Eugen Radu

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.

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.

miR-193 expression differentiates telocytes from other stromal cells

Telocytes (TCs) are a particular type of interstitial (stromal) cells defined by very long, moniliform telopodes. Their tissue location, between blood vessels and other cells such as cardiomyocytes (CMC) and neurons, suggests a role in intercellular signalling. In order to define a microRNA (miR) signature in cardiac TCs, we have found that miR‐193 is differentially expressed between TCs and other interstitial cells. Because miR‐193 regulates c‐kit, our data support the previous finding that TCs express c‐kit in certain circumstances. In addition, the miRs which are specific to CMC and other muscle cells (e.g. miR‐133a, miR‐208a) are absent in TCs. Overall the data reinforce the view that TCs are a particular type of interstitial (mesenchymal) cells.

Cajal-type cells from human mammary gland stroma: phenotype characteristics in cell culture.

We report here the in vitro isolation of Cajal‐like interstitial cells from human inactive mammary‐gland Stroma. Primary cell cultures examined in phase‐contrast microscopy or after vital methylene‐blue staining revealed a cell population with characteristic morphological phenotype: fusiforms, triangular or polygonal cell body and the corresponding (very) long, slender, moniliform cytoplasmic processes. Giremsa staining pointed out the typical knobbed aspect of cell prolongations. Immunofluorescence (IF) showed, like in situ immunohistochemistry, that Cajal‐type cells in vitro (primary culltures), expressed c‐kit/CD117 and vimentin. In conclusion, the images presented here reinforce our previous hypothesis that human mammary glands have a distinct population of Cajal‐like cells in non‐epithelial tissue compartments.

Biocompatibility Assessment of Novel Collagen-Sericin Scaffolds Improved with Hyaluronic Acid and Chondroitin Sulfate for Cartilage Regeneration

Cartilage tissue engineering (CTE) applications are focused towards the use of implantable biohybrids consisting of biodegradable scaffolds combined with in vitro cultured cells. Hyaluronic acid (HA) and chondroitin sulfate (CS) were identified as the most potent prochondrogenic factors used to design new biomaterials for CTE, while human adipose-derived stem cells (ASCs) were proved to display high chondrogenic potential. In this context, our aim was not only to build novel 3D porous scaffolds based on natural compounds but also to evaluate their in vitro biological performances. Therefore, for prospective CTE, collagen-sericin (Coll-SS) scaffolds improved with HA (5% or 10%) and CS (5% or 10%) were used as temporary physical supports for ASCs and were analyzed in terms of structural, thermal, morphological, and swelling properties and cytotoxic potential. To complete biocompatibility data, ASCs viability and proliferation potential were also assessed. Our studies revealed that Coll-SS hydrogels improved with 10% HA and 5% CS displayed the best biological performances in terms of cell viability, proliferation, morphology, and distribution. Thus, further work will address a novel 3D system including both HA 10% and CS 5% glycoproteins, which will probably be exposed to prochondrogenic conditions in order to assess its potential use in CTE applications.

Apoptosis in human embryo development: 3. Fas‐induced apoptosis in brian primary cultures

Fas (APO‐1/CD95) is an important apoptotic mediator for both immune and nervous systems. In the present study, we have investigated the expression and function of Fas in human embryonic/fetal brain primary cultures from 12 human embryos and fetuses with gestational ages between 5 to 22 weeks. Anti‐Fas fluorescent antibody was used for labeling of Fas positive cells and for quantitation of Fas expression in brain cultures. To demonstrate that Fas receptor is functional in human embryonic/fetal brain cells, anti‐Human‐Fas monoclonal antibody (0.5 μg/ml) was used to induce apoptosis in brain primary cultures. Apoptosis was investigated by flow‐cytometry and fluorescent microscopy using TUNEL and annexin V labeling. Fas was found to be expressed in the embryonic/fetal human primary brain cultures, on neuronal and glial cells or their precursors, varying with gestational ages. Cross‐linking of Fas induced apoptosis in brain cultures indicating that Fas receptor functions as a death receptor. We also showed that cell death triggered through Fas receptor was caspase dependent, hence it was blocked by a selective caspase‐8 inhibitor (IETD‐fmk).These results suggest that Fas is involved in neuronal apoptosis in the developing human brain.

CD117/c‐kit positive interstitial (Cajal‐like) cells in human pancreas

We provide evidence that interstitial Cajal‐like cells, previously described in human pancreas ‐ pICC (J Cel Mol Med, 9: 169, 2005), are positive for c‐kit irrespective of immunohistochemical procedures used. Various sample types (fresh cryosections or formalin‐fixed, paraffin‐embedded specimens), various slide pretreatments (with or without heat‐induced epitope retrieval) or different antibodies used (Dako polyclonal or Santa Cruz monoclonal), all showed CD117‐positive pICC.

Co-stimulatory and adhesion molecules of dendritic cells in rheumatoid arthritis

Dendritic cells (DCs) in the rheumatoid arthritis (RA) joint mediate the immunopathological process and act as a potent antigen presenting cell. We compared the expression of co‐stimulatory and adhesion molecules on DCs in RA patients versus controls with traumatic joint lesions and evalulated the correlation between the immunophenotypical presentation of DCs and the clinical status of the disease. Samples of peripheral venous blood, synovial fluid (SF) and synovial tissue (ST) were obtained from 10 patients with RA at the time of hip or knee replacement and from 9 control patients with knee arthroscopy for traumatic lesions. Clinical status was appreciated using the DAS28 score. Blood, SF and dissociated ST cell populations were separated by centrifugation and analyzed by flow cytometry. Cells phenotypes were identified using three‐color flow cytometry analysis for the following receptors HLA‐DR, CD80, CD83, CD86, CD11c, CD18, CD54, CD58, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD56. HLA‐DR molecules, co‐stimulatory receptors CD80, CD86, CD83 and adhesion molecules CD18, CD11c, CD54, CD58, were analyzed by two‐color immunofluorescence microscopy on ST serial sections. In patients with active RA (DAS28>5.1) we found a highly differentiated subpopulation of DCs in the ST and SF that expressed an activated phenotype (HLA‐DR, CD86+, CD80+, CD83+, CD11c+, CD54+, CD58+). No differences were found between circulating DCs from RA patients and control patients. Our data suggest an interrelationship between clinical outcome and the immunophenotypical presentation of DCs. Clinical active RA (DAS28>5.1) is associated with high incidence of activated DCs population in the ST and SF as demonstrated by expression of adhesion and co‐stimulatory molecules.

Apoptosis in the immune system: 1. Fas-induced apoptosis in monocytes-derived human dendritic cells.

Dendritic cells (DC) are cells of the hematopoietic system specialized in capturing antigens and initiating T cell‐mediated immune responses. We show here that human DC generated from adherent peripheral blood mononuclear cells (PBMC) after in vitro stimulation with granulocyte macrophage colony stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) express Fas antigen (APO‐1, CD95) and can undergo apoptosis upon triggering of Fas by monoclonal antibodies. Immature monocytes‐derived dendritic cells (MDDC) upregulate CD86 and HLA‐DR expression and develop dendrites and veiled processes. Flow cytometry analysis revealed CD95 expression in approx. 40% of these MDDC and incubation with anti‐CD95 mAb (0.5μg/ml) induced apoptosis when compared to untreated controls. The extent of apoptosis induced by the agonist anti‐Fas antibody strongly related to the percentage of cells expressing CD 95. Upon tumor necrosis factor α (TNF‐α) additional stimulation, MDDC assumed a characteristic mature dendritic cells morphology showing prolonged veils, CD83 expression, and high levels of HLA‐DR. These cells have downregulated their Fas receptors (to approx. 20%) and undergo apoptosis to a lesser extent when treated with anti‐CD 95, as demonstrated by the hardly noticeable effect of this antibody on the viability of cultured cells as compared to controls. Thus, upon TNF‐α induced maturation, MDDC became resistant to Fas‐induced apoptosis. The apoptotic episodes surrounding the earlier stage of DC differentiation appeared to be mediated by Fas. In contrast, a Fas independent pathway is probably responsible for the apoptotic events associated with terminally differentiated DC.