Levels Of Nestin Research Articles

Abstract 2457: Nestin regulates pancreatic cancer stem cell functions

Abstract Background: Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell/progenitor cell marker. Nestin expression has also been shown to be up-regulated in progenitor cells in muscle, testis, teeth and pancreas. In the pancreas, lineage-tracing experiments have indicated that exocrine cells are derived from Nestin-expressing progenitor cells. Moreover, activation of K-ras in the nestin cell lineage is sufficient for the initiation of premalignant pancreatic intraepithelial neoplasia lesions in mice. To examine cancer stem cell (CSC) functions, floating cultures in plates with a low-attachment surface have been widely employed. CSCs are reported to be able to grow in floating spheres in serum-free culture medium supplemented with epidermal growth factor (EGF) and/or fibroblast growth factor-2 (FGF-2), whereas non-CSCs, more differentiated non-CSCs cannot form spheres. In this study, we examined nestin expression in spheres and clarified whether nestin regulates sphere forming activity. Methods: Human pancreatic ductal adenocarcinoma (PDAC) cell lines, PANC-1, KLM-1, MIAPaCa-2 and PK-45H were used to perform sphere-forming assay in ultra-low attachment plates supplemented with EGF and FGF-2. Expression of CSC markers including CD133, CD44, CD24, epithelial specific antigen (ESA), and nestin in spheres were determined by real time RT-PCR and immunocytochemistry. Sphere-forming cells were dispersed with trypsin, and then cell growth and migration were assessed. Next, short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H, human PDAC cells, which express high levels of nestin, in order to assess the effects of decreased nestin expression on sphere-forming ability. Conversely, KLM-1 cells which express low level of nestin were stably transfected with a nestin expression vector and their sphere forming ability was then determined. Results: All the PDAC cell lines formed spheres, and all CSC markers examined were expressed in the spheres. The sphere cells showed low cell growth rates and increased cell migration as compared to non-sphere cells. Expression levels of nestin in the spheres of PDAC cells were higher than those of non-sphere cells. Nestin shRNA transfected PANC-1 and PK-45H cells showed a marked decrease in sphere number and diameter. By contrast, nestin-gene-transfected KLM-1 cells exhibited increased sphere formation. Conclusion: Nestin is a pancreatic CSC marker that promotes the sphere-forming ability of CSCs. Therefore, nestin may represent a novel therapeutic target for CSCs in PDAC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2457. doi:10.1158/1538-7445.AM2011-2457

Neuroepithelial stem cell marker nestin regulates the migration, invasion and growth of human gliomas

Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell marker during central nervous system development. Nestin is expressed in gliomas, and its expression levels are higher in gliomas with high WHO histopathological classification grades than in those with low grades. In the present study, we examined whether nestin regulates the biological activities of human glioma cells. Immunohistochemically, the nestin expression patterns in 10 human glioblastoma patients were examined. The expression levels of nestin in A172, a human high-grade glioma cell line, and KG-1-C, a human low-grade glioma cell line, were examined using real-time PCR, Western blot and immunofluorescence analyses. An expression vector carrying a short hairpin RNA targeting nestin was stably transfected into A172 (Sh) cells. The effects of decreased expression levels of nestin in Sh cells on cell growth, migration, invasion, adhesion to extracellular matrices and fibrillar actin expression on three-dimensional culture plates were examined. The nestin expression vector was transiently transfected into KG-1-C (Nes) cells, and the effects of the nestin overexpression on cell growth and migration were examined. Nestin was expressed in the cytoplasm of the glioblastoma cells in all cases examined. Sh cells showed marked decreases in the expression levels of nestin mRNA and protein, and the growth rate of Sh cells was lower than that of sham (Sc) cells. In contrast, the adhesion activity of Sh cells to types I and IV collagens, fibronectin and laminin was higher than that of Sc cells. Fibrillar actin was clearly detected at the periphery of colonies of Sh cells at the attachment sites on three-dimensional culture plates. The migration and invasion of Sh cells were markedly inhibited compared with those of Sc cells. In contrast, the levels of nestin expression markedly increased in the Nes cells, which were transiently transfected with the nestin expression vector. The growth rate and motility of Nes cells were higher than those of the mock cells. In conclusion, nestin plays important roles in cell growth, migration, invasion and adhesion to extra-cellular matrices in glioma cells. Nestin may serve as a novel candidate for molecular-targeted therapy for gliomas, including glioblastomas.

Abstract 1421: The stem cell marker nestin is a novel therapeutic target to suppress tumor growth and invasion of malignant melanoma

Abstract BACKGROUND: Nestin, a class VI intermediate filament protein, was first described as a neural stem cell/progenitor cell marker expressed during central nervous system development. Recently, nestin was detected in various neoplasms such as glioblastomas, gastrointestinal stromal tumors, prostate cancers, breast cancers, pancreatic cancers and malignant melanomas. An abundance of nestin has been identified in metastatic sites and the dermal parts of nodular melanomas, and the expression level of nestin has relevance to poor prognosis in malignant melanomas. These findings suggest that nestin plays important roles in the invasion of melanoma cells. Furthermore, recent studies have shown that nestin is one of the cancer stem cell markers in melanomas. Therefore, we hypothesized that nestin may be a novel therapeutic target for malignant melanomas. In this study, we used a silencing strategy to clarify the effectiveness of nestin-targeting therapy in malignant melanomas. METHODS: To determine the expression patterns of nestin, we performed immunohistochemical analysis of nestin in human melanoma tissues. Next, we stably knocked down nestin expression in A375 human malignant melanoma cells by short hairpin RNA (shRNA) transfection in vitro. We also transfected sham vectors to A375 cells as negative controls. Cell proliferation, invasion, morphology, and sphere-forming ability were compared with nestin-shRNA-transfected cells (Sh) and sham cells (Sc). RESULTS: Immunohistochemical staining demonstrated that nestin was strongly expressed in melanoma cells in the invasive front of human melanoma tissues. Reduced expression level of nestin mRNA and protein in Sh cells was confirmed by quantitative RT-PCR, western blot and immunocytochemical analyses. Sh and Sc cells did not show characteristic morphological differences; however, stress fiber formation of F-actin was markedly increased in Sh cells. Cell growth of Sh cells was slower than that of Sc cells at 48 hrs as determined by cell counts and MTT assay. On the cell invasion assay using a modified Boyden chamber coated with matrigel on the inner surface of the inserts, the invasion of Sh cells was significantly attenuated in comparison with that of Sc cells. Sphere-forming ability using ultra-low attachment plates supplemented with EGF and FGF-2 showed that Sh cells formed fewer spheres than Sc cells. CONCLUSION: Nestin is expressed in melanoma cells at the invasive front of melanoma tissues, and decreased expression level of nestin inhibits the growth and invasion of melanoma cells. Furthermore, nestin regulates the functions of cancer stem cells of malignant melanoma as determined by sphere-forming ability. Our findings imply that nestin is a promising novel therapeutic target in malignant melanomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1421. doi:10.1158/1538-7445.AM2011-1421

Expression of the Stem Cell Marker Nestin in Pre/Hypertrophic Chondrocytes in Osteochondroma

The pathogenesis of osteochondroma, the most common benign bone tumour, is not fully known. To date, regulation of nestin protein levels in osteochondromas and normal growth plates has not been reported. This study used immunohistochemical analysis to detect nestin protein levels in 48 osteochondromas and 13 normal growth plates (eight fetal and five postnatal). Nestin protein was mainly restricted to prehypertrophic and hypertrophic chondrocytes in osteochondroma. Nestin levels were significantly higher in osteochondromas in adolescents (age range 4 - 18 years, n = 32) than in osteochondromas in young adults (age range 20 - 28 years, n = 11), and significantly higher in osteochondromas in young adults than in those in older adults (age range 40 - 77 years; n = 5). Nestin protein was not present in normal growth plates. The presence of nestin protein parallels the biological behaviour of osteochondromas and is restricted to prehypertrophic and hypertrophic chondrocytes, indicating that these nestin-positive cells may be central to osteochondroma development.

Induction-dependent neural marker expression and electrophysiological characteristics of bone marrow mesenchymal stem cells that naturally express high levels of nestin

Under certain experimental conditions, bone marrow mesenchymal stem cells (MSCs) express neuronal phenotypes and neuronal markers, which suggests that they could be used to treat various neurological diseases. In the present study, MSCs were isolated from adult rat bone marrow, cultivated, and evaluated for neurotrophin expression profiles, as well as the potential to differentiate into functional neuronal-like cells in vitro. MSCs from passage 5 were pre-induced with DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 10 ng/mL bFGF (fibroblast growth factor-2). Subsequently, a chemical inductor containing Dimethyl Sulphoxide (DMSO), Butylated Hydroxyanisole (BHA) and forskolin were used to induce neural expression of MSCs. Expression patterns of nestin, NF-200, and GFAP at time points before and after induction were detected by immunofluorescence. Nerve Growth Factor (NGF), brain-derived neurotrophic factor (BDNF) expressions in MSCs were evaluated by RT-PCR. The whole-cell patch clamp technique was utilized to elucidate the electrical behavior of MSC before and after 24-h differentiation induction. Immunofluorescence analysis revealed that MSCs expressed nestin (57.1% ± 6.9%), but not NF-200 or GFAP. Following neural induction, the cells exhibited a neuronal-like appearance. Nestin and NF-200 expression was positive in the neuronal-like cells, but GFAP expression was negative. After 6-, 12- and 24-h induction, the ratio of nestin-positive cells was 96.5% ± 1.9%, 88.1% ± 5.4%, and 33.5% ± 5.4%. NF-200 positive cells were 90.1% ± 2.9%, 97.5% ± 1.3%, and 98.1% ± 1.6%, respectively. However, prior to induction, MSCs already expressed NGF and BDNF. With a stimulus impulse of 40 mV, the density of the transient outward K current was (9.95 ± 4.85) pA/pF (n = 9) and (328.50 ± 30.62) pA/pF (n = 9) before and after induction, and the density of transient calcium ion currents was (−0.059 ± 0.027) pA/pF (n = 7) and (−6.66 ± 0.50) pA/pF (n = 7), respectively. Transient outward potassium currents and calcium ions currents gradually increased following induction. In addition, MSCs isolated from bone marrow exhibited characteristics of neuronal progenitor cells and expressed neurotrophins. These cells exhibited the capacity to differentiate into functional neuronal-like cells in vitro. These results suggested that MSCs express high levels of nestin and could be utilized for therapeutic strategies to treat nervous system diseases.

Nestin is a novel target for suppressing pancreatic cancer cell migration, invasion and metastasis

Nestin, is a class VI intermediate filament (IF) that is expressed in 30% of pancreatic ductal adenocarcinoma (PDAC) cases, and its expression in PDAC positively correlates with peripancreatic invasion. An expression vector carrying a short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high nestin levels. Alterations in morphology and alignment of actin filaments and α-tubulin were examined by phase-contrast and immunocytochemistry. Effects on cell growth, migration in scratch and Boyden chamber assays, invasion, cell adhesion, and in vivo growth were determined. Differences in mRNA levels were examined by arrays. Nestin shRNA-transfected cells exhibited decreased nestin expression, a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous F-actin and E-cadherin, and attenuated migration and invasion, both of which were enhanced following nestin re-expression. Expression of α-tubulin, and in vitro cell growth and adhesion were not altered by nestin down-regulation, whereas hepatic metastases were decreased. Thus, nestin plays important roles in pancreatic cancer cell migration, invasion and metastasis by selectively modulating the expression of actin and cell adhesion molecules, and may therefore be a novel therapeutic target in PDAC.</br> See commentary: Pancreatic cancer with Nest-in tendencies

Nestin as a regulator of Cdk5 in differentiating myoblasts.

Many types of progenitor cells are distinguished by the expression of the intermediate filament protein nestin, a frequently used stem cell marker, the physiological roles of which are still unknown. Whereas myogenesis is characterized by dynamically regulated nestin levels, we studied how altering nestin levels affects myoblast differentiation. Nestin determined both the onset and pace of differentiation. Whereas depletion of nestin by RNAi strikingly accelerated the process, overexpression of nestin completely inhibited differentiation. Nestin down-regulation augmented the early stages of differentiation, at the level of cell-cycle withdrawal and expression of myogenic markers, but did not affect proliferation of undifferentiated dividing myoblasts. Nestin regulated the cleavage of the Cdk5 activator protein p35 to its degradation-resistant form, p25. In this way, nestin has the capacity to halt myoblast differentiation by inhibiting sustained activation of Cdk5 by p25, which is critical for the progress of differentiation. Our results imply that nestin regulates the early stages of myogenesis rather than maintains the undifferentiated state of progenitor cells. In the bidirectional interrelationship between nestin and Cdk5, Cdk5 regulates the organization and stability of its own nestin scaffold, which in turn controls the effects of Cdk5. This nestin-Cdk5 cross-talk sets the pace of muscle differentiation.

Transplantation of Enteric Cells Expressing p75 in the Rodent Stomach

Neural crest stem cells may hold potential as a cellular therapeutic to repopulate the enteric nervous system. The expression of the low-affinity nerve growth factor receptor, p75, may enrich enteric cells isolated from the neonatal rodent intestinal tract for neural crest stem cells. While these cells have shown tremendous promise in vitro, it remains to be determined whether they will survive and differentiate after in vivo transplantation. Cells isolated from the neonatal rodent intestinal muscularis were sorted according to their degree of p75 expression. The parent, p75-high, and p75-low expressing populations were injected into the rodent stomach for 7 d in vivo. Cells that expressed high levels of p75 also expressed high levels of nestin. Evaluation of the parent, p75-high, and p75-low populations of cells showed 420-, 130-, and 20-fold growth, respectively, after 11 d of culture. Transplantation of these populations of cells into syngeneic rodent stomachs showed cell survival in the injection site after 7 d. Cells expressing either the neuronal marker, peripherin, or the glial marker, S100, were present in and around the injection site when the parent and the p75-high populations were transplanted. Enteric cells survive transplantation into the rodent stomach and induce the expression of differentiation markers in and around the injection site. Based on these results, enteric cells may hold potential as a cellular therapeutic in a variety of gastrointestinal disorders.

Emerging new sites for adult neurogenesis in the mammalian brain: a comparative study between the hypothalamus and the classical neurogenic zones

In adult mammalian brain, two main germinative regions located in the subventricular zone of the lateral ventricle and in the subgranular cell layer of the hippocampal dentate gyrus have been considerably documented and are still under intense scrutiny. However, new neuron formation has recently been reported in various other brain areas including the hypothalamus. This central structure, responsible for the control of many major neuroendocrine functions such as reproduction, expresses high levels of PSA-NCAM and nestin, both proteins being involved in structural and morphological plasticity mechanisms. Cell proliferation and new neuron production have been demonstrated in the adult hypothalamus of numerous species, although not hitherto described in non-human primates and humans. Similarly to the subventricular zone and in the subgranular cell layer, the adult hypothalamic neurogenesis process is subject to dynamic regulation by various physiological and pharmacological signals. Several pieces of evidence support the hypothesis that a stem cell niche-like architecture exist in the hypothalamus region lining the third ventricle thereby enabling adult neural stem cells to continuously generate neurons in vivo throughout life. Furthermore, recent data indicating that new hypothalamic neurons may become functionally implicated in sensory information processing endorse the assumption that the hypothalamus might be a neurogenic region.

Inducible expression of stem cell associated intermediate filament nestin reveals an important role in glioblastoma carcinogenesis

The intermediate filament nestin is transiently expressed in neural stem/progenitor cells during the development of central nervous system. Recently, increasing evidence has shown that upregulation of nestin is related to malignancy of several cancers, especially glioblastoma. However, the function of nestin in carcinogenesis remains unclear. In this study, we investigated the role of nestin in glioblastoma carcinogenesis by comparing subclones of rat C6 glioblastoma cells that were either high or low for nestin expression. We found that while nestin expression did not influence the in vitro proliferation of glioblastoma cells, subclones characterized by high levels of nestin formed tumors in vivo at significantly faster rates than subclones with low expression. Importantly, C6 subclones that expressed nestin at low levels in vitro were also found to give rise to tumors highly positive for the protein, suggesting that induction of nestin plays an important role in glioblastoma carcinogenesis. Derivation of nestin positive tumors from nestin negative human U87 glioblastoma cells in immunodeficient mice further confirmed that a switch to positive expression of nestin is fundamental to the course of glioblastoma development. Blocking the expression of nestin in glioblastoma tumors via intratumor injection of shRNA significantly slowed tumor growth and volume. These results demonstrated that nestin plays a crucial role in development of glioblastoma and may potentially be targeted for treatment of the disease.

Nestin, a neuroectodermal stem cell marker, is expressed by bovine sertoli cells

Nestin, an intermediate filament protein is expressed by neuroectodermal stem cells and tumors originating from cells of neuroectodermal and mesenchymal lineages. Nestin expression is prominent in embryos and remains upregulated until 3–6 weeks after birth but is downregulated afterward. Sertoli cells are nucleated somatic cells that are spanned in the seminiferous epithelium and play a critical role in supporting and controlling germ-cell development. In this context, we employed immunocytochemical, Western blot, and Flow cytometric analyses to demonstrate nestin expression in bovine sertoli cells. Immunostaining clearly showed that setoli cells express high levels of nestin, a result which was confirmed by Western blot analysis of purified cells. Intracellular staining of sertoli cells by flow cytometry revealed that around 74% of the cells express this marker. Given the high expression of vimentin by sertoli cells, it is proposed that the expression of nestin in these cells might be required for the formation of stable vimentin/nestin intermediate filament network. In light of these findings, it seems that sertoli cells of mature bull have potentiality of proliferation.

Analysis of side population cells derived from dental pulp tissue

Kenmotsu M, Matsuzaka K, Kokubu E, Azuma T, Inoue T. Analysis of side population cells derived from dental pulp tissue. International Endodontic Journal.AimTo investigate the characteristics of side population (SP) cells derived from the dental pulp of young and aged rats.MethodologyMaxillary and mandibular incisors were extracted from 5-week-old (young) rats and 60- to 80-week-old (aged) rats. Coronal pulp tissue was removed mechanically, and single-cell suspensions were prepared using collagenase and dispase. Cells were stained with Hoechst 33342 and sorted with an fluorescence-activated cell sorter (FACS). Isolated SP and main population (MP) cells were analysed by real-time reverse transcription polymerase chain reaction, immunohistochemical localization and cell cycle determination. Two-way analysis of variance and the multiple comparison Scheffè test were used for statistical analysis (P < 0.05).ResultsApproximately 0.40% of pulp cells in young rats and 0.11% in aged rats comprised SP cells. SP cells expressed a higher mRNA level of ATP-binding cassette transporter G2 (ABCG2), but lower mRNA levels of nestin, alkaline phosphatase, p16 and p57 than MP cells in both age groups. Immunohistochemical observation revealed ABCG2-positive cells localized in the cell-rich zone and nestin in the odontoblastic layer in both groups. Furthermore, the majority of both young and aged SP and MP cells were in growth arrest of the G0/G1 phase.ConclusionThe FACS analysis revealed a decrease in the proportion of SP cells with age, whilst p16 mRNA expression indicated an increase in cell senescence. The cell cycles of SP and MP cells from both young and aged dental pulp were generally in the G0/G1 phase.

Nestin and SOX9 and SOX10 transcription factors are coexpressed in melanoma

Nestin is an intermediate filament expressed in proliferating neural progenitor cells and has been considered as a stem cell marker. Nestin is also found in melanoma and we recently demonstrated that its expression in melanoma cell lines is regulated by the transcription factors SOX9 and SOX10, but not BRN2. In this study, the expression levels of nestin, BRN2, SOX9 and SOX10 were analysed in tissues of melanoma (n = 78) and melanocytic nevi (n = 26) by immunohistochemistry. All proteins were highly expressed in primary and metastatic melanomas and, apart from BRN2, showed much lower levels in melanocytic nevi. Significant coexpression of nestin with SOX9 and SOX10 was found in primary melanoma confirming our in vitro data. Correlation analysis with clinicopathological data revealed that nestin was significantly associated with presence of ulceration in primary tumors and SOX9 with more advanced stage of disease. Our data reveal that SOX9 and SOX10 are highly expressed in melanoma and seem to have a regulatory role in nestin expression. The association with ulceration and advanced-stage tumors, respectively, suggests that nestin and SOX9 may be negative prognostic markers in melanoma.

A Novel Population of Myeloid Cells Responding to Coxsackievirus Infection Assists in the Dissemination of Virus within the Neonatal CNS

Enterovirus infection in newborn infants is a significant cause of aseptic meningitis and encephalitis. Using a neonatal mouse model, we previously determined that coxsackievirus B3 (CVB3) preferentially targets proliferating neural stem cells located in the subventricular zone within 24 h after infection. At later time points, immature neuroblasts, and eventually mature neurons, were infected as determined by expression of high levels of viral protein. Here, we show that blood-derived Mac3(+) mononuclear cells were rapidly recruited to the CNS within 12 h after intracranial infection with CVB3. These cells displayed a myeloid-like morphology, were of a peripheral origin based on green fluorescent protein (GFP)-tagged adoptive cell transplant examination, and were highly susceptible to CVB3 infection during their migration into the CNS. Serial immunofluorescence images suggested that the myeloid cells enter the CNS via the choroid plexus, and that they may be infected during their extravasation and passage through the choroid plexus epithelium; these infected myeloid cells ultimately penetrate into the parenchyma of the brain. Before their migration through the ependymal cell layer, a subset of these infected myeloid cells expressed detectable levels of nestin, a marker for neural stem and progenitor cells. As these nestin(+) myeloid cells infected with CVB3 migrated through the ependymal cell layer, they revealed distinct morphological characteristics typical of type B neural stem cells. The recruitment of these novel myeloid cells may be specifically set in motion by the induction of a unique chemokine profile in the CNS induced very early after CVB3 infection, which includes upregulation of CCL12. We propose that intracranial CVB3 infection may lead to the recruitment of nestin(+) myeloid cells into the CNS which might represent an intrinsic host CNS repair response. In turn, the proliferative and metabolic status of recruited myeloid cells may render them attractive targets for CVB3 infection. Moreover, the migratory ability of these myeloid cells may point to a productive method of virus dissemination within the CNS.

Pre‐activation of retinoid signaling facilitates neuronal differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into neurons in an appropriate cellular environment. Retinoid signaling pathway is required in neural development. However, the effect and mechanism through retinoid signaling regulates neuronal differentiation of MSCs are still poorly understood. Here, we report that all-trans-retinoic acid (ATRA) pre-induction improved neuronal differentiation of rat MSCs. We found that, when MSCs were exposed to different concentrations of ATRA (0.01-100 micromol/L) for 24 h and then cultured with modified neuronal induction medium (MNM), 1 micromol/L ATRA pre-induction significantly improved neuronal differentiation efficiency and neural-cell survival. Compared with MNM alone induced neural-like cells, ATRA/MNM induced cells expressed higher levels of Nestin, neuron specific enolase (NSE), microtubule-associated protein-2 (MAP-2), but lower levels of CD68, glial fibrillary acidic protein (GFAP), and glial cell line-derived neurotrophic factor(GDNF), also exhibited higher resting membrane potential and intracellular calcium concentration, supporting that ATRA pre-induction promotes maturation and function of derived neurons but not neuroglia cells from MSCs. Endogenous retinoid X receptors (RXR) RXRalpha and RXRgamma (and to a lesser extent, RXRbeta) were weakly expressed in MSCs. But the expression of RARalpha and RARgamma was readily detectable, whereas RARbeta was undetectable. However, at 24 h after ATRA treatment, the expression of RARbeta, not RARalpha or RARgamma, increased significantly. We further found the subnuclear redistribution of RARbeta in differentiated neurons, suggesting that RARbeta may function as a major mediator of retinoid signaling during neuronal differentiation from MSCs. ATRA treatment upregulated the expression of Vimentin and Stra13, while it downregulated the expression of Brachyury in MSCs. Thus, our results demonstrate that pre-activation of retinoid signaling by ATRA facilitates neuronal differentiation of MSCs.

An improved, standardised protocol for the isolation, enrichment and targeted neural differentiation of Nestin+ progenitors from adult human dermis

Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.

Abstract 4185: Isolation of cancer stem-like cells from a novel orthotopic xenograt model of supratentorial primitive neuroectodermal tumor that is susceptible to oncolytic Seneca Valley Virus

Abstract Purpose: Supratentorial primitive neuroectodermal tumor (PNET) accounts for 2-3% of all pediatric brain tumors. Despite multimodality therapies, the clinical outcome remains poor, and the 5-year progression free survival rate is less than 40%. Clinically relevant animal models are needed for understanding tumor biology and developing more effective therapies. Here we report our recent development of a clinically relevant animal model, isolation and characterization of cancer stem-like cells in the xenograft tumors, and preclinical investigation of the antitumor efficacy of a novel picorna oncolytic virus Seneca Valley Virus-001 (SVV-001) both in vitro and vivo, especially in cancer stem-like cells. Experimental Design: Orthotopic xenograft mouse model was developed by direct injection of 105 tumor cells from a freshly resected brain PNET tumor specimen into the right cerebrum of Rag2/severe combined immunodeficient mice. CD133+ cells were quantitatively analyzed and isolated with FACS using human-specific antibodies against CD133. Antitumor activity of SVV-001was evaluated in vitro in cultured xenograft tumor cells and in vivo in mice bearing pre-formed xenograft tumors. Results: A novel transplantable PNET xenograft model IC-2664PNT was established. The xenograft tumors not only replicate the biologic phenotypes but also preserve cancer stem-like cell pools. The IC-2664PNET neurospheres, which expressed high levels of CD133 and nestin, were efficiently infected and killed by SVV-001 (0.5-25 virus particle/ cell). A single tail vein injection of SVV-001 (5×1012 virus particle/kg) prolonged survival time of PNET xenograft model. Conclusions: We have established the first clinically relevant animal model for childhood supratentorial PNET. It replicated the major histopathological features of the original patient tumor, and preserved a CD133+ stem cells pool during serial subtransplantations. SVV-001 has potent antitumor efficacy in PNET, especially selectively kill cancer stem-like cells, and prolong survival in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4185.

Abstract 408: Stem cell marker Nestin regulated the migration and invasion of pancreatic cancer cells

Abstract Background: Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell/progenitor cell marker. Nestin expression has also been shown to be up-regulated in progenitor cells in muscle, testis, teeth and pancreas. In the pancreas, lineage-tracing experiments have indicated that exocrine cells are derived from Nestin-expressing progenitor cells. Moreover, activation of Kras in the Nestin cell lineage is sufficient for the initiation of premalignant pancreatic intraepithelial neoplasia lesions in mice. We have reported that Nestin was expressed in 30% of human pancreatic ductal adenocarcinoma (PDAC) cases, and Nestin expression in PDAC positively correlated with nerve involvement and invasion of the peripancreatic tissue margin. Therefore, we hypothesized that Nestin may play an important role in the migration and invasive potential of PDAC cells. In this study, we used a silencing strategy to clarify the roles of Nestin in human pancreatic cancer cells. Methods: An expression vector carrying a short hairpin RNA (shRNA) targeting Nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high levels of Nestin. Changes in the morphology and alignment of actin filaments were analyzed using phase-contrast images and immunocytochemistry. Effects of decreased expression of Nestin on cell growth, migration in scratch and Boyden chamber assays, invasion through matrigel, and cell adhesion on extracellular matrices were examined. Differences in mRNA levels of selected signaling molecules were examined by PCR arrays. Results: Targeting Nestin with shRNA caused a marked decrease in Nestin mRNA and protein levels. Nestin shRNA-transfected cells (Nes-sh cells) exhibited a sheet-like appearance with tight cell-cell adhesion, and increased expression of filamentous (F)-actin by comparison with sham-transfected cells, whereas anchorage-dependent and -independent cell growth and cell-extracellular matrix adhesion of Nes-sh cells did not differ from those of sham cells. By contrast, migration and invasion of Nes-sh cells were markedly attenuated. To clarify the mechanisms underlying this observation, we analyzed differences in selected signaling molecules by PCR arrays that could determine the expression of mRNA closely related to tumor metastasis. The gene expressing the greatest value in Nes-sh cells was E-cadherin. Real-time PCR and western blotting confirmed that E-cadherin expression was increased in Nes-sh cells by comparison with sham cells. Conclusion: Nestin participates in the regulation of pancreatic cancer cell migration and invasion, in part, by altering F-actin and E-cadherin expression. These observations suggest that Nestin may modulate the migration of Nestin-positive progenitor cells during pancreatic development, and may serve as a novel target for suppression of invasion and metastasis in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 408.

A Comparison of Differentiation Protocols for RGC-5 Cells

PURPOSE. Although the RGC-5 cell line is widely used in retinal ganglion cell (RGC) research, recent data have raised questions about the nature of these cells. The authors performed a systematic analysis of RGC-5 cells to determine which RGC or neuronal markers are expressed after treatment with known differentiating agents, thus providing further insight into the nature of these cells and assisting in defining their future use. METHODS. RGC-5 cells were treated for 5 days with staurosporine (STSN; 316 nM), trichostatin A (TSA; 500 nM), or succinyl-concanavalin A (sConA; 50 microg/mL), after which they were assayed for specific marker antigen/mRNA expression. Treated cells were also assayed for excitotoxic responsiveness. RESULTS. Neither treated nor untreated RGC-5 cells expressed any specific RGC marker mRNAs or proteins (Brn-3, neurofilaments, Thy-1) or calbindin, calretinin, synaptophysin, PKCalpha, or glial fibrillary acidic protein. However, control RGC-5 cells did express the neuronal markers tau, betaIII-tubulin, microtubule-associated protein (MAP)-1b, MAP2, and PGP9.5. Although treatment with sConA had no effect on the expression of these markers, STSN and (dose dependently) TSA increased their expression and induced excitotoxic responsiveness. All cells, treated or not, expressed high levels of nestin but no other progenitor cell markers. All cells also expressed cone-specific, but not rod-specific, opsin indicative of cone photoreceptor lineage. CONCLUSIONS. RGC-5 cells expressed neuronal, but not RGC-specific, markers that were dose dependently upregulated by TSA. Hence, TSA provided the best tested means to terminally differentiate the cells to a neuronal phenotype from a precursor-like lineage.

Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia-like cells, actively proliferating cells, and label-retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow-dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100beta), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA-NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow-cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA-positive cells in fast domains further display a morphology distinct from radial glia and co-express PSA-NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA-NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ.