Neural Crest Stem Cell Markers Research Articles

Widespread Distribution of Luteinizing Hormone/Choriogonadotropin Receptor in Human Juvenile Angiofibroma: Implications for a Sex-Specific Nasal Tumor.

Juvenile angiofibroma (JA) is a rare, sex-specific, and highly vascularized nasal tumor that almost exclusively affects male adolescents, but its etiology has been controversial. The G protein-coupled hormone receptor LHCGR [luteinizing hormone (LH)/choriogonadotropin (hCG) receptor] represents a promising new candidate for elucidating the underlying mechanisms of sex specificity, pubertal manifestation, and JA progression. We used highly sensitive RNAscope technology, together with immunohistochemistry, to investigate the cellular expression, localization, and distribution of LHCGR in tissue samples from JA patients. Our results provide evidence for LHCGR expression in subsets of cells throughout JA tissue sections, with the majority of LHCGR+ cells located in close vicinity to blood vessels, rendering them susceptible to endocrine LH/hCG signaling, but LHCGR+ cells were also detected in fibrocollagenous stroma. A majority of LHCGR+ cells located near the vascular lumen co-expressed the neural crest stem cell marker CD271. These results are intriguing as both LH and hCG are produced in a time- and sex-dependent manner, and are known to be capable of inducing cell proliferation and angiogenesis. Our results give rise to a new model that suggests endocrine mechanisms involving LHCGR and its ligands, together with autocrine and paracrine signaling, in JA vascularization and cell proliferation.

Adult hair follicle stem cells differentiate into neuronal cells in explanted rat intestinal tissue.

Hair follicle stem cells (HFSCs) are adult stem cells located in the outer root sheath of the follicle bulge with high neural plasticity, which promise a potential for the stem cell therapy for neurological diseases. Hirschsprung's disease (HD) is characterized by the absence of ganglia in the distant bowel. In this study, we elucidated the capacity of HFSCs to differentiate into neuronal cells in the aganglionic colon from embryonic rat. HFSCs were isolated from adult Sprague-Dawley (SD) rats and formed spheres that could be passaged. The cultured HFSCs expressed neural crest stem cells (NCSCs) markers such as SOX10, CD34, and nestin, which indicated their neural crest lineage. Subsequent differentiation assays demonstrated that these cells could give rise to neural progeny that expressed neuronal or glial markers. The aganglionic colon from the embryonic intestine was applied as in vitro explant to test the capacity of proliferation and differentiation of HFSCs. The HFSCs expressing GFP or RFP integrated in intestinal explants and maintained proliferative capacity. Moreover, the HFSCs differentiated into Tuj1- or S100β-positive cells in the cultured intestinal explants. The results proposed that the HFSCs might be an alternative source of neural stem cells for the HD therapy.

The Lamina Propria of the Oral Mucosa Harbors a Neural Crest-Like Stem Cell Population Resistant to Hyperglycemia Induced by Diabetes Type II

Oral mucosa stem cells (OMSC) are contained within niches in the lamina propria of the oral mucosa. In vitro, OMSC exhibit a primitive neural crest-stem cell phenotype and differentiate into neural crest-derived cell lineages. Clinical evidence indicate that wound healing of oral mucosa is scarless and negligibly affected by diabetes. Since stem cells play an important role in wound healing we hypothesized that in contrast to adult mesenchymal stem cells, OMSC are negligibly affected by diabetes type II hyperglycemia. The capacity of murine OMSC (mOMSC) obtained from diabetic db+/db+ mice (DmOMSC) to proliferate, express pluripotency-associated markers (PAM) (oct4, sox2, nanog) and neural crest stem cell markers (NCSM) (Klf4, c-Myc, twist, nestin), and differentiate along neural crest cell lineages was compared to that of OMSC derived from background WT C57BL/6 mice (WT-mOMSC) and to those of murine adipose tissue stem cells (mASC) derived from the same animals used for generating mOMSC. WT-mOMSC capacity to proliferate, express PAM and NCSM and differentiate was higher than that of WT-mASC. Diabetes reduced the proliferation rat of DmOMSC and db+/db+ derived mASC (DmASC) by 35% and 42%, respectively. Diabetes did not affect the expression of PAM and NSCSM in DmOMSC and nor their differentiation as compared to WT-mOMSC, but the diabetic state substantially reduced these parameters in DmASC as compared to WT-mASC and DmOMSC. We demonstrated that WT-mOMSC administration enhances wound healing in the db+/db+ diabetic wound skin model. To test the effect of diabetes on the therapeutic functionality of DmOMSC, the ability of these cells to enhance diabetic wound healing was assessed in this model. The results indicate that DmOMSC increased the rate of wound healing in db+/db+ mice as compared to placebo treated controls. The magnitude of this therapeutic effect is statistically equivalent to that of WT-mOMSC. Collectively, these data demonstrate that: i) diabetes decreased moderately DmOMSC proliferative ability; ii) in contrast to mASC, mOMSC withstand the detrimental effects of diabetes; and iii) the therapeutic properties of DmOMSC are mildly affected by long standing diabetes.

Role of dual specificity phosphatases (DUSPs) in melanoma cellular plasticity and drug resistance

Melanoma cells exhibit phenotypic plasticity that allows transition from a proliferative and differentiated phenotype to a more invasive and undifferentiated or transdifferentiated phenotype often associated with drug resistance. The mechanisms that control melanoma phenotype plasticity and its role in drug resistance are not fully understood. We previously demonstrated that emergence of MAPK inhibitor (MAPKi)-resistance phenotype is associated with decreased expression of stem cell proliferation genes and increased expression of MAPK inactivation genes, including dual specificity phosphatases (DUSPs). Several members of the DUSP family genes, specifically DUSP1, -3, -8 and -9, are expressed in primary and metastatic melanoma cell lines and pre-and post BRAFi treated melanoma cells. Here, we show that knockdown of DUSP1 or DUSP8 or treatment with BCI, a pharmacological inhibitor of DUSP1/6 decrease the survival of MAPKi-resistant cells and sensitizes them to BRAFi and MEKi. Pharmacological inhibition of DUSP1/6 upregulated nestin, a neural crest stem cell marker, in both MAPKi-sensitive cells and cells with acquired MAPKi-resistance. In contrast, treatment with BCI resulted in upregulation of MAP2, a neuronal differentiation marker, only in MAPKi-sensitive cells but caused downregulation of both MAP2 and GFAP, a glial marker, in all MAPKi-resistant cell lines. These data suggest that DUSP proteins are involved in the regulation of cellular plasticity cells and melanoma drug resistance and are potential targets for treatment of MAPKi-resistant melanoma.

P75 neurotrophin receptor positively regulates the odontogenic/osteogenic differentiation of ectomesenchymal stem cells via nuclear factor kappa-B signaling pathway

ABSTRACT p75NTR, a neural crest stem cell marker, is continuously expressed in mesenchymal cells during tooth development. Importantly, high expression of p75NTR in the late bell stage implicates its involvement in odontogenesis and mineralization. However, the regulatory mechanisms underlying p75NTR involvement in odonto/osteogenic differentiation remain unclear. Here, we investigate the effect and potential mechanisms underlying p75NTR involvement in odonto/osteogenic differentiation. We dissected EMSCs from the first branchial arches of mice embryo and compared the proliferation and migration of p75NTR+/+ and p75NTR−/−EMSCs by transwell, scratch and cell counting kit 8(CCK8)assays. The differentiation ability and the involvement of nuclear factor kappa-B (NF-κB) pathway were investigated through alkaline phosphatase and immunofluorescence assay, real-time PCR, and western blot. During induction of dental epithelium conditioned medium, p75NTR+/+ EMSCs exhibited deeper Alkaline phosphatase (ALP) staining and higher expression of odonto/osteogenic genes/proteins (e.g., dentin sialoprotein (DSPP) than p75NTR+/+ EMSCs. Moreover, p75NTR+/+ EMSCs exhibited higher nuclear P65 expression than p75NTR−/−EMSCs. Inhibition of NF-κB pathway with Bay11-7082 in p75NTR+/+EMSCs substantially decreased DSPP expression level. However, activation of NF-κB pathway with Bay11-7082 in p75NTR−/−EMSCs enhanced DSPP expression level. Thus, p75NTR possibly plays a paramount role in the proliferation and differentiation of EMSCs via NF-κB pathway.

Discovering the Potential of Dental Pulp Stem Cells for Corneal Endothelial Cell Production: A Proof of Concept.

Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques.

Neural crest stem cells can be induced in vitro from human-induced pluripotent stem cells using a novel protocol free of feeder cells.

Objective: Our knowledge of human neural crest stem cells (NCSCs) is expanding, owing to recent advances in technologies utilizing human-induced pluripotent stem cells (hiPSCs) that generate NCSCs. However, the clinical application of these technologies requires the reduction of xeno-materials. To overcome this significant impediment, this study aimed to devise a novel method to induce NCSCs from hiPSCs without using a feeder cell layer.Materials and Methods: hiPSCs were cultured in feeder-free maintenance media containing the Rho-associated coiled-coil forming kinase inhibitor Y-27632. When the cells reached 50–70% confluence, differentiation was initiated by replacing the medium with knockout serum replacement (KSR) medium containing Noggin and SB431542. The KSR medium was then gradually replaced with increasing concentrations of Neurobasal medium from day 5 to 11.Results: Immunocytochemistry and flow cytometry were performed 12 days after induction of differentiation and revealed that the cells generated from hiPSCs expressed the NCSC markers p75 and HNK-1, but not the hiPSC marker SOX2.Conclusion: These findings demonstrate that hiPSCs were induced to differentiate into NCSCs in the absence of feeder cells.

Immunocytochemical characterization of primary teeth pulp stem cells from three stages of resorption in serum-free medium.

Dental pulp stem cells from primary teeth cultured in serum-free conditions may have clinical use for the repair and regeneration of teeth as well as other complex tissues and organs. The aim of this study was to test the change in the stem cell markers expression/ stem cell population in human primary pulp cells at the different stages of root resorption. Caries-free human primary canines at defined stages of physiological root resorption were included (n=9). In vitro cultures were established in xeno-free, serum-free Essential 8™ medium with human truncated vitronectin for cell attachment. An embryonic stem cell line (GENEA002) was used as a positive control. The expression of embryonic stem cell markers (Oct4, Nanog and Sox2), neural crest stem cell markers (nestin and Dlx2) and mesenchymal stem cell surface markers (CD90, CD73 and CD105) were investigated by immunocytochemistry. Mesenchymal stem cell markers CD105, CD73 and CD90 and haematopoietic markers: CD45, CD34, CD11b, CD19 and HLA-DR were quantified with flow cytometry. The early neural progenitor markers nestin and Dlx2 were detected in most serum-free cultured dental pulp stem cells, regardless of the tooth resorption stage from which they were harvested. Only isolated cells were found that expressed the embryonic stem cell transcription factors Oct4A, Nanog and Sox2, and in the late stages of resorption, no Oct4A was detected. The majority expressed the mesenchymal stem cell markers CD90, CD73 and CD105. Flow cytometry found positive signals for CD90>97.3%, CD73>99.6% and CD105>82.5%, with no detectable differences between resorption stages. This study identified populations of dental pulp cells in vitro with markers characteristically associated with embryonic stem cells, neural crest-derived cells and mesenchymal stem cells. Flow cytometry found CD105 expressed at lower levels than CD90 and CD73. The consistency of stem cell marker expression in cells cultured from teeth at different resorption stages suggests that pre-exfoliated primary teeth that are free of caries may provide a convenient source of multipotent stem cells for use in regenerative medicine.

Successful culture of human transition zone cells.

Human corneal endothelial cells undergo very little or no proliferation and respond to cell loss by migration and cellular enlargement. Significant cell loss or damage may result in corneal oedema, opacity and loss of vision. In vitro expansion of corneal endothelial cells (CECs) is a promising strategy for corneal regeneration. The transition zone (TZ) may be an alternative source of CECs. The objective of this study was to establish a protocol for TZ cell culture, and to determine their potential to proliferate and differentiate into cells that resemble CECs in vitro. An explant culture protocol for the human TZ was established. Cell proliferation was assessed using 5-ethynyl-2'-deoxyuridine (EdU) assay. The expression of stem cell and endothelial markers was assessed using immunohistochemistry and quantitative polymerase chain reaction. TZ cells can be passaged up to 12 times; cells became polygonal 3 to 4 passages before senescence. An average of 41% of cells incorporated EdU over a 5-day period. TZ cells expressed the corneal endothelial proteins ZO-1 and Na+ /K+ ATPase, Col8A2, the periocular mesenchyme marker PITX2, and the neural crest stem cell markers Nestin and Sox10. TZ cells expressed mRNA of a range of neural crest, periocular mesenchyme, and corneal endothelial genes. TZ cells can proliferate and differentiate into cells that resemble CECs, demonstrating their potential to be an alternative cell source for corneal endothelial cell therapy.

Progenitor cells expressing nestin, a neural crest stem cell marker, differentiate into outer root sheath keratinocytes.

Nestin, which was originally described as a neural crest stem cell marker, is known to be expressed in bulge follicle cells of human, canine and murine anagen hairs. However, the capacity of nestin-expressing cells to differentiate into the components of the hair follicle or the epidermis has been insufficiently investigated. To determine whether nestin-expressing cells are capable of differentiating into keratinocytes. A double-transgenic mouse line Nes-Cre/CAG-CAT-EGFP, in which enhanced green fluorescent protein (EGFP) is expressed upon Cre-based recombination driven by the nestin promoter. The tissue distribution of EGFP+ and nestin+ cells in the skin of the mouse line was analysed by immunofluorescence and immunohistochemical analyses. EGFP+ cells were recognized in the outer epithelial cell layers of anagen and telogen hair follicles, but rarely seen in the interfollicular epidermis. The EGFP+ cells in the outer layers of the hair follicles coexpressed keratin 14, a marker of the outer root sheath (ORS) keratinocytes, but not trichohyalin granules, an inner root sheath keratinocyte cell marker. Immunostaining for nestin failed to detect its expression in the majority of hair follicle epithelial cells, suggesting that the EGFP+ cells in the ORS were derived from nestin-expressing progenitor cells that had become further committed along the epithelial cell lineage, where nestin is no longer expressed. These results suggest that progenitor cells that differentiate into ORS keratinocytes are distinct from those for other hair follicle or epidermal components and provide implications for regenerative medicine and the molecular classification of hair follicle tumours.

Induced pluripotent stem cells with NOTCH1 gene mutation show impaired differentiation into smooth muscle and endothelial cells: Implications for bicuspid aortic valve-related aortopathy

ObjectiveThe NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. MethodsThe iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1−/− (NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1−/− and wild type cells. ResultsThe expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1−/−NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1−/− NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1−/−CVPCs, the expression of ISL1, NKX2.5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1−/−ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. ConclusionsNOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve.

MYB fusions and CD markers as tools for authentication and purification of cancer stem cells from salivary adenoid cystic carcinoma

Cancer stem cells (CSC) are considered the major cause of aggressive tumor behavior, recurrence, metastases, and resistance to radiation, making them an attractive therapeutic target. However, isolation of CSC from tumor tissue and their characterization are challenging due to uncertainty about their molecular markers and conditions for their propagation. Adenoid cystic carcinoma (ACC), which arises predominantly in the salivary glands, is a slow-growing but relentless tumor that frequently invades nerves and metastasizes. New effective treatment approaches for ACC have not emerged over the last 40years. Previously, based on a highly conserved SOX10 gene signature that we identified in the majority of ACC tumors, we suggested the existence in ACC of SOX10+ cells with neural stem properties and corroborated this hypothesis via isolation from ACC tissue a novel population of CSC, termed ACC-CSC. These cells activated NOTCH1 signaling and co-expressed SOX10 and other ACC-intrinsic neural crest stem cell markers with CD133, a CSC cell surface marker, suggesting that ACC is driven by a previously uncharacterized population of SOX10+/CD133+ cells with neural stem cell properties. Here, we authenticated ACC identity of our primary cultures by demonstrating that most of them harbor MYB-NFIB fusions, which are found in 86% of ACC. We demonstrated using CyTOF, a novel mass cytometry technology, that these cells express high β-catenin and STAT3 levels and are marked by CD24 and CD44. Finally, to streamline development of ACC cell lines, we developed RT-PCR tests for distinguishing mouse and human cells and used immunomagnetic cell sorting to eliminate mouse cells from long-term cell cultures. Overall, this study describes a new population of CSC that activates signaling pathways associated with poor prognosis, validates their ACC identity, and optimizes approaches that can be used for purification of ACC-CSC and generation of cell lines.

The Differentiation of Skin Mesenchymal Stem Cells Towards a Schwann Cell Phenotype: Impact of Sigma-1 Receptor Activation.

Neural crest stem cells (NCSCs) are the source of mature Schwann cells in the peripheral nervous system (PNS). The NCSC population resides in the bulge of hair follicles and in the dermis. Recently, it was shown that 2-3% of the human dermis mesenchymal stem cell (MSC) population expresses the NCSC marker CD271, thus enabling the use of skin MSCs for studying Schwann cell differentiation in vitro. The aims of this study were to establish a protocol for human skin MSC differentiation towards Schwann cell-like cells (SC-lcs) and to analyse the expression of sigma-1 receptor (S1R) in SC-lcs. The impact of S1R ligands, namely the selective agonist PRE-084, the positive allosteric modulator E1R and the selective antagonist NE-100, on Schwann cell differentiation was assessed. The expression of the neuron-specific genes Tubulin-βIII and Integrin-6α, the Schwann cell-specific gene S100b, MBP and the NCSC-specific genes p75NTR, Sox10, Notch1, Integrin-4α, Ap2α and Pax6 was analysed in MSCs and SC-lcs by real-time RT-PCR. BDNF secretion was evaluated by ELISA. The effect of S1R ligands on SC-lc differentiation was measured using BDNF ELISA and MBP flow cytometry. After MSC differentiation, NCSC markers p75NTR and Integrin-4α were downregulated 3.5-fold and 2-fold, respectively. To the contrary, MBP and S100b were significantly upregulated in SC-lcs. S1R ligands showed a tendency to increase the secretion of BDNF by the SC-lc population. PRE-084 and E1R increased MBP expression in the SC-lc population, whereas 3μM NE-100 inhibited MBP expression in SC-lcs. In conclusion, our data demonstrate that S1R plays an important role in skin MSC differentiation towards myelinating Schwann cells.

Human dental follicle cells express embryonic, mesenchymal and neural stem cells markers

ObjectiveThis study was conducted to identify and characterize dental follicle stem cells (DFSCs) by analyzing expression of embryonic, mesenchymal and neural stem cells surface markers. Design Dental follicle cells (DFCs) were evaluated by immunocytochemistry using embryonic stem cells markers (OCT4 and SOX2), mesenchmal stem cells (MSCs) markers (Notch1, active Notch1, STRO, CD44, HLA-ABC, CD90), neural stem cells markers (Nestin and β-III-tubulin), neural crest stem cells (NCSCs) markers (p75 and HNK1) and a glial cells marker (GFAP). RT-PCR was performed to identify the expression of OCT4 and NANOG in DFCs and dental follicle tissue. ResultsImmunocytochemistry and RT-PCR analysis revealed that a significant proportion of the DFCs evaluated expressed human embryonic stem cells marker OCT4 (75%) whereas NANOG was weakly expressed. A considerable amount of MSCs (90%) expressed Notch1, STRO, CD44 and HLA-ABC. However, they were weakly positive for CD90. Moreover, it was possible to demonstrate that dental follicle contains a significant proportion of neural stem/progenitors cells, expressing β-III-tubulin (90%) and nestin (70%). Interestingly, immunocytochemistry showed DFCs positive for p75 (50%), HNK1 (<10%) and a small proportion (<20%) of GFAP-positive cells. This is the first study reporting the presence of NCSCs and glial-like cells in the dental follicle. ConclusionsThe results of the present study suggest the occurrence of heterogeneous populations of stem cells, particularly neural stem/progenitor cells, in the dental follicle, Therefore, the human dental follicle might be a promising source of adult stem cells for regenerative purposes.

Simulated Microgravity Disrupts Cytoskeleton Organization and Increases Apoptosis of Rat Neural Crest Stem Cells Via Upregulating CXCR4 Expression and RhoA-ROCK1-p38 MAPK-p53 Signaling.

Neural crest stem cells (NCSCs) are a population of multipotent stem cells that are distributed broadly in many tissues and organs and are capable of differentiating into a variety of cell types that are dispersed throughout three germ layers. We are interested in studying the effects of simulated microgravity on the survival and self-renewal of NCSCs. NCSCs extracted from the hair follicle bulge region of the rat whisker pad were cultured in vitro, respectively, in a 2D adherent environment and a 3D suspension environment using the rotatory cell culture system (RCCS) to simulate microgravity. We found that rat NCSCs (rNCSCs) cultured in the RCCS for 24 h showed disrupted organization of filamentous actin, increased globular actin level, formation of plasma membrane blebbing and neurite-like artifact, as well as decreased levels of cortactin and vimentin. Interestingly, ∼70% of RCCS-cultured rNCSCs co-expressed cleaved (active) caspase-3 and neuronal markers microtubule-associated protein 2 (MAP2) and Tuj1 instead of NCSC markers, suggesting stress-induced formation of neurite-like artifact in rNCSCs. In addition, rNCSCs showed increased C-X-C chemokine receptor 4 (CXCR4) expression, RhoA GTPase activation, Rho-associated kinase 1 (ROCK1) and p38 mitogen-activated protein kinase (MAPK) phosphorylation, and p53 expression in the nucleus. Incubation of rNCSCs with the Gα protein inhibitor pertussis toxin or CXCR4 siRNA during RCCS-culturing prevented cytoskeleton disorganization and plasma membrane blebbing, and it suppressed apoptosis of rNCSCs. Taken together, we demonstrate for the first time that simulated microgravity disrupts cytoskeleton organization and increases apoptosis of rNCSCs via upregulating CXCR4 expression and the RhoA-ROCK1-p38 MAPK-p53 signaling pathway.

Feasibility Study of Canine Epidermal Neural Crest Stem Cell Transplantation in the Spinal Cords of Dogs.

This pilot feasibility study aimed to determine the outcome of canine epidermal neural crest stem cell (cEPI-NCSC) grafts in the normal spinal cords of healthy bred-for-research dogs. This included developing novel protocols for (a) the ex vivo expansion of cEPI-NCSCs, (b) the delivery of cEPI-NCSCs into the spinal cord, and (c) the labeling of the cells and subsequent tracing of the graft in the live animal by magnetic resonance imaging. A total of four million cEPI-NCSCs were injected into the spinal cord divided in two locations. Differences in locomotion at baseline and post-treatment were evaluated by gait analysis and compared with neurological outcome and behavioral exams. Histopathological analyses of the spinal cords and cEPI-NCSC grafts were performed at 3 weeks post-transplantation. Neurological and gait parameters were minimally affected by the stem cell injection. cEPI-NCSCs survived in the canine spinal cord for the entire period of investigation and did not migrate or proliferate. Subsets of cEPI-NCSCs expressed the neural crest stem cell marker Sox10. There was no detectable expression of markers for glial cells or neurons. The tissue reaction to the cell graft was predominantly vascular in addition to a degree of reactive astrogliosis and microglial activation. In the present study, we demonstrated that cEPI-NCSC grafts survive in the spinal cords of healthy dogs without major adverse effects. They persist locally in the normal spinal cord, may promote angiogenesis and tissue remodeling, and elicit a tissue response that may be beneficial in patients with spinal cord injury. It has been established that mouse and human epidermal neural crest stem cells are somatic multipotent stem cells with proved innovative potential in a mouse model of spinal cord injury (SCI) offering promise of a valid treatment for SCI. Traumatic SCI is a common neurological problem in dogs with marked similarities, clinically and pathologically, to the syndrome in people. For this reason, dogs provide a readily accessible, clinically realistic, spontaneous model for evaluation of epidermal neural crest stem cells therapeutic intervention. The results of this study are expected to give the baseline data for a future clinical trial in dogs with traumatic SCI.

Perivascular-derived stem cells with neural crest characteristics are involved in tendon repair.

Tendons and ligaments exhibit limited regenerative capacity following injury, with damaged tissue being replaced by a fibrotic scar. The physiological role of scar tissue is complex and has been studied extensively. In this study, we demonstrate that rat tendons contain a unique subpopulation of cells exhibiting stem cell characteristics, including clonogenicity, multipotency, and self-renewal capacity. Additionally, these putative stem cells expressed markers consistent with neural crest stem cells (NCSCs). Using immunofluorescent labeling, we identified P75(+) (p75 neurotrophin receptor) cells in the perivascular regions of the native rat tendon. Importantly, P75(+) cells were frequently localized near vascular cells and increased in number within the peritenon after injury. Ultrastructural analysis showed that perivascular cells detached from vessels in response to injury, migrated into the interstitial space, and deposited extracellular matrix. Characterization of P75(+) cells isolated from the scar tissue indicated that this population also expressed the NCSC markers, Vimentin, Sox10, and Snail. In conclusion, our results suggest that neural crest-like stem cells of perivascular origin reside within the rat peritenon and give rise to scar-forming stromal cells following tendon injury.

Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells

Neural crest stem cells (NCSCs), a population of multipotent cells that migrate extensively and give rise to diverse derivatives, including peripheral and enteric neurons and glia, craniofacial cartilage and bone, melanocytes and smooth muscle, have great potential for regenerative medicine. Non-human primates provide optimal models for the development of stem cell therapies. Here, we describe the first derivation of NCSCs from cynomolgus monkey embryonic stem cells (CmESCs) at the neural rosette stage. CmESC-derived neurospheres replated on polyornithine/laminin-coated dishes migrated onto the substrate and showed characteristic expression of NCSC markers, including Sox10, AP2α, Slug, Nestin, p75, and HNK1. CmNCSCs were capable of propagating in an undifferentiated state in vitro as adherent or suspension cultures, and could be subsequently induced to differentiate towards peripheral nervous system lineages (peripheral sympathetic neurons, sensory neurons, and Schwann cells) and mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). CmNCSCs transplanted into developing chick embryos or fetal brains of cynomolgus macaques survived, migrated, and differentiated into progeny consistent with a neural crest identity. Our studies demonstrate that CmNCSCs offer a new tool for investigating neural crest development and neural crest-associated human disease and suggest that this non-human primate model may facilitate tissue engineering and regenerative medicine efforts.

Abstract 2045: Identifying key molecular events at the onset of melanoma using a neural crest stem cell marker

Abstract An ongoing challenge in cancer biology is understanding the earliest, initiating events that switch a premalignant cell into a fully competent cancer cell. Driven by this challenge, we focused on the zebrafish gene crestin that marks the embryonic neural crest from the onset of somitogenesis through 72 hours post fertilization when it normally turns off. Surprisingly, our lab previously showed that crestin mRNA is reexpressed in melanoma tumors in adult zebrafish, reading out a neural crest progenitor state. We have developed a fluorescent reporter using the crestin promoter/enhancer that recapitulates the embryonic expression pattern of crestin, and when bred into our BRAFV600E/p53 mutant melanoma zebrafish model, marks grossly visible melanoma tumors. Remarkably, we found that crestin:EGFP becomes active when lesions are only a few cells in number, potentially in the first cell of the melanoma. Using a crestin:CreERt2 inducible recombinase together with a lox-dependent fluorescent lineage tracer line ubi:switch, multiple neural crest descendant lineages including melanocytes can be traced. Further by expressing the melanocyte lineage master transcription factor mitf under the control of the crestin promoter, we can rescue melanocyte formation in mitf null embryos, and in the Na/BRAFV600E/p53 melanoma-prone mutant background, some adult fish develop darkly pigmented patches and melanoma tumors. Finally, we isolated small patches of crestin:EGFP positive cells and show that these precursor lesions are enriched for expression of the neural crest progenitor transcription factor sox10. These data support a model in which BRAFV600E/p53 mutated melanocytes stochastically reactivate an early melanocyte or neural crest progenitor program as read out by crestin reactivation at the initiation of melanoma formation. Using our novel reporter, we are able to analyze in vivo the earliest steps of cancer formation as well as the neural crest lineage program. Citation Format: Charles K. Kaufman, Christian Mosimann, Richard M. White, Dominic Matos, Leonard I. Zon. Identifying key molecular events at the onset of melanoma using a neural crest stem cell marker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2045. doi:10.1158/1538-7445.AM2014-2045

Feline Coccygeal Teratoma: Immunohistochemical Characterization of Immature Tissue Components

Congenital teratomas arising in the coccygeal region have not been reported in domestic animals. This report describes a congenital coccygeal teratoma in a male Burmese cat. A round to oval expansile mass with solid and cystic areas was found ventral to the 5th to 7th caudal vertebrae. Microscopically, the tumour was composed of derivatives of all three primordial germ layers with neuroectodermal predominance. Immunohistochemical double labelling identified immature tissue components in combination with Sox2, a regulator of stem cell proliferation and differentiation. Few Sox2-positive cells co-expressed the neural crest stem cell markers vimentin and neurotrophin receptor p75(NTR). Not all Sox2- and p75(NTR)-positive cells expressed vimentin. An overlapping expression of vimentin-negative and Sox2-, p75(NTR)-positive cells and GFAP- and p75(NTR)-positive cells may indicate a transition stage from immature to mature non-myelinating Schwann cells. Periaxin-positive myelinating Schwann cells surrounding neurofilament-positive axons were observed. Sox2 was additionally expressed in immature odontogenic epithelial cells and in immature cells of endodermal origin. Sox2 was not observed in mature cells, with the exception of satellite glial cells and mucous glands. Despite the presence of immature tissue components, no recurrence was observed 1 year after surgical removal.