Dynamics of telopodes (telocyte prolongations) in cell culture depends on extracellular matrix protein.

Telocytes (TC) are cells with telopodes (Tp), very long prolongations (up to 100 µm) with an uneven caliber ( www.telocytes.com ). Factors determining the dynamics of cellular prolongations are still unknown, although previous studies showed telopode motility in TC cultures. We comparatively investigated, by time-lapse videomicroscopy, the dynamics of Tp of mouse heart TC seeded on collagen, fibronectin, and laminin. Under our experimental conditions, TC and fibroblasts (cell line L929) behaved differently in terms of adherence, spreading, and prolongation extension. Fibroblasts showed lower spreading on the matrix proteins used. The time needed for spreading was 2-4 h for TC, versus 8-10 h for fibroblasts. The values for final cell surface area after spreading were between 200 and 400 µm(2) for fibroblasts and 800-2,000 µm(2) for TC. TC showed a more than three times higher ability to spread on the tested matrix proteins. An extremely low capacity to extend prolongations with lengths shorter than cell bodies was noted for fibroblasts, while TC extended prolongations longer than the cell body length, with a moniliform appearance. The stronger adherence and spreading were noted for TC seeded on fibronectin, while the lowest were on laminin. Collagen determined an intermediate adherence and spreading for TC, but the highest dynamics in Tp extensions. In conclusion, TC behave differently than fibroblasts in terms of adherence, spreading, and cell prolongation extension when seeded on various matrix proteins in cell culture.

Human cardiac telocytes: 3D imaging by FIB-SEM tomography.

Telocyte (TC) is a newly identified type of cell in the cardiac interstitium (www.telocytes.com). TCs are described by classical transmission electron microscopy as cells with very thin and long telopodes (Tps; cellular prolongations) having podoms (dilations) and podomers (very thin segments). TCs' three-dimensional (3D) morphology is still unknown. Cardiac TCs seem to be particularly involved in long and short distance intercellular signalling and, therefore, their 3D architecture is important for understanding their spatial connections. Using focused ion beam scanning electron microscopy (FIB-SEM) we show, for the first time, the whole ultrastructural anatomy of cardiac TCs. 3D reconstruction of cardiac TCs by FIB-SEM tomography confirms that they have long, narrow but flattened (ribbon-like) telopodes, with humps generated by the podoms. FIB-SEM tomography also confirms the network made by TCs in the cardiac interstitium through adherens junctions. This study provides the first FIB-SEM tomography of a human cell type.

Genetic comparison of mouse lung telocytes with mesenchymal stem cells and fibroblasts.

Telocytes (TCs) are interstitial cells with telopodes - very long prolongations that establish intercellular contacts with various types of cells. Telocytes have been found in many organs and various species and have been characterized ultrastructurally, immunophenotypically and electrophysiologically (www.telocytes.com). Telocytes are distributed through organ stroma forming a three-dimensional network in close contacts with blood vessels, nerve bundles and cells of the local immune system. Moreover, it has been shown that TCs express a broad range of microRNAs, such as pro-angiogenic and stromal-specific miRs. In this study, the gene expression profile of murine lung TCs is compared with other differentiated interstitial cells (fibroblasts) and with stromal stem/progenitor cells. More than 2000 and 4000 genes were found up- or down-regulated, respectively, in TCs as compared with either MSCs or fibroblasts. Several components or regulators of the vascular basement membrane are highly expressed in TCs, such as Nidogen, Collagen type IV and Tissue Inhibitor of Metalloproteinase 3 (TIMP3). Given that TCs locate in close vicinity of small vessels and capillaries, the data suggest the implication of TCs in vascular branching. Telocytes express also matrix metalloproteases Mmp3 and Mmp10, and thus could regulate extracellular matrix during vascular branching and de novo vessel formation. In conclusion, our data show that TCs are not fibroblasts, as the ultrastructure, immunocytochemistry and microRNA assay previously indicated. Gene expression profile demonstrates that TCs are functionally distinct interstitial cells with specific roles in cell signalling, tissue remodelling and angiogenesis.

Recent advances in synthesis, characterization of hydroxyapatite/polyurethane composites and study of their biocompatible properties.

The development of engineered biomaterials that mimic bone tissues is a promising research area that benefits from a growing interest. Polymers and polymer-ceramic composites are the principle materials investigated for the development of synthetic bone scaffolds thanks to their proven biocompatibility and biostability. Several polymers have been combined with calcium phosphates (mainly hydroxyapatite) to prepare nanocomposites with improved biocompatible and mechanical properties. Here, we report the hydrothermal synthesis in high pressure conditions of nanostructured composites based on hydroxyapatite and polyurethane functionalized with carboxyl and thiol groups. Cell-material interactions were investigated for potential applications of these new types of composites as coating for orthopedic implants. Physical-chemical and morphological characteristics of hydroxyapatite/polyurethane composites were evaluated for different compositions, showing their dependence on synthesis parameters (pressure, temperature). In vitro experiments, performed to verify if these composites are biocompatible cell culture substrates, showed that they are not toxic and do not affect cell viability.

Telocytes in human skin--are they involved in skin regeneration?

Telocytes (TCs), a particular interstitial cell type, have been recently described in a wide variety of mammalian organs (www.telocytes.com). The TCs are identified morphologically by a small cell body and extremely long (tens to hundreds of µm), thin prolongations (less than 100 nm in diameter, below the resolving power of light microscopy) called telopodes. Here, we demonstrated with electron microscopy and immunofluorescence that TCs were present in human dermis. In particular, TCs were found in the reticular dermis, around blood vessels, in the perifollicular sheath, outside the glassy membrane and surrounding sebaceous glands, arrector pili muscles and both the secretory and excretory portions of eccrine sweat glands. Immunofluorescence screening and laser scanning confocal microscopy showed two subpopulations of dermal TCs; one expressed c-kit/CD117 and the other was positive for CD34. Both subpopulations were also positive for vimentin. The TCs were connected to each other by homocellular junctions, and they formed an interstitial 3D network. We also found TCs adjoined to stem cells in the bulge region of hair follicles. Moreover, TCs established atypical heterocellular junctions with stem cells (clusters of undifferentiated cells). Given the frequency of allergic skin pathologies, we would like to emphasize the finding that close, planar junctions were frequently observed between TCs and mast cells. In conclusion, based on TC distribution and intercellular connections, our results suggested that TCs might be involved in skin homeostasis, skin remodelling, skin regeneration and skin repair.

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.

Experimental acute myocardial infarction: telocytes involvement in neo-angiogenesis.

We used rat experimental myocardial infarction to study the ultrastructural recovery, especially neo-angiogenesis in the infarction border zone. We were interested in the possible role(s) of telocytes (TCs), a novel type of interstitial cell very recently discovered in myocardim (see http://www.telocytes.com). Electron microscopy, immunocytochemistry and analysis of several proangiogenic microRNAs provided evidence for TC involvement in neo-angiogenesis after myocardial infarction. Electron microscopy showed the close spatial association of TCs with neoangiogenetic elements. Higher resolution images provided the following information: (a) the intercellular space between the abluminal face of endothelium and its surrounding TCs is frequently less than 50 nm; (b) TCs establish multiple direct nanocontacts with endothelial cells, where the extracellular space seems obliterated; such nanocontacts have a length of 0.4-1.5 µm; (c) the absence of basal membrane on the abluminal face of endothelial cell. Besides the physical contacts (either nanoscopic or microscopic) TCs presumably contribute to neo-angiognesis via paracrine secretion (as shown by immunocytochemistry for VEGF or NOS2). Last but not least, TCs contain measurable quantities of angiogenic microRNAs (e.g. let-7e, 10a, 21, 27b, 100, 126-3p, 130a, 143, 155, 503). Taken together, the direct (physical) contact of TCs with endothelial tubes, as well as the indirect (chemical) positive influence within the 'angiogenic zones', suggests an important participation of TCs in neo-angiogenesis during the late stage of myocardial infarction.

Identification of telocytes in skeletal muscle interstitium: implication for muscle regeneration.

Skeletal muscle interstitium is crucial for regulation of blood flow, passage of substances from capillaries to myocytes and muscle regeneration. We show here, probably, for the first time, the presence of telocytes (TCs), a peculiar type of interstitial (stromal) cells, in rat, mouse and human skeletal muscle. TC features include (as already described in other tissues) a small cell body and very long and thin cell prolongations-telopodes (Tps) with moniliform appearance, dichotomous branching and 3D-network distribution. Transmission electron microscopy (TEM) revealed close vicinity of Tps with nerve endings, capillaries, satellite cells and myocytes, suggesting a TC role in intercellular signalling (via shed vesicles or exosomes). In situ immunolabelling showed that skeletal muscle TCs express c-kit, caveolin-1 and secrete VEGF. The same phenotypic profile was demonstrated in cell cultures. These markers and TEM data differentiate TCs from both satellite cells (e.g. TCs are Pax7 negative) and fibroblasts (which are c-kit negative). We also described non-satellite (resident) progenitor cell niche. In culture, TCs (but not satellite cells) emerge from muscle explants and form networks suggesting a key role in muscle regeneration and repair, at least after trauma.

Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure.

Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC-CM), which exhibit cardiac-like transmembrane action potentials, intracellular Ca(2+) transients and contractions. While major features of the excitation-contraction coupling of iPSC-CM have been well-described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31-day-old (post-plating) iPSC-CM generated from human hair follicle keratinocytes (HFKT-iPSC-CM) were analysed by electron microscopy, and compared with those of human embryonic stem-cell-derived cardiomyocytes (hESC-CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT-iPSC-CM and hESC-CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT-iPSC-CM. We also found in both cell types: (1) 'Ca(2+)-release units', which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.

Cardiomyocyte precursors and telocytes in epicardial stem cell niche: electron microscope images.

A highly heterogeneous population of stem and progenitor cells has been described by light immunohistochemistry in the mammalian adult heart, but the ultrastructural identity of cardiac stem cells remains unknown. Using electron microscopy, we demonstrate the presence of cells with stem features in the adult mouse heart. These putative cardiac stem cells are small (6-10 microm), round cells, with an irregular shaped nucleus, large nucleolus, few endoplasmic reticulum cisternae and mitochondria, but numerous ribosomes. Stem cells located in the epicardial stem cell niche undergo mitosis and apoptosis. Cells with intermediate features between stem cells and cardiomyocyte progenitors have also been seen. Moreover, electron microscopy showed that cardiomyocyte progenitors were added to the peripheral working cardiomyocytes. Telocytes make a supportive interstitial network for stem cells and progenitors in the stem cell niche. This study enhances the hypothesis of a unique type of cardiac stem cell and progenitors in different stages of differentiation. In our opinion, stem cells, cardiomyocyte progenitors and telocytes sustain a continuous cardiac renewal process in the adult mammalian heart.

Telocytes in endocardium: electron microscope evidence.

The term TELOCYTES was very recently introduced, for replacing the name Interstitial Cajal-Like Cells (ICLC). In fact, telocytes are not really Cajal-like cells, they being different from all other interstitial cells by the presence of telopodes, which are cell-body prolongations, very thin (under the resolving power of light microscopy), extremely long (tens up to hundreds of micrometers), with a moniliform aspect (many dilations along), and having caveolae. The presence of telocytes in epicardium and myocardium was previously documented. We present here electron microscope images showing the existence of telocytes, with telopodes, at the level of mouse endocardium. Telocytes are located in the subendothelial layer of endocardium, and their telopodes are interposed in between the endocardial endothelium and the cardiomyocytes bundles. Some telopodes penetrate from the endocardium among the cardiomyocytes and surround them, eventually. Telopodes frequently establish close spatial relationships with myocardial blood capillaries and nerve endings. Because we may consider endocardium as a 'blood-heart barrier', or more exactly as a 'blood-myocardium barrier', telocytes might have an important role in such a barrier being the dominant cell population in subendothelial layer of endocardium.

Cardiac telocytes: serial dynamic images in cell culture.

Telocytes (TC) are interstitial cells with telopodes (Tp). These prolongations (Tp) are quite unique: very long (several tens of micrometres) and very thin (≤0.5 µm), with moniliform aspect: thin segments (podomeres) alternating with dilations (podoms). To avoid any confusion, TC were previously named interstitial Cajal-like cells (ICLC). Myocardial TC were repeatedly documented by electron microscopy, immunohistochemistry and immunofluorescence. TC form a network by their Tp, either in situ or in vitro. Cardiac TC are (completely) different of 'classic' fibroblasts or fibrocytes. We hereby present a synopsis of monitoring, by time-lapse videomicroscopy, of Tp network development in cell culture. We used a protocol that favoured interstitial cell selection from adult mouse myocardium. Videomicroscopy showed dynamic interactions of neighbour TC during the network formation. During their movement, TC leave behind distal segments (podomeres) of their Tp as guiding marks for the neighbouring cells to follow during network rearrangement.

Regenerative medicine: then and now--an update of recent history into future possibilities.

The fields of tissue engineering (TE) and regenerative medicine (RegMed) are yet to bring about the anticipated therapeutic revolution. After two decades of extremely high expectations and often disappointing returns both in the medical as well as in the financial arena, this scientific field reflects the sense of a new era and suggests the feeling of making a fresh start although many scientists are probably seeking reorientation. Much of research was industry driven, so that especially in the aftermath of the recent financial meltdown in the last 2 years we have witnessed a biotech asset yard sale. Despite any monetary shortcomings, from a technological point of view there have been great leaps that are yet to find their way to the patient. RegMed is definitely bound to play a major role in our life because it embodies one of the primordial dreams of mankind, such as: everlasting youth, flying, remote communication and setting foot on the moon. The Journal of Cellular and Molecular Medicine has been at the frontier of these developments in TE and RegMed from its beginning and reflects recent scientific advances in both fields. Therefore this review tries to look at RegMed through the keyhole of history which might just be like looking 'back to the future'.

TELOCYTES - a case of serendipity: the winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to TELOCYTES.

Ramon y Cajal discovered a particular cell type in the gut, which he named 'interstitial neurons' more that 100 years ago. In the early 1970s, electron microscopy/electron microscope (EM) studies showed that indeed a special interstitial cell type corresponding to the cells discovered by Cajal is localized in the gut muscle coat, but it became obvious that they were not neurons. Consequently, they were renamed 'interstitial cells of Cajal' (ICC) and considered to be pace-makers for gut motility. For the past 10 years many groups were interested in whether or not ICC are present outside the gastrointestinal tract, and indeed, peculiar interstitial cells were found in: upper and lower urinary tracts, blood vessels, pancreas, male and female reproductive tracts, mammary gland, placenta, and, recently, in the heart as well as in the gut. Such cells, now mostly known as interstitial Cajal-like cells (ICLC), were given different and confusing names. Moreover, ICLC are only apparently similar to canonical ICC. In fact, EM and cell cultures revealed very particular features of ICLC, which unequivocally distinguishes them from ICC and all other interstitial cells: the presence of 2-5 cell body prolongations that are very thin (less than 0.2 mum, under resolving power of light microscopy), extremely long (tens to hundreds of mum), with a moniliform aspect (many dilations along), as well as caveolae. Given the unique dimensions of these prolongations (very long and very thin) and to avoid further confusion with other interstitial cell types (e.g. fibroblast, fibrocyte, fibroblast-like cells, mesenchymal cells), we are proposing the term TELOCYTES for them, and TELOPODES for their prolongations, by using the Greek affix 'telos'.

Telocytes in human isolated atrial amyloidosis: ultrastructural remodelling.

The human heart can be frequently affected by an organ-limited amyloidosis called isolated atrial amyloidosis (IAA). IAA is a frequent histopathological finding in patients with long-standing atrial fibrillation (AF). The aim of this paper was to investigate the ultrastructure of cardiomyocytes and telocytes in patients with AF and IAA. Human atrial biopsies were obtained from 37 patients undergoing cardiac surgery, 23 having AF (62%). Small fragments were harvested from the left and right atrial appendages and from the atrial sleeves of pulmonary veins and processed for electron microscopy (EM). Additional fragments were paraffin embedded for Congo-red staining. The EM examination certified that 17 patients had IAA and 82% of them had AF. EM showed that amyloid deposits, composed of characteristic 10-nm-thick filaments were strictly extra-cellular. Although, under light microscope some amyloid deposits seemed to be located within the cardiomyocyte cytoplasm, EM showed that these deposits are actually located in interstitial recesses. Moreover, EM revealed that telopodes, the long and slender processes of telocytes, usually surround the amyloid deposits limiting their spreading into the interstitium. Our results come to endorse the presumptive association of AF and IAA, and show the exclusive, extracellular localization of amyloid fibrils. The particular connection of telopodes with amyloid deposits suggests their involvement in isolated atrial amyloidosis and AF pathogenesis.

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.

A distinct type of cell in myocardium: interstitial Cajal-like cells (ICLCs).

The existence of a novel type of interstitial cells in the heart, interstitial Cajal-like cells (ICLCs), had been described for the first time in 2005. Their identification was mainly based on ultrastructural criteria: very long (tens up to hundreds of micrometres) and moniliform prolongations, which are extremely thin (less than 0.2 microm), below the resolving power of light microscopy. Myocardial ICLCs were also identified by methylene-blue vital staining, silver impregnation, and immunoreactivity for CD 34, vimentin, CD117/c-kit, etc. Although a series of studies provided evidence for the existence of ICLCs in human atria and rat ventricles, further investigations in other laboratories, using additional techniques, are required to substantiate the consistency of these findings. Here we provide further evidence for the existence of ICLCs in human and mammalian hearts (by transmission and scanning electron microscopy, as well as confocal laser scanning microscopy). Noteworthy, we confirm that ICLCs communicate with neighbouring cells via shedding (micro)vesicles. Although these so-called ICLCs represent a distinct type of cells, different from classical interstitial cells of Cajal, or fibroblasts, their role(s) in myocardium remain(s) to be established. Several hypotheses are proposed: (i) adult stromal (mesenchymal) stem cells, which might participate in cardiac repair/remodelling; (ii) intercellular signalling (e.g. via shedding microvesicles); (iii) chemo-mechanical transducers and (iv) players in pacemaking and/or arrhytmogenesis, and so on.

Human epicardium: ultrastructural ancestry of mesothelium and mesenchymal cells.

The human sub-epicardial area contains an unexplored cellular population under a layer of mesothelial cells. Transmission electron microscopy revealed the existence of interstitial Cajal-like cells (ICLCs), isolated smooth muscle cells (iSMC) and mesenchymal cells besides other well-known cells. The presence of iSMC in the sub-epicardial space is quite unique and could explain why epicardial-derived cells isolated from human epicardium generate smooth muscle cells in culture. Mesenchymal cells, guided by ICLCs, were found migrating from sub-epicardial area in the mesothelial layer. These findings suggest that epithelial-mesenchymal transition is not a common process involved in cardiac regeneration in vivo.

Myocardial interstitial Cajal-like cells (ICLC) in caveolin-1 KO mice.

Abstract We compared, by transmission electron microscopy (TEM), the ultrastructure of interstitial Cajal-like cells (ICLC) in normal mammalian myocardium versus caveolin-1 null mice. TEM showed that myocardial ICLCs of caveolin-1-deficient mice retain their main ultrastructural characteristics, for example, location among cardiomyocytes, close vicinity to nerves and/or blood capillaries, specialized cell-to-cell junctions, presence of 2-3 typical processes, which are very long (several tens of micrometres), but are very thin (0.1-0.2 microm) and moniliform. However, the most striking modification of myocardial ICLC in caveolin-1 KO mice was the absence of caveolae. Beyond this main observation, three other findings could be reported: (1) the absence of caveolae in capillary endothelium, (2) persistence of (some) caveolae at the level of cardiomyocte sarcolemma or vascular smooth muscle cell sarcolemma and (3) (un)expected ultrastructural modifications such as increased thickness of capillary basement membrane and increased autophagy of several cardiomyocytes.

Cardiac renewing: interstitial Cajal-like cells nurse cardiomyocyte progenitors in epicardial stem cell niches.

Recent studies suggested that various cell lineages exist within the subepicardium and we supposed that this area could host cardiac stem cell niches (CSCNs). Using transmission electron microscopy, we have found at least 10 types of cells coexisting in the subepicardium of normal adult mice: adipocytes, fibroblasts, Schwann cells and nerve fibres, isolated smooth muscle cells, mast cells, macrophages, lymphocytes, interstitial Cajal-like cells (ICLCs) and cardiomyocytes progenitors (CMPs). The latter cells, sited in the area of origin of coronary arteries and aorta, showed typical features of either very immature or developing cardiomyocytes. Some of these cells were connected to each other to form columns surrounded by a basal lamina and embedded in a cellular network made by ICLCs. Complex intercellular communication occurs between the ICLCs and CMPs through electron-dense nanostructures or through shed vesicles. We provide here for the first time the ultrastructural description of CSCN in the adult mice myocardium, mainly containing ICLCs and CMPs. The existence of resident CMPs in different developmental stages proves that cardiac renewing is a continuous process. We suggest that ICLCs might act as supporting nurse cells of the cardiac niches and may be responsible for activation, commitment and migration of the stem cells out of the niches. Briefly, not only resident cardiac stem cells but also ICLCs regulate myocyte turnover and contribute to both cardiac cellular homeostasis and endogenous repair/remodelling after injuries.