quantify SK3 mRNA expression in myometrium from pregnant and non‐pregnant women. Myometrial biopsies were obtained from pregnant (n = 11) and non‐pregnant (n = 11) women. The expression of SK3 channels was assessed using immunohistochemistry and SK3 mRNA was determined by qRT‐PCR. In non‐pregnant myometrium SK3 immunoreactivity was observed in CD34 positive (CD34⁺) interstitial Cajal‐like cells (ICLC), now called telocytes. Although CD34⁺ cells were also present in pregnant myometrium, they lacked SK3 immunoreactivity. Furthermore, the immunohistochemical results showed that SK3 expression in vascular endothelium was similar between the two groups. CD117 immunoreactivity was only detected in small round cells that resemble mast cells. Compared to non‐pregnant myometrium we found significantly less SK3 mRNA in pregnant myometrium. We demonstrate that SK3 channels are localized solely in CD34⁺ cells and not in smooth muscle cells, and that the molecular expression of SK3 channels is higher in non‐pregnant compared to pregnant myometrium. On the basis of our previous study and the present findings, we propose that SK3 activators reduce contractility in human myometrium by modulating telocyte function. This is the first report to provide evidence for a possible role of SK3 channels in human uterine telocytes.     

Ultrastructure of Cajal-like interstitial cells in the human detrusor

The aim of this ultrastructural study was to examine the human detrusor for interstitial cells of Cajal (ICC)-like cells (ICC-L) by conventional transmission electron microscopy (TEM) and immuno-transmission electron microscopy (I-TEM) with antibodies directed towards CD117 and CD34. Two main types of interstitial cells were identified by TEM: ICC-L and fibroblast-like cells (FLC). ICC-L were bipolar with slender (0.04 μm) flattened dendritic-like processes, frequently forming a branching labyrinth network. Caveolae and short membrane-associated dense bands were present. Mitochondria, rough endoplasmic reticulum and Golgi apparatus were observed in the cell somata and cytoplasmic processes. Intermediate filaments were abundant but no thick filaments were found. ICC-L were interconnected by close appositions, gap junctions and peg-and-socket junctions (PSJ) but no specialised contacts to smooth muscle or nerves were apparent. FLC were characterised by abundant rough endoplasmic reticulum but no caveolae or membrane-associated dense bands were observed; gap junctions and PSJ were absent and intermediate filaments were rare. By I-TEM, CD34 gold immunolabelling was present in long cytoplasmic processes corresponding to ICC-L between muscle fascicles but CD117 gold immunolabelling was negative. Thus, ICC-like cells are present in the human detrusor. They are CD34-immunoreactive and have a myoid ultrastructure clearly distinguishable from fibroblast-like cells. ICC-L may be analogous to interstitial cells of Cajal in the gut.     

Telocytes in the human kidney cortex

Renal interstitial cells play an important role in the physiology and pathology of the kidneys. As a novel type of interstitial cell, telocytes (TCs) have been described in various tissues and organs, including the heart, lung, skeletal muscle, urinary tract, etc. ( www.telocytes.com ). However, it is not known if TCs are present in the kidney interstitium. We demonstrated the presence of TCs in human kidney cortex interstitium using primary cell culture, transmission electron microscopy (TEM) and in situ immunohistochemistry (IHC). Renal TCs were positive for CD34, CD117 and vimentin. They were localized in the kidney cortex interstitial compartment, partially covering the tubules and vascular walls. Morphologically, renal TCs resemble TCs described in other organs, with very long telopodes (Tps) composed of thin segments (podomers) and dilated segments (podoms). However, their possible roles (beyond intercellular signalling) as well as their specific phenotype in the kidney remain to be established.     

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 approximately 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 microm), very thin (0.1-0.5 microm 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 MitoTracker 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/ 1 microm 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.     

Phenotypic characterization of GPR120-expressing cells in the interstitial tissue of pancreas

GPR120 functions as a plasma membrane receptor for unsaturated long-chain free fatty acids and involves in GLP-1 secretion, adipogenesis and the control of energy balance. Pancreas is the key organ in fuel and energy metabolism. Here GPR120 expression in human and rat pancreas was observed by RT-PCR, and the distribution and phenotypes of GPR120-positive cells in human and rat pancreas were shown by immunohistochemical staining. GPR120 mRNA expression was found in human and rat pancreas. GPR120-positive cells were scattered mainly in the interstitial tissues of human and rat pancreas, and they were not co-localized with nestin, vimentin, alpha-SMA and glucagon, respectively. However, GPR120 was distributed on the cells positively stained by CD68, the specific marker of macrophages, and on the cells positive stained by CD34 and CD117, the markers of interstitial cells. In conclusion, this study demonstrates the expression of GPR120 in pancreas and shows the distribution of GPR120 in human and rat pancreas.     

Myocardial telocytes: a specific new cellular entity

The existence of a new type of interstitial cells in the heart namely, interstitial Cajal-like cells (ICLC), has been described for the first time by Hinescu and Popescu in 2005. This study was then followed by an ascending trend of publications regarding the morphology, phenotype and distribution of myocardial ICLC in diverse species including human patients. Recently the new term ‘telocytes’ has been proposed for cells formerly known as ICLC, and the term ‘telopodes’ has been proposed for the prolongations of these cells. The identification of these cells is based on ultrastructural criteria. In addition, telocyters/telyopodes can be identified by several complementary approaches including methylene blue vital staining, silver impregnation and immunoreactivity against CD117/c-kit, vimentin, etc. This point of view presents critical data existing in literature, as well as own results, which unequivocally provide compelling evidence that telocytes are a new distinct cellular entity of myocardial interstitium. Several presumable functions of the myocardial telocytes are discussed: (i) intercellular signalling, (ii) cardiac repair/remodelling and (iii) stem cell nursing in cardiac renewal.     

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 MitoTracker-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 lamina propria and smooth muscle meshwork. t-ICC appeared located beneath of epithelium, in a 10-15 microm thick 'belt', where 18+/-2% of cells were t-ICC. In the whole lamina propria, t-ICC were about 9%, and in muscularis approximately 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. TEM showed that the diagnostic 'gold standard' (Huizinga et al., 1997) is fulfilled by 'our' t-ICC. However, we suggest a 'platinum standard', adding a new defining criterion- characteristic cytoplasmic processes (number: 1-5; length: tens of microm; thickness: < or =0.5 microm; aspect: moniliform; branching: dichotomous; organization: network, labyrinthic-system). Quantitatively, the ultrastructural architecture of t-ICC is: nucleus, 23.6+/-3.2% of cell volume, with heterochromatin 49.1+/-3.8%; mitochondria, 4.8+/-1.7%; rough and smooth endoplasmic-reticulum (1.1+/-0.6%, 1.0+/-0.2%, respectively); caveolae, 3.4+/-0.5%. We found more caveolae on the surface of cell processes versus cell body, as confirmed by IF for caveolins. Occasionally, the so-called 'Ca2+-release units' (subplasmalemmal close associations of caveolae+endoplasmic reticulum+mitochondria) were detected in the dilations of cell processes. Electrophysiological single unit recordings of t-ICC in primary cultures indicated sustained spontaneous electrical activity (amplitude of membrane potentials: 57.26+/-6.56 mV). Besides the CD117/c-kit marker, t-ICC expressed variously CD34, caveolins 1&2, alpha-SMA, S-100, vimentin, nestin, desmin, NK-1. t-ICC were negative for: CD68, CD1a, CD62P, NSE, GFAP, chromogranin-A, PGP9.5, but IHC showed the possible existence of (neuro)endocrine cells in tubal interstitium. We call them 'JF cells'. In conclusion, the identification of t-ICC might open the door for understanding some tubal functions, e.g. pace-making/peristaltism, secretion (auto-, juxta- and/or paracrine), regulation of neurotransmission (nitrergic/purinergic) and intercellular signaling, via the very long processes. Furthermore, t-ICC might even be uncommitted bipotential progenitor cells.     

Human placenta: de visu demonstration of interstitial Cajal-like cells

Traditional interstitial cells of Cajal (ICC) are present in the digestive tube and are supposed to act as pacemakers and neuromodulators. However, interstitial Cajal-like cells (ICLCs) were found outside the gastrointestinal tract, in various organs (e.g. ureter, bladder, fallopian tube, uterus, pancreas, mammary gland, myocardium etc.) and looking for such ICLC is a priority in our laboratories.We report here unequivocal visual evidence that ICLCs are present in the mesenchymal tissue of the villi from human term placenta.The following methods were used: a. vital staining with methylene blue (cryosections); b. silver impregnation (paraffin sections); c. Epon-embedded sections (approximately 1 microm) of glutaraldehyde/osmium fixed tissue, stained with toluidine blue; d. primary cell cultures (or second-passage cells) to reveal the characteristic, very long, moniliform cell processes and mitochondrial localization at dilations (molecular fluorescence probe: Mito Tracker Green); e. immunofluorescence for c-kit/CD117 marker or other characteristic proteins; f. transmission electron microscopy to establish the identity of ICLC.