Mihail Eugen Hinescu

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.

Telocytes in Human Term Placenta: Morphology and Phenotype

In the last few years, a new cell type – interstitial Cajal-like cell (ICLC) – has been described in digestive and extra-digestive organs. The name has recently been changed to telocytes (TC) and their typical thin, long processes have been named telopodes (TP). To support the hypothesis that TC may also be present in human placenta and add to the information already available, we provide evidence on the ultrastructure, immunophenotype, distribution, and interactions with the surrounding stromal cells of TC in the villous core of human term placenta. We used phase-contrast microscopy, light microscopy of semithin sections, transmission electron microscopy, immunohistochemistry, and immunofluorescence of tissue sections or cell cultures, following a pre-established diagnostic algorithm. Transmission electron microscopy showed cells resembling TC, most (∼76%) having 2–3 very thin, longprocesses (tens to hundreds of micrometers), with an uneven calibre(≤0.5 µm thick) and typical branching pattern. The dilations of processes accommodate caveolae, endoplasmic reticulum cisternae, and mitochondria. These TC have close contacts with perivascular SMC in stem villi. In situ, similar cells are positive for c-kit, CD34, vimentin, caveolin-1, vascular endothelial growth factor (VEGF), and inducible nitric oxide synathase (iNOS). The c-kit-positive cells inconsistently co-express CD34, CD44, αSMA, S100, neuron-specific enolase, and nestin. Among cells with a morphologic TC profile in cell cultures, about 13% co-express c-kit, vimentin, and caveolin-1; 70% of the c-kit-positive cells co-express CD34 and 12% co-express iNOS or VEGF. In conclusion, this study confirms the presence of TC in human term placenta and provides their ultrastructural and immunophenotypic characterization.

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.

Telocytes in human epicardium

The existence of the epicardial telocytes was previously documented by immunohistochemistry (IHC) or immunofluorescence. We have also demonstrated recently that telocytes are present in mice epicardium, within the cardiac stem‐cell niches, and, possibly, they are acting as nurse cells for the cardiomyocyte progenitors. The rationale of this study was to show that telocytes do exist in human (sub)epicardium, too. Human autopsy hearts from 10 adults and 15 foetuses were used for conventional IHC for c‐kit/CD117, CD34, vimentin, S‐100, τ, Neurokinin 1, as well as using laser confocal microscopy. Tissue samples obtained by surgical biopsies from 10 adults were studied by digital transmission electron microscopy (TEM). Double immunolabelling for c‐kit/CD34 and, for c‐kit/vimentin suggests that in human beings, epicardial telocytes share similar immunophenotype features with myocardial telocytes. The presence of the telocytes in human epicardium is shown by TEM. Epicardial telocytes, like any of the telocytes are defined by telopodes, their cell prolongations, which are very long (several tens of μm), very thin (0.1–0.2 μm, below the resolving power of light microscopy) and with moniliform configuration. The interconnected epicardial telocytes create a 3D cellular network, connected with the 3D network of myocardial telocytes. TEM documented that telocytes release shed microvesicles or exocytotic multivesicular bodies in the intercellular space. The human epicardial telocytes have similar phenotype (TEM and IHC) with telocytes located among human working cardiomyocyte. It remains to be established the role(s) of telocytes in cardiac renewing/repair/regeneration processes, and also the pathological aspects induced by their ‘functional inhibition’, or by their variation in number. We consider telocytes as a real candidate for future developments of autologous cell‐based therapy in heart diseases.

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.±45% working myocytes, ˜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 (˜420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.150±0.1 μm), very long for a non‐nervous cell (several tens of μm) 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 ˜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 α‐smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S‐100.

Mesenchymal stem cells and cardiac repair

•  Introduction •  MSC isolation, characterization and standardization ‐  Isolation from different sources ‐  Isolation under different culture conditions ‐  Characterization of MSCs ‐  Standardization of MSCs •  Mechanisms of cardiac repair ‐  Differentiation of MSCs towards cardiomyocytes ‐  Paracrine effect of MSCs ‐  MSCs and blood vessel regeneration ‐  MSC integration into the injured myocardium •  Ex vivo manipulation of MSCs ‐  Pre‐treatment with growth factors ‐  Genetic engineering ‐  Hypoxia preconditioning ‐  Pharmacological interventions •  Pre‐clinical application on cardiovascular disease •  Clinical application: where are we? •  Summary

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.

Interstitial Cajal‐like cells (ICLC) in human atrial myocardium

We present here visual evidence for the existence of a new type of interstitial cells in human atrial myocardium: interstitial Cajal‐like cells (ICLC). These cells fulfil the so‐called ‘platinum standard’(a set of 10 ultrastructural criteria for the positive diagnosis of ICLC). Conventional transmission electron microscopy (TEM), followed by reconstructions from serial photomicrographs, revealed typical ICLC with 2 or 3 long, moniliform processes (several tens of micrometers long and 0.1–0.5 μm thick), emerging from the (small) cell body. Cell processes dichotomously branch and have mitochondria (at the level of dilations), caveolae and Ca2+ release units. Cell prolongations establish close spatial relationships between each other, as well as with capillaries, myocardial cells, and other connective tissue cells. Our preliminary data suggest that ICLC exist in rat ventricular myocardium, too.

Interstitial Cajal‐like cells (ICLC) in myocardial sleeves of human pulmonary veins

We present here evidence for the existence of a new type of interstitial cell in human myocardial sleeves of pulmonary veins: interstitial Cajal‐like cell (ICLC). This cell fulfils the criteria for positive diagnosis of ICLC, including CD 117/c‐kit positivity. Transmission electron microscopy revealed typical ICLC with 2 or 3 very long processes (several tens of mm) suddenly emerging from the cellular body. Also, these processes appear moniliform but extremely thin (0.1–0.4 mm) under the resolving power of the usual microscopy. Cell processes establish close spatial relationships between each other, as well as with capillaries and nerve endings. ICLC appear located among the myocardial cells and particularly at the border between the myocardial sleeve and pulmonary vein wall.