RAPID COMMUNICATION |
Correspondence to: R. Scott Heller, Hagedorn Research Institute, DK 2820 Gentofte, Denmark. E-mail: shll@hagedorn.dk
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Summary |
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In this study we examined the expression of nestin in islets, the exocrine part, and the big ducts of the adult human pancreas by immunofluorescent double staining. Two different anti-nestin antisera in combination with various pancreatic and endothelial markers were employed. Nestin-immunoreactive cells were found in islets and in the exocrine portion. All nestin-positive cells co-expressed the vascular endothelial markers PECAM-1 (CD31), endoglin (CD105), and CD34 as well as vimentin. Endocrine, acinar, and duct cells did not stain for nestin. We also demonstrated that in the area of big pancreatic ducts, nestin-positive cells represent small capillaries scattered in the connective tissue surrounding the duct epithelium and do not reside between the duct cells. We detected nestin-expressing endothelial cells located adjacent to the duct epithelium where endocrine differentiation occurs. We have shown that nestin is expressed by vascular endothelial cells in human pancreas, and therefore it is unlikely that nestin specifically marks a subpopulation of cells representing endocrine progenitors in the adult pancreas.
(J Histochem Cytochem 51:697706, 2003)
Key Words: CD34, endoglin, nestin, PECAM-1, pancreas, vascular endothelium, vimentin
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Introduction |
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NESTIN, a class VI intermediate filament, was originally described as a neuronal stem/progenitor cell marker during CNS development (
In several in vitro differentiation assays using embryonic and adult stem cells, nestin has been used as a selection marker for neuronal (
The aim of this study was to thoroughly investigate nestin immunoreactivity in the adult human pancreas by using double fluorescence staining to further clarify the type of cells expressing nestin.
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Materials and Methods |
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Human Materials
Human adult pancreases from heart-beating cadaveric non-diabetic donors were procured at European hospitals associated with the Eurotransplant Foundation (Leiden; The Netherlands) and with the beta cell bank of the JDRF Center, a European multicenter program involving beta-cell transplantation in type 1 diabetic patients. The data reported in this study are based on tissue samples taken from the body of two different pancreata. The donors were female with ages 45 years and 30 years.
Fixation and Tissue Preparation
Materials used for histological analysis were fixed either in 4% paraformaldehyde (PFA) in Ca2+- and Mg2+-free PBS (CMFPBS) for 4 hours at room temperature (RT) or overnight in formalin at 4C before paraffin embedding or cryoprotection, respectively. Paraffin sections of 4 µm were cut with a microtome and stored at RT. Frozen tissue was generated by transferring the fixed specimen in 30% sucrose (Merck; Darmstadt, Germany) in CMFPBS at 4C for 1 day (cryoprotection) followed by embedding in Tissue tek (Sakura Finetek Europe; Zoeterwoude, The Netherlands) and storage at -80C. Cryosections of 68-µm thickness were cut on a cryostat, mounted on poly-lysine-coated slides (Menzel-Gläser; Braunscheig, Germany), and stored at -80C. All pictures shown in this study were done on formalin-fixed frozen sections.
Immunohistochemistry
Cryosections were dried at RT for 20 min, washed in CMF and blocked with 10% donkey or goat non-immune serum (Jackson ImmunoResearch Laboratories; West Chester, PA) for 30 min. For paraffin sections, the slides were dewaxed in xylene and rehydrated through a descending ethanol series. Antigen retrieval was accomplished by microwave treatment (two times for 5 min at 600 W in 0.01 mol/liter citrate buffer, pH 6.0) followed by three washes in CMFPBS. The dilution of the blocking sera and the antibodies was carried out by using CMFPBS supplemented with 0.3% bovine serum albumin (Sigma, St Louis, MO), 0.3% Triton X-100 (Sigma), and 0.05% saponin (Sigma). After blocking of nonspecific binding, the primary antibodies were applied at the proper concentration (Table 1) for 2 hr at RT followed by three washes in CMFPBS. Sections were then incubated with the secondary antibodies (Table 2) together with 100 ng/ml 4-diamidino-2-phenylindole (DAPI; Merck) for 30 min at RT and washed three times in CMFPBS. Slides were coverslipped with mounting medium (KPL) for fluorescence microscopy. Images were collected by using an Olympus BX51 microscope (Olympus Optical; Tokyo, Japan) equipped with a Hamamatsu chilled color 3CCD camera (Hamamatsu Photonics; Solna, Sweden), and images were processed using the IMAGE-Pro 4.5 PC software package (Media Cybernetics; Silver Spring, MD). No specific signal was detected when the primary antibodies were omitted (data not shown).
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The two anti-nestin antisera (
By testing the antisera on differently fixed tissues we could observe that especially formalin-fixed, frozen and, to a certain extent, paraffin-fixed tissue showed the most convincing results concerning staining intensity as well as the specific filamentous staining pattern of nestin. Using PFA-fixed frozen and paraffin pancreas from the same specimen the nestin immunoreactivity was moderate to very weak and revealed a more unspecific pattern throughout the whole pancreas.
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Results |
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Nestin Expression in Pancreatic Islets of Langerhans
We first examined for nestin localization in pancreatic islets. Nestin immunoreactivity could be found in a subpopulation of cells in and around the islets. The cells expressing nestin were either single or more often organized in small longitudinal groups of cells (Fig 1A, Fig 1E, and Fig 1I). The numbers of nestin cells and their localization within and around the islet varied. Some islet sections did not show any nestin immunoreactivity. No co-expression of nestin and chromogranin A, a marker of endocrine cells, could be detected (Fig 1H). Further immunofluorescent double staining revealed that, in the pancreatic islets, nestin-positive cells co-expressed the vascular endothelial markers PECAM-1 (platelet/endothelial cell adhesion molecule-1), endoglin, and CD34 (Fig 1A1C, Fig 1E1G, and Fig 1I1K). Notably, the staining pattern of nestin and PECAM-1 was very similar. We found no evidence to suggest that nestin-positive/PECAM-1-negative cells were present. Nestin also co-localized with endoglin and CD34, although not all endoglin and CD34 immunoreactive cells stained positive for nestin (Fig 1E1G and Fig 1I1K). We observed that in islets nestin was co-localized with vimentin, which mainly marks cells of mesenchymal origin, but we also found several vimentin-positive/nestin-negative cells next to the double-labeled cells (data not shown). Fig 1C and Fig 1D illustrate that the nestin/PECAM-1 double-stained cells mark a distinct subpopulation of the vimentin-expressing cells on the adjacent islet stained section. Chromogranin A immunoreactivity on adjacent sections was used to demonstrate the islet area of the nestin staining (Fig 1D, Fig 1H, and Fig 1L).
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Expression of Nestin in the Exocrine Part of the Pancreas
To further investigate nestin immunoreactivity in the pancreas, we performed a closer examination of the exocrine part. Nestin was co-localized neither with the acinar marker amylase (Fig 2A and Fig 2D) nor with cytokeratin 7 (CK7), a marker of pancreatic duct cells (Fig 2L2N). We could find nestin expression frequently in capillary-like structures as well as in some single cells scattered in the exocrine region and often located next to lobules and acini (Fig 2A, Fig 2E, Fig 2I, and Fig 2L). All nestin-positive cells co-expressed PECAM-1 (Fig 2A2C), CD34 (Fig 2I2K), vimentin (Fig 2E2G), and endoglin (data not shown). Occasionally, we could detect endoglin (data not shown) as well as CD34-expressing cells and very rarely PECAM-1 (data not shown)-positive cells that did not stain for nestin in the exocrine part of the pancreas (Fig 2I2K). In contrast, vimentin was detected in a broader population of cells, which did not express nestin (Fig 2E2G).
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Nestin Expression in the Large Ducts
In the large interlobular pancreatic ducts, the duct epithelium is surrounded by a thick layer of connective tissue, bordering the exocrine lobules. This is illustrated by double staining for vimentin and amylase (Fig 3M). On the adjacent sections we examined nestin expression. Using anti-nestin antiserum I, we detected nestin immunoreactivity in single cells and in small cell groups that were dispersed in the connective tissue and in several cases were very close to the duct epithelium (Fig 3A, Fig 3E, and Fig 3I). Similar to our previous observations in the exocrine and endocrine part of the pancreas, the nestin-positive cells co-expressed PECAM-1 (Fig 3) as well as endoglin, CD34, and vimentin (data not shown). Using a second anti-nestin antiserum (antiserum II), we obtained comparable results as with antiserum I (Fig 3I3K). Endothelial cells of larger vessels in the periphery of the connective tissue did not show any nestin immunoreactivity (data not shown). Double immunostaining on consecutive sections revealed no co-expression of nestin and the duct marker CK7. However, in several cases nestin-positive cells were in close proximity to the duct epithelium and sometimes they were immediately adjacent to it (Fig 3D, Fig 3H, and Fig 3L). We could not detect any nestin expression in cells integrated in the duct epithelium or delaminating from it.
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Next we investigated if nestin is transiently expressed in duct regions that give rise to endocrine cells. For that reason we examined areas where developing endocrine cells occasionally bud off from the epithelium in the large ducts. Double immunofluorescence staining showed that in these local regions chromogranin A-immunoreactive (endocrine) cells were mostly seen as a second cell layer on the peripheral side of the CK7-positive duct epithelium but still attached to it (Fig 4A4C). Some cells co-expressed both markers, often in an arrangement where chromogranin A was expressed only or to a stronger extent distally, whereas CK7 immunoreactivity was found more proximal to the epithelium. Analysis of nestin and chromogranin A immunoreactivity on consecutive sections showed that nestin was not expressed in the maturing cell types but was frequently located in cells of the connective tissue neighboring the areas of budding endocrine cells (Fig 4D4F). These adjacent nestin-positive cells co-expressed PECAM-1 (data not shown).
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We also detected nestin in later stages of the duct-endocrine budding process, where it was expressed either in cells aligning endocrine aggregates still attached to the duct or was found in large, already vascularized clusters placed in the outside edge of the connective tissue (data not shown).
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Discussion |
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Using two different anti-human nestin antisera and various established endothelial markers, we show by double staining that nestin is expressed in endothelial cells of the adult human pancreas. Nestin-positive cells could be found in islets of Langerhans, the exocrine region, and scattered in the connective tissue surrounding large interlobular pancreatic ducts. In all cases, nestin immunoreactivity was co-localized with the endothelial markers PECAM-1, endoglin, and CD34, as well as vimentin. In large blood vessels nestin was expressed at low levels or was absent. Both nestin antisera gave a comparable immunoreactivity pattern. Occasional discrepancies in the staining intensity might be explained by the varying affinity of the antisera.
Apart from the larger blood vessels, we observed that the expression pattern of nestin was very similar to PECAM-1 in the different parts of the pancreas. PECAM-1 is a cell adhesion molecule which is strongly expressed by vascular endothelial cells of normal tissue and in proliferating blood vessels of a variety of tumors (
The endothelial localization of nestin was further substantiated by staining for vimentin, a class III intermediate filament protein, which is mainly expressed in cells of mesenchymal origin, such as endothelial cells, fibroblasts, and smooth muscle cells (
PECAM-1, CD34, and recently endoglin and nestin have been used as promising prognostic markers in a variety of tumor types because of their enhanced expression in activated and proliferating endothelial cells (
Recently, nestin expression has been described in the developing mouse, rat and human pancreas. In all studies the absence of nestin in the developing pancreatic epithelium was demonstrated and suggested that nestin-positive cells do not serve as pancreatic precursor cells during development (
In the adult pancreas the duct epithelium is generally believed to be one source to replenish the beta-cell mass during the turnover process of endocrine cells (
To explain this discrepancy we tried to investigate specific nestin expression in regions where endocrine cells develop from the large duct epithelium. Examination of these focal areas revealed that nestin was co-expressed neither with CK7 nor with chromogranin A. Instead, we found nestin immunoreactivity in small capillaries frequently aligning the differentiating cells from the stromal side. The co-expression of both ductal and endocrine markers in some of the differentiating cells developing from the large duct epithelium suggests that CK7 positive duct cells represent the endocrine progenitors. Similar observations have been reported previously by Bouwens and coworkers, who demonstrated further that especially in human pancreas also small intercalated duct cells of the exocrine part give rise to minor units of beta-cells (
Recently,
Thus, in agreement with the studies on the developing pancreas our results support the hypothesis that nestin is not a marker for endocrine precursors of the ducts but nestin cells might indirectly be involved in the endocrine differentiation process by releasing initiation signals to the duct epithelia since they represent adjacent capillaries.
Potential progenitor cells residing in adult pancreatic islets have been discussed as another source for new beta cells (
In our study we noted that the occurrence of nestin varied in the different islet areas and that nestin expression was more frequently seen in larger islet sections, where more endothelial cells were present compared to the small islet regions presenting a lower occurrence of capillaries. Usually nestin expression was observed in islets as small groups of endothelial cells lying scattered between the endocrine cells. In certain instances, islets were sectioned in a region where whole nestin-positive capillaries infiltrating the islets could be seen. Nestin immunoreactivity could not be detected in islet sections where vascular endothelium was absent. Our findings suggest that using nestin as a marker for isolation of endocrine progenitors would lead to a specific accumulation of endothelial cells mainly from microvessels and it is not unlikely that potential islet cell progenitors adjoining capillaries might be carried along in the isolation procedure.
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Acknowledgments |
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Supported by grants from the Danish Research Council (Center for Transgenic Animals) and from the Juvenile Diabetes Research Foundation (JDRF Center of Beta Cell Therapy in Europe).
We are grateful to Urban Lendahl and Toshiyuki Takeuchi for supplying us with the anti-nestin antisera.
Received for publication February 24, 2003; accepted February 26, 2003.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balza E, Castellani P, Zijlstra A, Neri D, Zardi L, Siri A (2001) Lack of specificity of endoglin expression for tumor blood vessels. Int J Cancer 94:579-585[Medline]
Bertelli E, Regoli M, Lucattelli M, Bastianini A, Fonzi L (2002) Nestin expression in rat adrenal gland. Histochem Cell Biol 117:371-377[Medline]
Bonner-Weir S (2000) Perspective: postnatal pancreatic beta cell growth. Endocrinology 141:1926-1929
Bouwens L, Pipeleers DG (1998) Extra-insular beta cells associated with ductules are frequent in adult human pancreas. Diabetologia 41:629-633[Medline]
Cheifetz S, Bellon T, Cales C, Vera S, Bernabeu C, Massague J, Letarte M (1992) Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells. J Biol Chem 267:19027-19030
Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper JH (1984) Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 133:157-165
Clarke SR, Shetty AK, Bradley JL, Turner DA (1994) Reactive astrocytes express the embryonic intermediate neurofilament nestin. Neuroreport 5:1885-1888[Medline]
Egerbacher M, Böck P (1997) Morphology of the pancreatic duct system in mammals. Microsc Res Tech 37:407-417[Medline]
Eliasson C, Sahlgren C, Berthold CH, Stakeberg J, Celis JE, Betsholtz C, Eriksson JE et al. (1999) Intermediate filament protein partnership in astrocytes. J Biol Chem 274:23996-24006
Engerman RL, Pfaffenbach D, Davis MD (1967) Cell turnover of capillaries. Lab Invest 17:738-743[Medline]
Fina L, Molgaard HV, Robertson D, Bradley NJ, Monaghan P, Delia D, Sutherland DR et al. (1990) Expression of the CD34 gene in vascular endothelial cells. Blood 75:2417-2426[Abstract]
Frojdman K, Pelliniemi LJ, Lendahl U, Virtanen I, Eriksson JE (1997) The intermediate filament protein nestin occurs transiently in differentiating testis of rat and mouse. Differentiation 61:243-249[Medline]
Gougos A, Letarte M (1990) Primary structure of endoglin, an RGD-containing glycoprotein of human endothelial cells. J Biol Chem 265:8361-8364
Grigelioniené G, Blennow M, Török C, Fried G, Dahlin I, Lendahl U, Lagercrantz H (1996) Cerebrospinal fluid of newborn infants contains a deglycosylated form of the intermediate filament nestin. Pediatr Res 40:809-814[Abstract]
Guz Y, Nasir I, Teitelman G (2001) Regeneration of pancreatic beta cells from intra-islet precursor cells in an experimental model of diabetes. Endocrinology 142:4956-4968
Hunziker E, Stein M (2000) Nestin-expressing cells in the pancreatic islets of Langerhans. Biochem Biophys Res Commun 271:116-119[Medline]
Kachinsky AM, Dominov JA, Miller JB (1995) Intermediate filaments in cardiac myogenesis: nestin in the developing mouse heart. J Histochem Cytochem 43:843-847
Kim HS, Kang HS, Messam CA, Min KW, Park CS (2002) Comparative evaluation of angiogenesis in gastric adenocarcinoma by nestin and CD34. Applied Immunohistochem and Mol Morphol 10:121-127
Kumar P, Wang JM, Bernabeu C (1996) CD 105 and angiogenesis. J Pathol 178:363-366[Medline]
Lammert E, Cleaver O, Melton D (2001) Induction of pancreatic differentiation by signals from blood vessels. Science 294:564-567
Lardon J, Rooman I, Bouwens L (2002) Nestin expression in pancreatic stellate cells and angiogenic endothelial cells. Histochem Cell Biol 117:535-540[Medline]
Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD (2000) Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nature Biotechnol 18:675-679[Medline]
Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585-595[Medline]
Liao F, Huynh HK, Eiroa A, Greene T, Polizzi E, Muller WA (1995) Migration of monocytes across endothelium and passage through extracellular matrix involve separate molecular domains of PECAM-1. J Exp Med 182:1337-1343[Abstract]
Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay RD (2001) Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292:1389-1394
Matsumoto K, Yoshitomi H, Rossant J, Zaret KS (2001) Liver organogenesis promoted by endothelial cells prior to vascular function. Science 294:559-563
Mokry J, Nemecek S (1998) Angiogenesis of extra- and intraembryonic blood vessels is associated with expression of nestin in endothelial cells. Folia Biol 44:155-161
Nakayama H, Enzan H, Miyazaki E, Kuroda N, Naruse K, Hiroi M (2000) Differential expression of CD34 in normal colorectal tissue, peritumoral inflammatory tissue, and tumour stroma. J Clin Pathol 53:626-629
Niki T, Pekny M, Hellemans K, Bleser PD, Berg KV, Vaeyens F, Quartier E et al. (1999) Class VI intermediate filament protein nestin is induced during activation of rat hepatic stellate cells. Hepatology 29:520-527[Medline]
O'Connell PJ, McKenzie A, Fisicaro N, Rockman SP, Pearse MJ, d'Apice AJ (1992) Endoglin: a 180-kD endothelial cell and macrophage restricted differentiation molecule. Clin Exp Immunol 90:154-159[Medline]
Osborn M, Debus E, Weber K (1984) Monoclonal antibodies specific for vimentin. Eur J Cell Biol 34:137-143[Medline]
Parums DV, Cordell JL, Micklem K, Heryet AR, Gatter KC, Mason DY (1990) JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Path 43:752-757[Abstract]
Petropavlovskaia M, Rosenberg L (2002) Identification and characterization of small cells in the adult pancreas: potential progenitor cells? Cell Tissue Res 310:51-58[Medline]
Piper K, Ball SG, Turnpenny LW, Brickwood S, Wilson DI, Hanley NA (2002) Beta-cell differentiation during human development does not rely on nestin-positive precursors: implications for stem cell-derived replacement therapy. Diabetologia 45:1045-1047[Medline]
Scholz D, Schaper J (1997) Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is localized over the entire plasma membrane of endothelial cells. Cell Tissue Res 290:623-631[Medline]
Sejersen T, Lendahl U (1993) Transient expression of the intermediate filament nestin during skeletal muscle development. J Cell Sci 106:1291-1300
Selander L, Edlund H (2002) Nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas. Mech Dev 113:189-192[Medline]
Serluca FC, Drummond IA, Fishman MC (2002) Endothelial signaling in kidney morphogenesis: a role for hemodynamic forces. Curr Biol 12:492-497[Medline]
Steinert PM, Chou YH, Prahlad V, Parry DA, Marekov LN, Wu KC, Jang SI et al. (1999) A high molecular weight intermediate filament-associated protein in BHK- 21 cells is nestin, a type VI intermediate filament protein. Limited co-assembly in vitro to form heteropolymers with type III vimentin and type IV alpha-internexin. J Biol Chem 274:9881-9890
Sugawara K, Kurihara H, Negishi M, Saito N, Nakazato Y, Sasaki T, Takeuchi T (2002) Nestin as a marker for proliferative endothelium in gliomas. Lab Invest 82:345-351[Medline]
Tanaka F, Otake Y, Yanagihara K, Kawano Y, Miyahara R, Li M, Yamada T et al. (2001) Evaluation of angiogenesis in non-small cell lung cancer: comparison between anti-CD34 antibody and anti-CD1015 antibody. Clin Cancer Res 7:3410-3415
Tanigawa N, Amaya H, Matsumura M, Shimomatsuya T (1997) Association of tumour vasculature with tumour progression and overall survival of patients with non-early gastric carcinomas. Br J Cancer 75:566-571[Medline]
Terling C, Rass A, Mitsiadis TA, Fried K, Lendahl U, Wroblewski J (1995) Expression of the intermediate filament nestin during rodent tooth development. Int J Dev Biol 39:947-956[Medline]
Tsujimura T, MakiishiShimobayashi C, Lundkvist J, Lendahl U, Nakasho K, Sugihara A, Iwasaki T et al. (2001) Expression of the intermediate filament nestin in gastrointestinal stromal tumors and interstitial cells of Cajal. Am J Pathol 158:817-823
Vaittinen S, Lukka R, Sahlgren C, Hurme T, Rantanen J, Lendahl U, Eriksson JE et al. (2001) The expression of intermediate filament protein nestin as related to vimentin and desmin in regenerating skeletal muscle. J Neuropathol Exp Neurol 60:588-597[Medline]
Vindigni C, Miracco C, Spina D, Presenti L, Gallorini M, Vatti R, de Stefano A et al. (1997) Cell proliferation, cell death and angiogenesis in early and advanced gastric cancer of intestinal type. Int J Cancer 74:637-641[Medline]
Yang J, Bian W, Gao X, Chen L, Jing N (2000) Nestin expression during mouse eye and lens development. Mech Dev 94:287-291[Medline]
Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M et al. (2001) Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 50:521-533