Distribution and characteristics of telocytes as nurse cells in the architectural organization of engineered heart tissues

Interstitial Cajal-like cells are a distinct type of interstitial cell with a wide distribution in mammalian organs and tissues,and have been given the name"telocytes".Recent studies have demonstrated the potential roles of telocytes in heart development,renewal,and repair.However,further research on the functions of telocytes is limited by the complicated in vivo environment.This study was designed to construct engineered heart tissue(EHT)as a three-dimensional model in vitro to better understand the role of telocytes in the architectural organization of the myocardium.EHTs were constructed by seeding neonatal cardiomyocytes in collagen/Matrigel scaffolds followed by culture under persistent static stretch.Telocytes in EHTs were identified by histology,toluidine blue staining,immunofluorescence,and transmission electron microscopy.The results from histology and toluidine blue staining demonstrated widespread putative telocytes with compact toluidine blue-stained nuclei,which were located around cardiomyocytes.Prolongations from the cell bodies showed a characteristic dichotomous branching pattern and formed networks in EHTs.Immunofluorescence revealed positive staining of telocytes for CD34 and vimentin with typical moniliform prolongations.A series of electron microscopy images further showed that typical telocytes embraced the cardiomyocytes with their long prolongations and exhibited a marked appearance of nursing cardiomyocytes during the construction of EHTs.This finding highlights the great importance of telocytes in the architectural organization of EHTs.It also suggests that EHT is an appropriate physical and pathological model system in vitro to study the roles of telocytes during heart development and regeneration.     

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.     

Telocytes accompanying cardiomyocyte in primary culture: two- and three-dimensional culture environment

Recently, the presence of telocytes was demonstrated in human and mammalian tissues and organs (digestive and extra-digestive organs, genitourinary organs, heart, placenta, lungs, pleura, striated muscle). Noteworthy, telocytes seem to play a significant role in the normal function and regeneration of myocardium. By cultures of telocytes in two- and three-dimensional environment we aimed to study the typical morphological features as well as functionality of telocytes, which will provide important support to understand their in vivo roles. Neonatal rat cardiomyocytes were isolated and cultured as seeding cells in vitro in two-dimensional environment. Furthermore, engineered myocardium tissue was constructed from isolated cells in three-dimensional collagen/Matrigel scaffolds. The identification of telocytes was performed by using histological and immunohistochemical methods. The results showed that typical telocytes are distributed among cardiomyocytes, connecting them by long telopodes. Telocytes have a typical fusiform cell body with two or three long moniliform telopodes, as main characteristics. The vital methylene blue staining showed the existence of telocytes in primary culture. Immunohistochemistry demonstrated that some c-kit or CD34 immuno-positive cells in engineered heart tissue had the morphology of telocytes, with a typical fusiform cell body and long moniliform telopodes. Also, a significant number of vimentin+ telocytes were present within engineered heart tissue. We suggest that the model of three-dimensional engineered heart tissue could be useful for the ongoing research on the functional relationships of telocytes with cardiomyocytes. Because the heart has the necessary potential of changing the muscle and non-muscle cells during the lifetime, telocytes might play an active role in the heart regeneration process. Moreover, telocytes might be a useful tool for cardiac tissue engineering.     

Identification of telocytes in the lamina propria of rat duodenum: transmission electron microscopy

Recently the new term 'telocytes' has been proposed for cells formerly known as interstitial Cajal-like cells. 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, extremely long with a moniliform aspect. The identification of these cells is based on ultrastructural criteria. The presence of telocytes in others organs was previously documented. We reported for the first time, an ultrastructural study of telocytes in the lamina propria of rat duodenum. Our findings show that typical telocytes are present in the rat duodenum. Telocytes are located in the lamina propria, immediately below mucosal crypts. Telopodes frequently establish close spatial relationships with immune cells, blood vessels and nerve endings. On the basis of their distribution and morphology, we suggest that these cells may be involved in immune response and in our opinion, it may be possible that different locations of telocytes could be associated with different roles.     

Electron microscopy of human fascia lata: focus on telocytes

From the histological point of view, fascia lata is a dense connective tissue. Although extracellular matrix is certainly the most predominant fascia's feature, there are also several cell populations encountered within this structure. The aim of this study was to describe the existence and characteristics of fascia lata cell populations viewed through a transmission electron microscope. Special emphasis was placed on telocytes as a particular interstitial cell type, recently discovered in a wide variety of tissues and organs such as the heart, skeletal muscles, skin, gastrointestinal tract, uterus and urinary system. The conducted study confirmed the existence of a telocyte population in fascia lata samples. Those cells fulfil main morphological criteria of telocytes, namely, the presence of very long, thin cell processes (telopodes) extending from a relatively small cell body. Aside from telocytes, we have found fibroblasts, mast cells and cells with features of myofibroblastic differentiation. This is the first time it has been shown that telocytes exist in human fascia. Currently, the exact role of those cells within the fascia is unknown and definitely deserves further attention. One can speculate that fascia lata telocytes likewise telocytes in other organs may be involved in regeneration, homeostasis and intracellular signalling.     

Telocytes in liver: electron microscopic and immunofluorescent evidence

Hepatic interstitial cells play a vital role in regulating essential biological processes of the liver. Telocytes (TCs), a novel type of interstitial cells firstly identified by Popescu and his coworkers, have been reported in many tissues and organs, but not yet in liver (go to http://www.telocytes.com ). We used transmission electron microscopy and immunofluorescence (double labelling for CD34 and c‐kit/CD117, or vimentin, or PDGF Receptor‐α, or β) to provide evidence for the existence of TCs in mice liver. The distribution of TCs in liver was found to be of similar density in the four hepatic lobes. In conclusion, here we show the presence of TCs in mice liver. It remains to be determined the possible roles of TCs in the control of liver homeostasis and regeneration, the more so as a close special relationship was found between TCs and hepatic putative stem (progenitor) cells.     

Telocytes in the human sinoatrial node

The sinoatrial node (SAN) is composed mostly of pacemaker, transitional and Purkinje‐like cells. Pacemaker cells, especially in the centre of the SAN, are surrounded by dense fibrous tissue and do not have any contact with transitional cells. We hypothesize that the SAN contains telocytes that have contacts with pacemaker cells and contractile myocardium. Immunohistochemistry using antibodies against HCN4 and antibody combinations against CD34 and HCN4 was carried out on 12 specimens. Confocal laser scanning microscopy (CLSM) with two mixtures of primary antibodies, namely CD34/S100 and vimentin/S100, was performed in three cases. In two cases, CLSM was carried out with CD117 antibody. Specimens for electron microscopy and immunocytochemistry with HCN4 immunogold labelling were taken from another three patients. In our study, we found cells with the immunophenotype of telocytes in the SAN. There were twice as many of these cells in the centre of the SAN as in the periphery (20.3 ± 4.8 versus 10.8 ± 4.4 per high‐power field). They had close contact with pacemaker cells and contractile cardiomyocytes and expressed HCN4. The ultrastructural characteristics of these cells are identical to those of telocytes observed earlier in other organs. Our study provides evidence that telocytes are present in the SAN.     

Evidence for progressive reduction and loss of telocytes in the dermal cellular network of systemic sclerosis

Telocytes, a peculiar type of stromal cells, have been recently identified in a variety of tissues and organs, including human skin. Systemic sclerosis (SSc, scleroderma) is a complex connective tissue disease characterized by fibrosis of the skin and internal organs. We presently investigated telocyte distribution and features in the skin of SSc patients compared with normal skin. By an integrated immunohistochemical and transmission electron microscopy approach, we confirmed that telocytes were present in human dermis, where they were mainly recognizable by their typical ultrastructural features and were immunophenotypically characterized by CD34 expression. Our findings also showed that dermal telocytes were immunophenotypically negative for CD31/PECAM‐1 (endothelial cells), α‐SMA (myofibroblasts, pericytes, vascular smooth muscle cells), CD11c (dendritic cells, macrophages), CD90/Thy‐1 (fibroblasts) and c‐kit/CD117 (mast cells). In normal skin, telocytes were organized to form three‐dimensional networks distributed among collagen bundles and elastic fibres, and surrounded microvessels, nerves and skin adnexa (hair follicles, sebaceous and sweat glands). Telocytes displayed severe ultrastructural damages (swollen mitochondria, cytoplasmic vacuolization, lipofuscinic bodies) suggestive of ischaemia‐induced cell degeneration and were progressively lost from the clinically affected skin of SSc patients. Telocyte damage and loss evolved differently according to SSc subsets and stages, being more rapid and severe in diffuse SSc. Briefly, in human skin telocytes are a distinct stromal cell population. In SSc skin, the progressive loss of telocytes might (i) contribute to the altered three‐dimensional organization of the extracellular matrix, (ii) reduce the control of fibroblast, myofibroblast and mast cell activity, and (iii) impair skin regeneration and/or repair.     

FIB‐SEM tomography of human skin telocytes and their extracellular vesicles

We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non‐epithelial spaces (stroma) of many organs (see www.telocytes.com ). TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms (containing mitochondria, endoplasmic reticulum and caveolae) and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB‐SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB‐SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon‐like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) – the podoms. FIB‐SEM tomography also showed numerous extracellular vesicles (diameter 438.6 ± 149.1 nm, n = 30) released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc.     

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.     

Relationships between telocytes and cardiomyocytes during pre‐ and post‐natal life

Evidence has been given that the adult heart contains a specific population of stromal cells lying in close spatial relationship with cardiomyocytes and with cardiac stem cells in sub‐epicardial cardiogenic niches. Recently termed ‘telocytes’ because of their long cytoplasmic processes embracing the parenchymal cells, these cells have been postulated to be involved in heart morphogenesis. In our opinion, investigating the occurrence and morphology of telocytes during heart histogenesis may shed further light on this issue. Our findings show that typical telocytes are present in the mouse heart by early embryonic to adult life. These cells closely embrace the growing cardiomyocytes with their long, slender cytoplasmic processes. Hence, in the developing myocardium, telocytes may play nursing and guiding roles for myocardial precursors to form the correct three‐dimensional tissue architectural pattern, as previously suggested.     

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.     

A loss of telocytes accompanies fibrosis of multiple organs in systemic sclerosis

Systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of the skin and various internal organs. In SSc, telocytes, a peculiar type of stromal (interstitial) cells, display severe ultrastructural damages and are progressively lost from the clinically affected skin. The aim of the present work was to investigate the presence and distribution of telocytes in the internal organs of SSc patients. Archival paraffin‐embedded samples of gastric wall, myocardium and lung from SSc patients and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were studied on tissue sections subjected to CD34 immunostaining. CD34/CD31 double immunofluorescence was performed to unequivocally differentiate telocytes (CD34‐positive/CD31‐negative) from vascular endothelial cells (CD34‐positive/CD31‐positive). Few telocytes entrapped in the fibrotic extracellular matrix were found in the muscularis mucosae and submucosa of SSc gastric wall. In the muscle layers and myenteric plexus, the network of telocytes was discontinuous or even completely absent around smooth muscle cells and ganglia. Telocytes were almost completely absent in fibrotic areas of SSc myocardium. In SSc fibrotic lung, few or no telocytes were observed in the thickened alveolar septa, around blood vessels and in the interstitial space surrounding terminal and respiratory bronchioles. In SSc, the loss of telocytes is not restricted to the skin, but it is a widespread process affecting multiple organs targeted by the fibrotic process. As telocytes are believed to be key players in the regulation of tissue/organ homoeostasis, our data suggest that telocyte loss might have important pathophysiological implications in SSc.     

The primary cilium of telocytes in the vasculature: electron microscope imaging

Blood vessels are highly organized and complex structure, which are far more than simple tubes conducting the blood to almost any tissue of the body. The fine structure of the wall of blood vessels has been studied previously using the electron microscope, but the presence the telocytes associated with vasculature, a specific new cellular entity, has not been studied in depth. Interestingly, telocytes have been recently found in the epicardium, myocardium, endocardium, human term placenta, duodenal lamina propria and pleura. We show the presence of telocytes located on the extracellular matrix of blood vessels (arterioles, venules and capillaries) by immunohistochemistry and transmission electron microscopy. Also, we demonstrated the first evidence of a primary cilium in telocytes. Several functions have been proposed for these cells. Here, the telocyte-blood vessels cell proximity, the relationship between telocytes, exosomes and nervous trunks may have a special significance.     

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.     

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.     

Presence,localization and morphology of TELOCYTES in developmental gonads of fishes

Telocytes are a new defined type of interstitial cells, considered as a stem cell, with very long and thin cytoplasmic extensions. They are present in the vertebrates, and may participate in tissue remodeling. In fish, during gonadal development, the events that culminate with the germinal epithelium formation are well known. However, the interstitial compartment remains poorly explored, although it may have a great contribution to the morpho-functional changes that occur in the gonad. As in other organisms, in fish, the interstitium consists especially of connective tissue elements. However, until now, there are no reports of the presence and the action of the telocytes in the connective tissue of gonads of fish. Thus, this study aimed to detect the presence, localization and morphology of telocytes during the gonadal development of several species of fish. The gonads were analyzed by light microscopy, transmission electron microscopy and immunohistochemistry for localization of CD34, Vimentin, and metalloproteinases. The presence of two proteins characteristics of mesenchymal cell was detected in cells of the gonads of all species. In addition, they presented a typical morphology of telocytes, showing cellular extensions. Gonadal telocytes also presented positive response to metalloproteinases. In mammals, telocytes can undergo de-differentiation contributing to the reorganization of the extracellular matrix. This role may be performed by the metalloproteinases detected here. The detection of Vimentin and CD34 in the same cellular type, associated with its morphological characteristics, allows us to conclude that some interstitial cells in Teleostei are considered telocytes, identical to the ones already described in mammals and other vertebrates.     

Transmission electron microscope evidence of telocytes in canine dura mater

Telocytes (TCs) are a novel type of interstitial cells present in a wide variety of organs and tissues ( www.telocytes.com ). Telocytes are identified morphologically by a small cell body and specific long prolongations (telopodes) alternating thin segments (podomers) with dilations (podoms). The presence of TCs in rat meninges has been identified in previous research. We here present further evidence that TCs existed in canine dura mater, closed to capillary and surrounded by a great deal of collagen fibres under transmission electron microscope.     

Telocytes in pleura: two- and three-dimensional imaging by transmission electron microscopy

Information about the ultrastructure of connective (interstitial) cells supporting the pleural mesothelium is scarce. Our aim has been to examine whether telocytes (TCs) are present in pleura, as in epicardium and mesentery. TCs are a distinct type of cell, characterized by specific prolongations named telopodes (Tp). We have used transmission electron microscopy (TEM) and electron tomography (ET) to determine whether ultrastructural diagnostic criteria accepted for TCs are fulfilled by any of the cell subpopulations existing in the sub-mesothelial layer in mouse and human pleura. TCs have been identified with TEM by their characteristic prolongations. Tp appear long and moniliform, because of the alternation of podomeres (thin segments of less than 0.2 μm) and podoms (small dilations accommodating caveolae, mitochondria, and endoplasmic reticulum). Tp ramifications follow a dichotomic pattern and establish specialized cell-to-cell junctional complexes. TCs, via their Tp, seem to form an interstitial network beneath the mesothelium, covering about two-thirds of the abluminal mesothelial layer. ET has revealed complex junctional structures and tight junctions connecting pleural TCs, and small vesicles at this level in Tp. Thus, pleural TCs share significant similarities with TCs described in other serosae. Whether TCs are a (major) player in mesothelial-cell-induced tissue repair remains to be established. Nevertheless, the extremely long thin Tp and complex junctional structures that they form and the release of vesicles (or exosomes) indicate the participation of TCs in long-distance homo- or heterocellular communication.     

Telocytes and interstitial cells of Cajal in the biliary system

A novel type of interstitial tissue cells in the biliary tree termed telocytes (TCs), formerly known as interstitial Cajal‐like cells (ICLCs), exhibits very particular features which unequivocally distinguish these cells from interstitial cells of Cajal (ICCs) and other interstitial cell types. Current research substantiates the existence of TCs and ICCs in the biliary system (gallbladder, extrahepatic bile duct, cystic duct, common bile duct and sphincter of Oddi). Here, we review the distribution, morphology and ultrastructure of TCs and ICCs in the biliary tree, with emphasis on their presumptive roles in physiological and pathophysiological processes.