Expression of the intermediate filament nestin in gastrointestinal stromal tumors and interstitial cells of Cajal

It has recently been proposed that gastrointestinal stromal tumors (GISTs) originate from stem cells that differentiate toward a phenotype of interstitial cells of Cajal (ICCs). Nestin is a newly identified intermediate filament protein, and is predominantly expressed in immature cells, such as neuroectodermal stem cells and skeletal muscle progenitor cells, and tumors originating from these cells. In this study, we examined, using immunohistochemistry, the nestin expression in GISTs and ICCs to clarify the origin of GISTs. Strong immunoreactivity for nestin was observed in all 18 GISTs, and its expression was confirmed by Western blot and Northern blot analyses. In contrast, three leiomyomas and a schwannoma that developed in the gastrointestinal tract showed no apparent immunoreactivity for nestin. Among 17 mesenchymal tumors (seven leiomyosarcomas, five malignant peripheral nerve sheath tumors, and five fibrosarcomas) that occurred in sites other than the gastrointestinal tract, only two malignant peripheral nerve sheath tumors were moderately immunoreactive for nestin. Furthermore, with fluorescence double immunostaining of the normal small intestine, nestin expression was demonstrated in ICCs. These results show that nestin may be a useful marker for diagnosis of GISTs, and support the current hypothesis that GISTs are tumors of stem cells that differentiate toward an ICC phenotype.     

Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle

The intermediate filament proteins nestin, vimentin, and desmin show a specific temporal expression pattern during the development of myofibers from myogenic precursor cells. Nestin and vimentin are actively expressed during early developmental stages to be later down-regulated, vimentin completely and nestin to minimal levels, whereas desmin expression begins later and is maintained in mature myofibers, in which desmin participates in maintaining structural integrity. In this study we have analyzed the expression levels and distribution pattern of nestin in intact and denervated muscle in rat and in human. Nestin immunoreactivity was specifically and focally localized in the sarcoplasm underneath neuromuscular junctions (NMJs) and in the vicinity of the myotendinous junctions (MTJs), ie, in regions associated with acetylcholine receptors (AChRs). This association prompted us to analyze nestin in neurogenically and myogenically denervated muscle. Immunoblot analysis disclosed a marked overall increase of accumulated nestin protein. Similar to the extrajunctional redistribution of AChRs in denervated myofibers, nestin immunoreactivity extended widely beyond the NMJ region. Re-innervation caused complete reversion of these changes. Our study demonstrates that the expression levels and distribution pattern of nestin are regulated by innervation, ie, signal transduction into myofibers.     

Nestin expression by newly formed human blood vessels

Nestin is a type VI intermediate filament protein originally described in neural stem cells. Here we report that immature endothelial cells generated in the course of angiogenesis express nestin. Endothelial cells of embryonic capillaries destined to vascularize growing organs also express this intermediate filament protein. Whereas nestin was sporadically expressed in mature adult human endothelial cells sporadically express nestin, this protein was consistently expressed in adult angiogenic vasculature. Nestin expression was also detected in capillaries of the corpus luteum, which replenishes itself by angiogenesis. Nestin-immunoreactive vessels were also observed in the infarcted hearts where transient ischemia triggered regeneration accompanied with neovascularization of the myocardium. Nestinpositive endothelial cells lined vessels nourishing solid growing tumors, including melanoblastomas and glioblastomas. Our data provide definitive evidence that endothelial precursors express the neural stem cell marker nestin and that this protein participates in formation of the cytoskeleton of newly formed endothelial cells. Because nestin expression was recognized under all conditions of vascular development, nestin represents a novel and reliable marker of neovascularization.     

Re-expression of nestin in the myocardium of postinfarcted patients

Intact cardiac muscle cells in the adult heart do not express intermediate filament nestin. In this study, we report on widespread expression of intermediate filament nestin in human myocardium of patients who died from the myocardial infarction. Nestin was detected in cardiomyocytes, endothelial cells, and few interstitial cells. Elevated levels of nestin were observed in cardiac muscle cells in all specimens, although the intensity of immunoreactivity and distribution of the signal differed. The strongest immunoreactivity was observed from 4 days after myocardial infarction in the infarction border zone where nestin was distributed homogeneously in the entire sarcoplasm of cardiac muscle cells. Within the following week, nestin in immunoreactive cardiomyocytes was redistributed and restricted to small subsarcolemmal foci and to intercalated discs. Angiogenic capillaries that grew between vital nestin-positive cardiomyocytes and entered the necrotic area expressed also high levels of nestin. Nestin-positive endothelial cells were often observed in mutual interactions with nestin-positive cardiac muscle cells. These findings document a crucial role of nestin in remodeling cytoskeleton of cells in the human postinfarcted myocardium.     

Transient expression of keratin during neuronal development in the adult rabbit spinal ganglion

A few neurons of the adult rabbit spinal ganglion express keratin. To examine the characters of these keratin-positive neurons, six kinds of intermediate filament proteins, namely keratin 8, keratin 14, nestin, vimentin, neurofilament 68 (NF-L) and glial fibrillary acidic protein (GFAP), were investigated immunohistochemically in developing and adult rabbit spinal ganglia. At 15 days of gestation, the spinal ganglion increased rapidly in volume and mainly consisted of three kinds of cells: small cells expressing vimentin, spindle-shaped cells co-expressing vimentin and nestin, and ovoid cells with an eccentric nucleus expressing nestin. Since some ovoid cells co-expressed nestin with either NF-L or GFAP, the ovoid cell may be considered to be an embryonic neural stem cell of the ganglion. In addition, a few keratin-positive polymorphic cells could be observed among these three kinds of cells. These polymorphic cells expressed five kinds of intermediate filament proteins, namely keratin 8, keratin 14, nestin, NF-L and GFAP. These cells were also detected in newborn and adult ganglia. A few neurons in the adult ganglion also expressed these five kinds of proteins as a Golgi-associated network. However, neurons expressing these proteins could not be detected in embryonic and newborn ganglia. Therefore, it may be considered that the keratin-positive polymorphic cell is a postnatal neural stem cell of the ganglion and that neurons transiently express keratin when polymorphic cells differentiate into neurons.     

Patterns of nestin and other intermediate filament expression distinguish between gastrointestinal stromal tumors,leiomyomas and schwannomas

The KIT-positive specific gastrointestinal stromal tumors (GISTs), leiomyomas, and schwannomas are the three most common types of primary mesenchymal tumors of the gastrointestinal (GI) tract. The intermediate filaments are abundant cytoskeletal proteins commonly used as cell differentiation markers in diagnostic immunohistochemistry. Their patterns have not been fully characterized in GI mesenchymal tumors, and could offer differential diagnostically useful parameters. Very recently, nestin, a class VI intermediate filament expressed in neuroectodermal stem cells and skeletal muscle progenitor cells, has been shown in GISTs and suggested as a marker for these tumors. In this study we immunohistochemically examined the expression of nestin and other intermediate filament proteins, including desmin, keratins (Ks), glial fibrillary acidic protein (GFAP), neurofilament, and vimentin in GISTs of different sites, esophageal leiomyomas and GI schwannomas. Nestin was nearly consistently present in GISTs of different locations whether spindle cell or epithelioid, and benign or malignant. It was also detected in 23 of 24 (96%) GI schwannomas, whereas leiomyomas were uniformly negative. Vimentin was present in both GISTs and schwannomas, whereas it was typically absent in leiomyomas (25% positive, usually focally). Desmin was present in all leiomyomas, whereas only 3% of GISTs (4 of 140) were positive, and all schwannomas were negative. K18 was detected in a minority of GISTs, leiomyomas, and schwannomas. Malignant GISTs were more commonly keratin positive than the benign ones; there was 18% K18 positivity in malignant gastric and small intestinal GISTs, but 9% K18 positivity in benign gastric and small intestinal GISTs. Moreover, K8, albeit to a lesser degree, was detected in a minority of GISTs, but K7, K14, K19 and K20 were not detected. GFAP was present in the majority of schwannomas, whereas all GISTs were negative; some leiomyomas had weak cytoplasmic positivity. These results document distinctive patterns of intermediate filament proteins in GI mesenchymal tumors. Nestin is confirmed to be consistently expressed in GISTs but it is also present in most GI schwannomas; GFAP is helpful when separating GISTs and schwannomas, since only the latter are positive. The potential presence of K8 and K18 in GISTs should not lead to the misdiagnosis of carcinoma on biopsy.     

Nestin expression as a new marker in malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumor (MPNST) can be difficult to diagnose because it lacks specific immunohistochemical markers. S-100, which is a useful marker of MPNST, has limited diagnostic utility. Recent studies suggest that nestin, which is an intermediate filament protein, is expressed in neuroectodermal stem cells. The diagnostic utility of immunostains for nestin and three other neural markers (S-100, CD56 and protein gene product 9.5 (PGP 9.5)) were evaluated in 35 cases of MPNST and in other spindle cell tumors. All MPNST cases were strongly positive for nestin and had cytoplasmic staining. Stains for S-100, CD56, and PGP 9.5 were positive in fewer cases (17/35, 11/35, and 29/35 cases, respectively), and had less extensive staining. Nestin was negative in 10/10 leiomyomas, and weak nestin expression was seen in 10/10 schwannomas, 3/10 neurofibromas, 2/8 synovial sarcomas, 2/10 liposarcomas, 4/7 carcinosarcomas and 3/7 malignant fibrous histiocytomas. In contrast, strong nestin positivity was seen in 10/10 rhabdomyosarcomas, 15/19 leiomyosarcomas, and 9/9 desmoplastic melanomas. Nestin is more sensitive for MPNST than other neural markers and immunostains for nestin in combination with other markers could be useful in the diagnosis of MPNST.     

Nestin expression in odontoblasts and odontogenic ectomesenchymal tissue of odontogenic tumours

BACKGROUND: Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine. AIMS: The aim of this study was to investigate nestin expression in various odontogenic tumours and evaluate its usefulness for histopathological diagnosis. METHODS: We studied formalin fixed, paraffin embedded specimens from 129 cases of odontogenic tumours and 9 of mandibular intraosseous myxoma. After characterisation of odontogenic ectomesenchymal tissues in these tumours using antibodies to vimentin, desmin, neurofilament, and glial fibrillary acidic protein, we immunohistochemically examined nestin expression. RESULTS: No differentiation towards muscle and nervous tissues was found in the odontogenic ectomesenchymal tissues. Although almost all the ameloblastomas and malignant ameloblastomas were negative for nestin, odontogenic ectomesenchyme in the odontogenic mixed tumours demonstrated nestin immunolocalisation, particularly in the region adjacent to the odontogenic epithelium. Odontoblasts and their processes, pulp cells near the positive odontoblasts, and flat cells adhering to the dentine showed immunoreaction with nestin in the odontomas and odontoma-like component in the ameloblastic fibro-odontomas. Neoplastic cells in almost half cases of jaw myxoma and one case of odontogenic fibroma expressed nestin. CONCLUSIONS: The distribution of nestin in the odontogenic mixed tumours suggests that nestin expression in the odontogenic ectomesenchyme is upregulated by stimulation from odontogenic epithelium. In addition, nestin may also be involved in the differentiation from pulp cells to odontoblasts in odontogenic tumours. Therefore, nestin is a useful marker for the odontogenic ectomesenchyme and odontoblasts in odontogenic tumours. Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine.     

Effects of Growth Factors and Basement Membrane Proteins on the Phenotype of U-373 MG Glioblastoma Cells as Determined by the Expression of Intermediate Filament Proteins

Various growth factors and basement membrane proteins have been implicated in the pathobiology of astrocytomas. The goal of this study was to determine the relative contribution of these two factors in modulating the phenotype of U-373 MG glioblastoma cells as determined by the expression of the intermediate filament proteins glial fibrillary acidic protein, vimentin, and nestin. For these determinations, cells plated in serum-free medium were treated either with growth factors binding to tyrosine kinase receptors including transforming growth factor-α, epidermal growth factor, platelet-derived growth factor-AA, basic fibroblast growth factor, and insulin-like growth factor-1 or with basement membrane proteins including collagen IV, laminin, and fibronectin. The changes in the expression levels of intermediate filament proteins in response to these treatments were analyzed by quantitation of immunoblots. The results demonstrate that collagen IV and growth factors binding to tyrosine kinase receptors decrease the glial fibrillary acidic protein content of U-373 MG cells. Growth factors binding to tyrosine kinase receptors also decrease the vimentin content of these cells but do not affect their nestin content. On the other hand, basement membrane proteins decrease the nestin content of U-373 MG cells but do not affect their vimentin content. The significance of these results with respect to the role played by different factors in modulating the phenotype of neoplastic astrocytes during tumor progression is discussed.     

Immunohistochemical examination for the distribution of podoplanin-expressing cells in developing mouse molar tooth germs

We recently reported the expression of podoplanin in the apical bud of adult mouse incisal tooth. This study was aimed to investigate the distribution of podoplanin-expressing cells in mouse tooth germs at several developing stages. At the bud stage podoplanin was expressed in oral mucous epithelia and in a tooth bud. At the cap stage podoplanin was expressed on inner and outer enamel epithelia but not in mesenchymal cells expressing the neural crest stem cell marker nestin. At the early bell stage nestin and podoplanin were expressed in cervical loop and odontoblasts. At the root formation stage both nestin and podoplanin were weakly expressed in odontoblasts generating radicular dentin. Podoplanin expression was also found in the Hertwig epithelial sheath. These results suggest that epithelial cells of developing tooth germ acquire the ability to express nestin, and that tooth germ epithelial cells maintain the ability to express podoplanin in oral mucous epithelia. The expression of podoplanin in odontoblasts was induced as tooth germ development advanced, but was suppressed with the completion of the primary dentin, suggesting that podoplanin may be involved in the cell growth of odontoblasts. Nestin may function as an intermediate filament that binds podoplanin in odontoblasts.     

Nestin is a wide‐spectrum abluminal cell marker of salivary gland tumors

Nestin is an intermediate filament that was first identified in neural progenitor cells. It is expressed in various cell types in the nervous system as well as in other systems. In the present study, we investigated nestin expression in non‐neoplastic salivary gland tissue and in salivary gland tumors. In non‐neoplastic salivary glands, nestin expression was observed in only a few abluminal cells. In contrast, diffuse nestin staining was observed in the abluminal cells of pleomorphic adenoma (11 of 11 cases), basal cell adenoma (7 of 7 cases), and epithelial‐myoepithelial carcinoma (2 of 2 cases). The stromal cells in basal cell adenoma also expressed nestin. In adenoid cystic carcinoma (6 of 7 cases) and polymorphous low‐grade adenocarcinoma (3 of 3 cases), nestin positive cells were observed focally. Nestin was not detected in Warthin tumor (6 cases), classical acinic cell carcinoma (2 cases), mucoepidermoid carcinoma (5 cases), or salivary duct carcinoma (4 cases). Because the nestin expression pattern in each histological salivary gland tumor type is unique, nestin could be a very useful abluminal cell marker for the diagnosis of salivary gland tumors.     

Nestin-expressing cells in the human hippocampus

Nestin is a protein that belongs to a family of intermediate filament proteins which are typical for undifferentiated neural stem and progenitor cells. In this work nestin expression was studied in the hippocampus obtained from patients with epilepsy. Immunohistochemical investigation demonstrated five types of nestin-positive cells, differing in morphological and immunological phenotype. These included cell with a radial glia phenotype, bipolar cells, small dendritic cells, subependymal and astrocyte-like cells. Two types of these cells: radial glia of dentate gyrus and bipolar NG2+ cells can be considered as neural progenitor cells possessing different degrees of commitment.     

Nestin expression in repopulating mesangial cells promotes their proliferation

We investigated whether the intermediate filament protein and neural stem cell marker nestin characterizes the glomerular progenitor/reserve cell population immigrating the glomerulus after mesangial cell (MC) injury in the rat (anti-Thy1 nephritis). Nestin expression was investigated by immunohistochemistry and real-time PCR during anti-Thy1 nephritis. Migration and proliferation assays were used to characterize the function of nestin in isolated MCs after nestin knockdown by siRNA. After MC injury during anti-Thy1 nephritis, glomerular nestin was transiently increased during the repopulation phase. At the peak of mesangial proliferation and expansion (day 5) most OX-7-positive MCs expressed nestin largely colocalizing with the activation marker alpha-smooth muscle actin and the proliferation marker PCNA. In contrast to a healthy, non-injured mesangium in vivo, MCs in culture are considered to be in an 'activated, injured state' and express nestin in a generalized distribution with condensed localization around the nucleus as well as intensive staining of cell protrusions such as filopodia. During cell cycle, the percentage of MCs with high nestin levels was increased during S- aupnd G2-phase. Blocking of nestin using specific siRNA resulted in inhibition of cell proliferation but not cell migration. In conclusion, nestin is constitutively expressed in podocytes, but is a marker for repopulating MCs after experimental MC injury in vivo. Nestin promotes MC proliferation in vitro, suggesting a supporting role for nestin during repair reaction.     

Expression of nestin in rat and human glomerular podocytes

Nestin is a neuroepithelial precursor cell marker expressed in a variety of human cell types during development. However, no information exists on the expression of nestin in mature glomeruli as well as during the glomerular development. Here, we examined nestin expression in rat and human glomerular tissues in quiescent states using RT-PCR and immunohistochemical methods. Nestin mRNA was detected in the rat glomeruli in parallel with its expression in developing rat brains. In the normal mature rat glomeruli, WT-1 positive cells expressed nestin. Co-expression of nestin and vimentin was observed in mature rat podocytes. Immunoelectron microscopy revealed nestin localization in the cell bodies and primary processes of podocytes. A similar expression pattern was observed for vimentin. In matured glomeruli, nestin was not expressed by mesangial and endothelial cells. In the newborn rat, early developing glomeruli (metanephric cap, metanephric vesicle, comma-shaped vesicle and S-shaped body phases) expressed nestin. In the capillary loop stage, Bowman's capsules also expressed nestin. Immunoelectron microscopy demonstrated that developing podocytes and endothelial cells in S-shaped phase glomeruli expressed nestin. Additionally, in immature glomeruli, the mesangial cells in capillary stage of glomerulus also expressed nexin. As in the rat, WT-1 positive cells in human glomeruli also expressed nestin and immunoelectron microscopy confirmed nestin expression in human glomerular podocytes. These results reveal that in normal condition nestin is expressed in several glomerular cell types at early stage of development and becomes confined to podocytes in mature glomeruli, thus implicating nestin in podocyte functions.     

Heterogeneous expression of nestin in myofibroblasts of various human tissues

In this study we explored the distribution of nestin‐positive cells in extraneural human tissues with special reference to stromal myofibroblasts. Tissue microarrays were constructed from various tissues with normal histology and tissues with fibrosing disorders. Sections were immunostained for nestin, alpha‐smooth muscle actin (alpha‐SMA), desmin, vimentin, CD34, and other stromal markers. Nestin was expressed in the myoepithelium of the breast, podocytes of the renal glomerulus, and endothelial cells of most organs. Nestin was also expressed in the stroma of several organs, including the intestine, uterine cervix, and endometrium. Nestin‐positive fibroblast‐like cells appeared in the stroma of the kidney, pancreas, lung, and skin in fibrosing conditions. With the notable exception of endometrial stromal cells, most of these nestin‐positive stromal cells were alpha‐SMA‐positive. Interestingly, we observed a concomitant appearance of nestin‐ and CD34‐positive myofibroblasts under fibrosing conditions. Further investigation showed that nestin was expressed by stromal fibroblasts in cervical squamous cell carcinoma, but not in lung adenocarcinoma, pointing to heterogeneity of cancer stroma. In conclusion, nestin was expressed in variable proportions of stromal myofibroblasts in human tissues. The differential expression of nestin may indicate phenotypic and functional heterogeneity. Nestin‐positive myofibroblast may represent a relatively immature subpopulation of cells with multipotentiality.     

Astrogliogenesis in Heterotopic Allotransplants of Rat Embryonic Neocortex

We studied gliogenesis in transplants of rat embryonic neocortex (E14-15) in 3, 7, 15, and 30 days and 12-13 months after transplantation into the sciatic nerve of adult animals. Immunogistochemical reactions to intermediate filament proteins nestin, vimentin, glial fibrillary acidic protein were used. In transplants, vimentin- and nestin-positive precursor cells differentiate into astrocytes earlier that in the developing rat neocortex (in situ). One year after transplantation, some astrocytes start to express nestin and vimentin, which attests to the development of reactive gliosis in the transplants.     

Nestin as a marker for proliferative endothelium in gliomas

Nestin is one of the intermediate filaments abundantly produced in the developing central nervous system and somites in the embryonic stage. Nestin is also reportedly detected in gliomas/glioblastomas. We retested nestin expression in brain tumors having a range of malignancy grades using immunostaining. The intensity of nestin immunostaining roughly paralleled the malignancy grade of the gliomas. However, many tumors were negative for nestin immunostaining, while nestin immunostaining was invariably detected in tumor endothelium regardless of glioma malignancy grades or brain tumor types. We suspected that angiogenic epithelial cells may express nestin, and we found that nestin was highly positive in bovine aortic endothelial cells in static culture. However, nestin expression decreased when the endothelial cells underwent laminar shear stress flow, under which endothelial cells exhibit differentiated features and a decreased rate of growth. Because nestin is highly expressed in growing endothelial cells, we examined its expression in hemangioblastomas because hemangioblasts are thought to be a precursor for angiogenic epithelial cells. As expected, nestin immunostained strongly in all four samples of hemangioblastomas. We suggest that nestin is not only a marker for neuroepithelial stem cells and glioma cells but also for tumor endothelial cells during rapid growth.