Morphologic evidence of telocytes in human thyroid stromal tissue.

Despite the evidence accumulated over the past decade that telocytes (TCs) are a distinctive, though long neglected, cell entity of the stromal microenvironment of several organs of the human body, to date their localization in the endocrine glands remains almost unexplored. This study was therefore undertaken to examine the presence and characteristics of TCs in normal human thyroid stromal tissue through an integrated morphologic approach featuring light microscopy and ultrastructural analysis. TCs were first identified by immunohistochemistry that revealed the existence of an intricate network of CD34+ stromal cells spread throughout the thyroid interfollicular connective tissue. Double immunofluorescence allowed to clearly differentiate CD34+ stromal cells lacking CD31 immunoreactivity from neighbour CD31+ microvascular structures, and the evidence that these stromal cells coexpressed CD34 and platelet-derived growth factor receptor α further strengthened their identification as TCs. Transmission electron microscopy confirmed the presence of stromal cells ultrastructurally identifiable as TCs projecting their characteristic cytoplasmic processes (i.e., telopodes) into the narrow interstitium between thyroid follicles and blood microvessels, where telopodes intimately surrounded the basement membrane of thyrocytes. Collectively, these morphologic findings provide the first comprehensive demonstration that TCs are main constituents of the human thyroid stroma and lay the necessary groundwork for further in-depth studies aimed at clarifying their putative implications in glandular homeostasis and pathophysiology.

Scleroderma-like Impairment in the Network of Telocytes/CD34+ Stromal Cells in the Experimental Mouse Model of Bleomycin-Induced Dermal Fibrosis.

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34+ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34+ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31-/CD34+ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA+ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine.

Impairment in the telocyte/CD34+ stromal cell network in human rheumatoid arthritis synovium.

Telocytes (TCs)/CD34+ stromal cells have recently emerged as peculiar interstitial cells detectable in a variety of organs throughout the human body. TCs are typically arranged in networks establishing unique spatial relationships with neighbour cells and likely contributing to the maintenance of tissue homeostasis by both cell-to-cell contacts and releasing extracellular vesicles. Hence, TC defects are being increasingly reported in different pathologies, such as chronic inflammatory and fibrotic conditions. In this regard, TCs/CD34+ stromal cells have been shown to constitute an intricate interstitial network in the subintimal area of the normal human synovial membrane, but whether they are altered in chronic synovitis has yet to be explored. We therefore undertook a morphologic study to compare the distribution of TCs/CD34+ stromal cells between normal synovium and chronically inflamed synovium from patients with rheumatoid arthritis (RA) by using CD34 immunohistochemistry and CD31/CD34 double immunofluorescence. CD34 immunostaining revealed that, at variance with normal synovium, the inflamed and hyperplastic RA synovial tissue was nearly or even completely devoid of TCs/CD34+ stromal cells. Double immunofluorescence confirmed that almost all CD34+ tissue components detectable in RA synovium were blood vessels coexpressing CD31, while a widespread network of CD31- /CD34+ TCs was clearly evident in the whole sublining layer of normal synovium. In the context of the emerging diverse roles of TCs/CD34+ stromal cells in the regulation of tissue homeostasis and structure, the remarkable impairment in their networks herein uncovered in RA synovium may suggest important pathophysiologic implications that will be worth investigating further.

A Two-Step Immunomagnetic Microbead-Based Method for the Isolation of Human Primary Skin Telocytes/CD34+ Stromal Cells.

Telocytes (TCs), commonly referred to as TCs/CD34+ stromal cells, are a peculiar type of interstitial cells with distinctive morphologic traits that are supposed to exert several biological functions, including tissue homeostasis regulation, cell-to-cell signaling, immune surveillance, and reparative/regenerative effects. At present, the majority of studies investigating these cells are mainly descriptive and focus only on their morphology, with a consequent paucity of functional data. To gain relevant insight into the possible functions of TCs, in vitro analyses are clearly required, but currently, the protocols for TC isolation are only at the early stages and not fully standardized. In the present in vitro study, we describe a novel methodology for the purification of human primary skin TCs through a two-step immunomagnetic microbead-based cell separation (i.e., negative selection for CD31 followed by positive selection for CD34) capable of discriminating these cells from other connective tissue-resident cells on the basis of their different immunophenotypic features. Our experiments clearly demonstrated that the proposed method allows a selective purification of cells exhibiting the peculiar TC morphology. Isolated TCs displayed very long cytoplasmic extensions with a moniliform silhouette (telopodes) and presented an immunophenotypic profile (CD31-/CD34+/PDGFRα+/vimentin+) that unequivocally differentiates them from endothelial cells (CD31+/CD34+/PDGFRα-/vimentin+) and fibroblasts (CD31-/CD34-/PDGFRα+/vimentin+). This novel methodology for the isolation of TCs lays the groundwork for further research aimed at elucidating their functional properties and possible translational applications, especially in the field of regenerative medicine.

Telocytes and lymphatic endothelial cells: Two immunophenotypically distinct and spatially close cell entities.

Telocytes (TCs) have recently emerged as a peculiar type of stromal cells located in both perivascular and interstitial compartments of multiple anatomical sites in humans, other mammals and vertebrates. Pioneer electron microscopy studies have ultrastructurally defined TCs as "stromal cells with telopodes" (i.e. very long and thin cell processes with a moniliform morphology conferred by the irregular alternation of slender segments and small, bead-like, dilated portions), whereupon it has become apparent that TCs largely correspond to the CD34+ stromal/interstitial cells detectable by immunohistochemical assays. Besides CD34, TCs are also characterized by the expression of platelet-derived growth factor receptor (PDGFR)α. Interestingly, recent works recommended that lymphatic endothelial cell (LEC) markers should be routinely assessed to discriminate with certainty TCs from LECs, because these two cell types may exhibit similar morphological traits, especially when initial lymphatics are sectioned longitudinally and appear as vascular profiles with no obvious lumen. Furthermore, it has been argued that lymphatic microvessels immunostained for the small mucin-type transmembrane glycoprotein podoplanin (PDPN), which is widely used as lymphatic endothelial marker, can be easily misidentified as TCs. Nevertheless, surprisingly these assumptions were not based on double tissue immunostaining for TC and LEC markers. Therefore, the present morphological study was undertaken to precisely investigate the mutual spatial organization and putative relationships of TCs and lymphatic vessels in tissues from different human organs. For this purpose, we carried out a series of double immunofluorescence analyses simultaneously detecting the CD34 or PDGFRα antigen and a marker of LECs, either PDPN or lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). In the connective tissue compartment of different organs, TCs were CD34+/PDGFRα+/PDPN-/LYVE-1- while LECs were CD34-/PDGFRα-/PDPN+/LYVE-1+, thus representing two definitely distinct, though spatially close, cell entities. The arrangement of telopodes to intimately surround the abluminal side of LECs suggests a possible role of TCs in the regulation of lymphatic capillary functionality, which is worth investigating further.

Systemic Sclerosis Serum Significantly Impairs the Multi-Step Lymphangiogenic Process: in Vitro Evidence.

In systemic sclerosis (SSc), the possible involvement of lymphatic microcirculation and lymphangiogenesis has traditionally been overshadowed by the greater emphasis placed on dysfunctional blood vascular system and angiogenesis. In the present in vitro study, we explore for the first time whether the SSc microenvironment may interfere with lymphangiogenesis, a complex, multi-step process in which lymphatic microvascular endothelial cells (LMVECs) sprout, migrate, and proliferate to generate new lymphatic capillaries. Normal human adult dermal LMVECs from three donors were treated with serum from SSc patients (n = 8), serum from healthy individuals (n = 8), or recombinant human vascular endothelial growth factor (VEGF)-C as a positive control for lymphangiogenesis. Cell proliferation, Boyden chamber Matrigel chemoinvasion, wound healing capacity, and lymphatic capillary morphogenesis on Geltrex were assayed. VEGF-C serum levels were measured by enzyme-linked immunosorbent assay. Gene and protein expression levels of the lymphangiogenic orchestrators VEGF receptor-3 (VEGFR-3)/Flt-4 and neuropilin-2 (NRP-2) were determined by real-time PCR and Western blotting, respectively. Conditioning with SSc serum significantly inhibited LMVEC proliferation, Matrigel invasion, and wound healing capacity with respect to healthy serum. The ability of LMVECs to form lymphatic tubes on Geltrex was also severely compromised in the presence of SSc serum. VEGF-C levels were comparable in SSc and healthy sera. Treatment with SSc serum resulted in a significant downregulation of both VEGFR-3/Flt-4 and NRP-2 mRNA and protein levels. In SSc, the pathologic environment severely hampers every lymphangiogenesis step, likely through the reduction of pro-lymphangiogenic VEGFR-3/NRP-2 co-receptor signaling. The impairment of the lymphangiogenic process opens a new scenario underlying SSc vascular pathophysiology, which is worth investigating further.

Morphological evidence for telocytes as stromal cells supporting satellite cell activation in eccentric contraction-induced skeletal muscle injury.

Although telocytes (TCs) have been proposed to play a "nursing" role in resident satellite cell (SC)-mediated skeletal muscle regeneration, currently there is no evidence of TC-SC morpho-functional interaction following tissue injury. Hence, we explored the presence of TCs and their relationship with SCs in an ex vivo model of eccentric contraction (EC)-induced muscle damage. EC-injured muscles showed structural/ultrastructural alterations and changes in electrophysiological sarcolemnic properties. TCs were identified in control and EC-injured muscles by either confocal immunofluorescence (i.e. CD34+CD31- TCs) or transmission electron microscopy (TEM). In EC-injured muscles, an extended interstitial network of CD34+ TCs/telopodes was detected around activated SCs displaying Pax7+ and MyoD+ nuclei. TEM revealed that TCs invaded the SC niche passing with their telopodes through a fragmented basal lamina and contacting the underlying activated SCs. TC-SC interaction after injury was confirmed in vitro by culturing single endomysial sheath-covered myofibers and sprouting TCs and SCs. EC-damaged muscle-derived TCs showed increased expression of the recognized pro-myogenic vascular endothelial growth factor-A, and SCs from the same samples exhibited increased MyoD expression and greater tendency to fuse into myotubes. Here, we provide the essential groundwork for further investigation of TC-SC interactions in the setting of skeletal muscle injury and regenerative medicine.

Changes in the telocyte/CD34+ stromal cell and α-SMA+ myoid cell networks in human testicular seminoma.

Telocytes (TCs), also known as CD34+ stromal/interstitial cells, have recently been identified within the connective tissue of a variety of organs including the normal human testis. Testicular TCs appear to constitute a widespread reticular network distributed either in the peritubular or in the intertubular stromal spaces where they have been suggested to play different roles, such as participation to testis morphogenesis, postnatal preservation of the normal tissue/organ three-dimensional structure, and regulation of spermatogenesis and androgen hormone secretion and release. Although increasing evidence indicates that TCs may be involved in the pathophysiology of various diseases, no study has yet reported possible changes in these cells within the stromal compartment of seminoma, one of the most frequent malignant testicular cancers in humans. Therefore, here we carried out the first investigation of the presence and tissue distribution of TCs/CD34+ stromal cells in human testicular seminoma in comparison with normal human testis using either CD34 immunohistochemistry or CD34/CD31 and CD34/α-smooth muscle actin (α-SMA) double immunofluorescence analyses. In seminoma tissue sections, we observed an overall loss of TCs (CD34+/CD31- stromal cells) accompanying a severe degeneration of the normal architecture of seminiferous tubules and stromal tissue associated with dense cellularity increase and presence of interstitial fibrosis. Noteworthy, in the seminoma tissue the disappearance of TCs was paralleled by an expansion of α-SMA+ myoid cells. Moreover, the CD34+/CD31+ blood vessel network was greatly expanded, while steroidogenic Leydig cells were undetectable in seminoma specimens. Since TCs are emerging as important regulators of tissue and organ homeostasis, collectively the present findings indicate that the possible pathophysiologic implications of the loss of TCs in human testicular seminoma should not be further overlooked.

Systemic Sclerosis Serum Steers the Differentiation of Adipose-Derived Stem Cells Toward Profibrotic Myofibroblasts: Pathophysiologic Implications.

Systemic sclerosis (SSc; scleroderma) is characterized by life-threatening progressive multiorgan fibrosis orchestrated by profibrotic myofibroblasts originating from different sources. Because recent data demonstrated that the majority of myofibroblasts in a murine scleroderma model arise from adipocytic progenitors through the adipocyte-myofibroblast transition process, we sought to determine whether the SSc microenvironment may affect the differentiation potential of adipose-derived stem cells (ADSC). Normal human ADSC from three donors were treated with serum from SSc patients (n = 6), serum from healthy individuals (n = 6), or recombinant human transforming growth factor-ß1 (TGFß1) as positive control of myofibroblastic phenotype induction. ADSC were subjected to in vitro adipogenic differentiation for up to 21 days in the presence of different stimuli followed by lipid content quantification. In selected experiments, adipocytic and mesenchymal/myofibroblast marker gene and protein expression levels were assessed by Real-Time PCR, immunoblotting and immunofluorescence after administration of different stimuli for 72 and 96 h, respectively. Cell contractile phenotype was assayed by collagen gel contraction assay. Likewise stimulation with TGFß1, SSc serum was able to significantly inhibit the adipocyte differentiation of ADSC as testified by a strong decrease in red-colored lipid droplets after 21 days of adipogenic induction. Treatment of ADSC either with SSc serum or TGFß1 resulted in the acquisition of a myofibroblast-like phenotype characterized by a reduced expression of the adipocytic markers perilipin and adiponectin, a significant upregulation of the mesenchymal/myofibroblast markers α-SMA+ stress fibers, S100A4 and type I collagen, and an ability to effectively contract collagen gels. In SSc, the pathologic environment may favor the differentiation of ADSC into profibrotic and contractile myofibroblast-like cells. These findings strengthen the notion that the generation of myofibroblasts from ADSC may be relevant in SSc pathophysiology potentially representing a new target for the prevention/treatment of multiorgan fibrosis.

TNF-α/TNF-R System May Represent a Crucial Mediator of Proliferative Synovitis in Hemophilia A.

Hemophilic arthropathy (HA) typically begins with proliferative synovitis that shares some similarities with inflammatory arthritides, in which the proinflammatory cytokine tumor necrosis factor (TNF)-α has a crucial pathogenetic role. Inappropriate release of TNF-α was shown to contribute to arthropathy development following intra-articular bleeding in hemophilic mice. Here, we were interested in determining whether systemic levels of TNF-α and synovial tissue expression of the TNF-α/TNF receptor (TNF-R) system could be increased and related to joint damage in hemophilia A patients with severe HA. Serum levels of TNF-α measured by quantitative enzyme-linked immunosorbent assay (ELISA) were significantly increased in HA patients (n = 67) compared to healthy controls (n = 20). In HA patients, elevated TNF-α levels were significantly associated with the number of hemarthroses, the grade of synovial hypertrophy, and both the clinical World Federation of Hemophilia score and ultrasound score. The expression of TNF-α, TNF-R1, and TNF-R2 was strongly increased in HA synovium (n = 10) compared to the non-inflamed osteoarthritis control synovium (n = 8), as assessed by both immunohistochemistry and Western blotting. Increased protein levels of TNF-α, TNF-R1, and TNF-R2 were retained in vitro by HA fibroblast-like synoviocytes (n = 6) with respect to osteoarthritis control fibroblast-like synoviocytes (n = 6). Stimulation with TNF-α resulted in a significant increase in HA fibroblast-like synoviocyte proliferation quantified by the water-soluble tetrazolium (WST)-1 assay, while it had no relevant effect on osteoarthritis fibroblast-like synoviocytes. Quantification of active/cleaved caspase-3 by ELISA demonstrated that TNF-α did not induce apoptosis either in HA or in osteoarthritis fibroblast-like synoviocytes. The TNF-α/TNF-R system may represent a crucial mediator of proliferative synovitis and, therefore, a new attractive target for the prevention and treatment of joint damage in HA patients. Our findings provide the groundwork for further clinical investigation of anti-TNF-α therapeutic feasibility in hemophiliacs.

Telocytes constitute a widespread interstitial meshwork in the lamina propria and underlying striated muscle of human tongue.

Telocytes have recently emerged as unique interstitial cells defined by their extremely long, thin and moniliform prolongations termed telopodes. Despite growing evidence that these cells consistently reside in the stromal compartment of various organs from human beings, studies dealing with telocytes in structures of the oral cavity are scarce. Hence, the present morphologic study was undertaken to explore for the first time the presence and specific localization of telocytes within tissues of the normal human tongue, a complex muscular organ whose main functions include taste, speech, and food manipulation in the oral cavity. Telocytes were initially identified by CD34 immunostaining and confirmed by CD34/PDGFRα double immunofluorescence and transmission electron microscopy. CD34+/PDGFRα+ telocytes were organized in interstitial meshworks either in the tongue lamina propria or in the underlying striated muscle. Lingual telocytes were immunonegative for CD31, c-kit and α-SMA. Telopodes were finely distributed throughout the stromal space and concentrated beneath the lingual epithelium and around CD31+ vessels, skeletal muscle bundles/fibers, and intramuscular nerves and ganglia. They also enveloped salivary gland units outside the α-SMA+ myoepithelial cells and delimited lymphoid aggregates. These findings establish telocytes as a previously overlooked interstitial cell population worth investigating further in the setting of human tongue pathophysiology.

Angiogenic T cells in primary Sjögren's syndrome: a double-edged sword?

OBJECTIVES: The mechanisms underlying increased cardiovascular risk in primary Sjögren's syndrome (pSS) remain unclear. Since the recently discovered angiogenic T cells (Tang) may participate in endothelial repair by cooperating with endothelial progenitor cells (EPC), we aimed to quantify and characterise Tang in the peripheral blood and minor salivary glands (MSG) of pSS patients. METHODS: Tang (CD3+CD31+CXCR4+) and EPC (CD34+CD133+VEGFR-2+) were quantified by flow cytometry in peripheral blood samples from 36 pSS patients and 20 healthy donors. Tang subsets were assessed on the basis of CD4, CD8 and CD28 expression. Labial MSG sections from 10 pSS patients and 12 non-pSS sicca syndrome controls were subjected to immunofluorescence staining to investigate the presence of Tang and the expression of the CXCR4-ligand stromal cell-derived factor-1 (SDF-1)/CXCL12. RESULTS: Circulating Tang cells were expanded and directly correlated to EPC in pSS. Both Tang and EPC directly correlated with disease activity as calculated with the EULAR Sjögren's syndrome disease activity index (ESSDAI). In pSS, the majority of Tang cells were CD4-CD8- double negative (DN) and lacked CD28 revealing a senescent phenotype. A subset of CD4+, CD8+ and DN Tang cells produced interleukin-17. Immunohistology revealed the exclusive presence of periductal and perivascular infiltrating Tang cells along with increased SDF-1/CXCL12 expression in pSS MSG compared to non-pSS sicca syndrome controls. CONCLUSIONS: In pSS, Tang cells are expanded in peripheral blood and infiltrate MSG. Tang may be novel actors in pSS-related endothelial dysfunction and glandular neo-angiogenesis and inflammation.

Reappraising the microscopic anatomy of human testis: identification of telocyte networks in the peritubular and intertubular stromal space.

Telocytes are a recently described stromal cell type widely distributed in various organs including the female and male reproductive systems. This study was aimed to investigate for the first time the existence, distribution and characteristics of telocytes in normal human testis by an integrated morphological approach (immunohistochemistry, immunofluorescence and transmission electron microscopy). We found that telocytes displaying typical long and moniliform prolongations and coexpressing CD34 and PDGFRα formed networks in the outer layer of peritubular tissue and around Leydig cells and vessels in the intertubular stroma. Testicular telocytes were immunophenotypically negative for CD31, c-kit/CD117 as well as α-SMA, thus making them clearly distinguishable from myoid cells/myofibroblasts located in the inner layer of peritubular tissue. Transmission electron microscopy confirmed the presence of cells ultrastructurally identifiable as telocytes (i.e. cells with telopodes alternating podomers and podoms) in the aforementioned locations. Intercellular contacts between neighboring telocytes and telopodes were observed throughout the testicular stromal compartment. Telopodes intimately surrounded and often established close contacts with peritubular myoid cells/myofibroblasts, Leydig cells and vessels. Extracellular vesicles were also frequently detected near telopodes. In summary, we demonstrated that telocytes are a previously neglected stromal component of human testis with potential implications in tissue homeostasis deserving further investigation.

Slit2/Robo4 axis may contribute to endothelial cell dysfunction and angiogenesis disturbance in systemic sclerosis.

OBJECTIVE: In systemic sclerosis (SSc), early microvascular injury is followed by impaired angiogenesis and peripheral capillary loss. Here, we investigated the possible contribution of the neurovascular guidance molecule Slit2 and its Roundabout (Robo) receptors to SSc-related endothelial cell dysfunction. METHODS: Circulating Slit2 levels were measured in patients with SSc and healthy controls. Slit2, Robo1 and Robo4 expression was investigated in SSc and healthy skin biopsies and explanted dermal microvascular endothelial cells (MVECs). Slit2/Robo4 function in MVEC angiogenesis was studied by cell viability, wound healing and capillary-like tube formation assays. RESULTS: Circulating Slit2 was significantly increased in either SSc or patients with a very early diagnosis of SSc (VEDOSS) compared with controls. Interestingly, serum Slit2 levels were raised in patients with VEDOSS with nailfold videocapillaroscopy (NVC) abnormalities, while they were similar in VEDOSS with normal NVC and controls. In SSc, Slit2 and Robo4 expression was upregulated in clinically affected skin and explanted MVECs in respect to controls. The angiogenic performance of healthy MVECs was significantly reduced after challenge with recombinant human Slit2 or SSc sera. These inhibitory effects were significantly attenuated when SSc sera were preincubated with an anti-Slit2 blocking antibody. In vitro angiogenesis was severely compromised in SSc-MVECs and could be significantly ameliorated by Slit2 neutralisation or ROBO4 gene silencing. Slit2/Robo4 axis interfered with angiogenesis through the inhibition of Src kinase phosphorylation. CONCLUSIONS: In SSc, increased circulating levels of Slit2 and activation of the Slit2/Robo4 antiangiogenic axis may contribute to peripheral microangiopathy since the very early phase of the disease.

Telocytes in human fetal skeletal muscle interstitium during early myogenesis.

A new peculiar stromal cell type called telocyte (TC)/CD34-positive stromal cell (i.e. cell with distinctive prolongations named telopodes) has recently been described in various tissues and organs, including the adult skeletal muscle interstitium of mammals. By forming a resident stromal three-dimensional network, TCs have been suggested to participate in different physiological processes within the skeletal muscle tissue, including homeostasis maintenance, intercellular signaling, tissue regeneration/repair and angiogenesis. Since a continuous interplay between the stromal compartment and skeletal muscle fibers seems to take place from organogenesis to aging, the present study was undertaken to investigate for the first time the presence of TCs in the human skeletal muscle during early myogenesis. In particular, we describe the morphological distribution of TCs in human fetal lower limb skeletal muscle during early stages of myogenesis (9-12 weeks of gestation). TCs were studied on tissue sections subjected to immunoperoxidase-based immunohistochemistry for CD34. Double immunofluorescence was further performed to unequivocally differentiate TCs (CD34-positive/CD31-negative) from vascular endothelial cells (CD34-positive/CD31-positive). Our findings provide evidence that stromal cells with typical morphological features and immunophenotype of TCs are present in the human skeletal muscle during early myogenesis, revealing differences in either CD34 immunopositivity or TC numbers among different gestation ages. Specifically, few TCs weakly positive for CD34 were found between 9 and 9.5 weeks. From 10 to 11.5 weeks, TCs were more numerous and strongly reactive and their telopodes formed a reticular network in close relationship with blood vessels and primary and secondary myotubes undergoing separation. On the contrary, a strong reduction in the number and immunopositivity of TCs was observed in fetal muscle sections from 12 weeks of gestation, where mature myotubes were evident. The muscle stroma showed parallel changes in amount, density and organization from 9 to 12 weeks. Moreover, blood vessels appeared particularly numerous between 10 and 11.5 weeks. Taken together, our findings suggest that TCs might play a fundamental role in the early myogenetic period, possibly guiding tissue organization and compartmentalization, as well as angiogenesis and maturation of myotubes.

Telocytes in skeletal, cardiac and smooth muscle interstitium: morphological and functional aspects.

Telocytes (TCs) represent a new distinct type of cells found in the stromal compartment of many organs, including the skeletal, cardiac and smooth muscles. TCs are morphologically defined as interstitial cells with a small cellular body from which arise very long (up to hundreds of micrometers) and thin moniliform processes (named telopodes) featuring the alternation of slender segments (called podomers) and small dilated portions (called podoms) accommodating some organelles. Although these stromal cells are mainly characterized by their ultrastructural traits, in the last few years TCs have been increasingly studied for their immunophenotypes, microRNA profiles, and gene expression and proteomic signatures. By their long-distance spreading telopodes, TCs build a three-dimensional network throughout the whole stromal space and communicate with each other and neighboring cells through homocellular and heterocellular junctions, respectively. Moreover, increasing evidence suggests that TCs may exert paracrine functions being able to transfer genetic information and signaling molecules to other cells via the release of different types of extracellular vesicles. A close relationship between TCs and stem/progenitor cell niches has also been described in several organs. However, the specific functions of TCs located in the muscle interstitium remain to be unraveled. Here, we review the morphological and possible functional aspects of TCs in skeletal, cardiac and smooth muscle tissues. The potential involvement of TCs in muscle tissue pathological changes and future possibilities for targeting TCs as a novel promising therapeutic strategy to foster muscle tissue regeneration and repair are also discussed.

Morphological evidence of telocytes in human synovium.

A new cell type named telocyte (i.e. cell with distinctive prolongations called telopodes) has recently been identified in the stroma of various organs in humans. However, no study has yet reported the existence of telocytes in the synovial membrane of diarthrodial joints. This work was therefore undertaken to search for telocytes in the normal human synovium using transmission electron microscopy, immunohistochemistry and immunofluorescence. Ultrastructural analyses demonstrated the presence of numerous spindle-shaped telocytes in the whole synovial sublining layer. Synovial telocytes exhibited very long and thin moniliform telopodes and were particularly concentrated at the boundary between the lining and sublining layers and around blood vessels. Light microscopy confirmed the presence of CD34-positive telocytes in the aforementioned locations. Moreover, synovial telocytes coexpressed CD34 and platelet-derived growth factor receptor α. Double immunostaining further allowed to unequivocally differentiate synovial telocytes (CD34-positive/CD31-negative) from vascular endothelial cells (CD34-positive/CD31-positive). The in vitro examination of fibroblast-like synoviocyte primary cultures revealed the coexistence of different cell types, including CD34-positive telocytes projecting typical moniliform telopodes. In conclusion, our work provides the first evidence that telocytes do exist in the human synovium and lays the groundwork for future studies on synovial telocytes in a variety of degenerative and destructive joint diseases.

Targeting anticoagulant protein S to improve hemostasis in hemophilia.

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.