Neural Crest Markers Research Articles

Abstract A22: Differentiation of human embryonic stem cells to sympathetic neurons: A potential model for understanding neuroblastoma pathogenesis

Abstract Background and Aims: Neuroblastoma is an embryonal malignancy derived from neural crest cells which give rise to the sympathetic nervous system (SNS). One of the main therapeutic challenges in neuroblastoma continues to be the emergence of relapsed disease, which occurs in 50% of patients with high risk disease and is associated with resistance to therapy. Our aim was to differentiate human embryonic stem cells (hESCs) to sympathetic neurons (SN) to model normal human SNS development. Results: Using the stromal-derived inducing activity (SDIA) of murine PA6 cells in combination with BMP4 and B27 neuronal supplement, H9 and a newly derived hESC line Ncl(R)14, were induced to differentiate to neural crest stem cells and SN. After 7 days of PA6 cell co-culture, mRNA expression of SNAIL and SOX-9 neural crest specifier genes and the neural marker Peripherin increased. Q-RT-PCR showed that expression of the pluripotency marker OCT 4 decreased, whereas p53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A marked increase in expression of the catecholaminergic marker Tyrosine Hydroxylase (TH) and the noradrenergic marker Dopamine Beta Hydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH and Peripherin. SN were identified by immunofluorescence by co-expression of TH & Peripherin or DBH & PHOX2B in p75+ cells. Live cell analysis and imaging showed increased migration in p75+ cells compared with p75- cells, consistent with a migratory neural crest phenotype in-vitro. Conclusions: We have established a model of nor-adrenergic SNS development using two hESC lines to improve our understanding of normal human SNS development and in future studies the pathogenesis of neuroblastoma. Citation Format: Jane Carr-Wilkinson, Nilendran Prathalingam, Deepali Pal, Helen Forgham, Majlinda Lako, Mary Herbert, Deborah A. Tweddle. Differentiation of human embryonic stem cells to sympathetic neurons: A potential model for understanding neuroblastoma pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A22.

High glucose suppresses embryonic stem cell differentiation into neural lineage cells

Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system.

Differential Neuronal Plasticity of Dental Pulp Stem Cells From Exfoliated Deciduous and Permanent Teeth Towards Dopaminergic Neurons.

Based on early occurrence in chronological age, stem-cells from human exfoliated deciduous teeth (SHED) has been reported to possess better differentiation-potential toward certain cell-lineage in comparison to stem-cells from adult teeth (DPSCs). Whether this same property between them extends for the yield of functional central nervous system neurons is still not evaluated. Hence, we aim to assess the neuronal plasticity of SHED in comparison to DPSCs toward dopaminergic-neurons and further, if the difference is reflected in a differential expression of sonic-hedgehog (SHH)-receptors and basal-expressions of tyrosine-hydroxylase [TH; through cAMP levels]. Human SHED and DPSCs were exposed to midbrain-cues [SHH, fibroblast growth-factor8, and basic fibroblast growth-factor], and their molecular, immunophenotypical, and functional characterization was performed at different time-points of induction. Though SHED and DPSCs spontaneously expressed early-neuronal and neural-crest marker in their naïve state, only SHED expressed a high basal-expression of TH. The upregulation of dopaminergic transcription-factors Nurr1, Engrailed1, and Pitx3 was more pronounced in DPSCs. The yield of TH-expressing cells decreased from 49.8% to 32.16% in SHED while it increased from 8.09% to 77.47% in DPSCs. Dopamine release and intracellular-Ca(2+) influx upon stimulation (KCl and ATP) was higher in induced DPSCs. Significantly lower-expression of SHH-receptors was noted in naïve SHED than DPSCs, which may explain the differential neuronal plasticity. In addition, unlike DPSCs, SHED showed a down-regulation of cyclic adenosine-monophosphate (cAMP) upon exposure to SHH; possibly another contributor to the lesser differentiation-potential. Our data clearly demonstrates for the first time that DPSCs possess superior neuronal plasticity toward dopaminergic-neurons than SHED; influenced by higher SHH-receptor and lower basal TH expression. J. Cell. Physiol. 231: 2048-2063, 2016. © 2016 Wiley Periodicals, Inc.

On the development of extragonadal and gonadal human germ cells.

ABSTRACTHuman germ cells originate in an extragonadal location and have to migrate to colonize the gonadal primordia at around seven weeks of gestation (W7, or five weeks post conception). Many germ cells are lost along the way and should enter apoptosis, but some escape and can give rise to extragonadal germ cell tumors. Due to the common somatic origin of gonads and adrenal cortex, we investigated whether ectopic germ cells were present in the human adrenals. Germ cells expressing DDX4 and/or POU5F1 were present in male and female human adrenals in the first and second trimester. However, in contrast to what has been described in mice, where ‘adrenal’ and ‘ovarian’ germ cells seem to enter meiosis in synchrony, we were unable to observe meiotic entry in human ‘adrenal’ germ cells until W22. By contrast, ‘ovarian’ germ cells at W22 showed a pronounced asynchronous meiotic entry. Interestingly, we observed that immature POU5F1+ germ cells in both first and second trimester ovaries still expressed the neural crest marker TUBB3, reminiscent of their migratory phase. Our findings highlight species-specific differences in early gametogenesis between mice and humans. We report the presence of a population of ectopic germ cells in the human adrenals during development.

Purinergic P2Y Receptors Are Involved in <i>Xenopus</i> Head Formation

P2Y receptors belong to the family of G protein-coupled receptors and are activated by nucleotides in the extracellular space. We showed that Xenopus P2Y1 and P2Y11 were expressed in the dorsal marginal zone from early gastrula stage and enriched in the central nervous system from neurula stages. They were expressed in the prospective head region during early development. Knockdown of P2Y1 and P2Y11 caused head malformation, such as small eyes, brain atrophy, and defect in cartilage tissues, as well as reduced expression of neural, placode, and neural crest markers. Furthermore, the expression of neural plate and epidermal markers was affected by P2Y1 or P2Y11 depletion at early neurula stage, suggesting that P2Y1 or P2Y11 might be required for the neural induction. Our findings suggested that P2Y receptors might be involved in distinguishing between neural and non-neural fates. The results also suggested that P2Y1 or P2Y11 could play a role in neural induction and/or maintenance of neural tissues in the head formation processes.

Expression of neural crest markers by human embryonic stem cells: an introductory project

Abstract Introduction Neural crest cells make up a transient migratory population of cells found in all vertebrate embryos. Great advances have been made over the past 20 years in clarifying the molecular basis of neural crest induction and, although much still remains unclear, it appears that it is a process involving several factors acting at different stages of embryogenesis. In the future, an understanding of the precise mechanisms involved in orofacial development, even at the earliest stages, may well be of use to all clinicians interested in the management of these tissues. Aim The present study was designed to determine if the early addition of noggin (a bone morphogenetic protein (BMP) antagonist) and/or the late addition of BMP4 would increase the expression of the transcription factors: Msx-1, Snail, Slug and Pax-7. Method This involved an assessment of the effects of early addition ( Days 0 to 3) of noggin and/or the late addition (Days 4 to 7) of BMP4 on the expression of the neural crest markers by human embryonic stem cells, co-cultured for eight days on a feeder layer of mouse PA6 cells. Results and conclusions The expression of the neural crest markers Pax-7, Msx-1, Slug, and Snail by human embryonic stem cells is likely to be affected by the addition of noggin and BMP4. Not all of these effects will necessarily be significant. The late addition of BMP4 is likely to significantly increase the expression of Pax-7 by human embryonic stem cells (hESCs), when compared with the effects of co-culturing with stromal cell-derived inducing activity, alone. The early addition of noggin and the late addition of BMP4 are likely to significantly increase the expression of Msx-1 by hESCs, when compared with the late addition of BMP4, alone. The hESC results support those from animal ESC studies that the late addition of BMP4, especially, may result in the differentiation of neural crest precursors.

Histogenesis of cardiac myxoma: the potential role of the cardiac neural crest

Cardiac myxoma is the most common tumor of the heart. The myxoma arises in the region of the interatrial septum and the fossa ovalis, with the left atrium as the most common site of growth. The histogenesis of cardiac myxoma remains unclear. We have previously suggested that a clearer understanding of tumor origins can be achieved through a detailed investigation of heart development (1). In the heart, cardiac neural crest contributes to the septation of the outflow tract, the morphogenesis of the great arteries, and the maturation of the atrioventricular valves and the conduction system (2). We hypothesized that myxoma develops from resident cardiac neural crest cells, via a re-activation of the developmental programs that govern epithelial-mesenchymal transition. Immunohistochemical analysis for canonical markers calretinin, vimentin, α-SMA, CD31, CD34, S100, plus cardiac neural crest markers plexin A2 and semaphorin 3C was performed in one case of sporadic cardiac myxoma. Primary myxoma cell culture was obtained ex vivo via both single cell isolation and outgrowth methods. In vitro confocal microscopy confirms the presence of myxoma cells positive for calretinin, vimentin, α-SMA, CD31, CD34, S100, plexin A2 and semaphorin 3C. Expression of semaphorin 3C and plexin A2 was also confirmed by Western blot analysis. In order to characterize in vivo behaviour of cultured myxoma cells, 2,3x106 cells (cell culture passage 3) were subcutaneously inoculated in interscapular region of immunocompromised mouse. Newly formed tissue was processed for histological and immunohistochemical evaluations. Hematoxylin and eosin staining confirmed the myxoid background of the mass. Despite the presence of CD31-positive human cells, there was no evidence of neovascularization. Our preliminary findings confirm our hypothesis, suggesting that cardiac neural crest in adult heart could be involved in the pathogenesis of cardiac myxoma.

Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells.

Neural crest (NC) cells are a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. Cranial NC cells migrate to the branchial arches and give rise to the majority of the craniofacial region, whereas trunk and tail NC cells contribute to the heart, enteric ganglia of the gut, melanocytes, sympathetic ganglia, and adrenal chromaffin cells. Positional information is indispensable for the regulation of cranial or trunk and tail NC cells. However, the mechanisms underlying the regulation of positional information during human NC induction have yet to be fully elucidated. In the present study, supplementation of bone morphogenetic protein (BMP) 4 in defined serum-free culture conditions including fibroblast growth factor-2 and Wnt3a from day 8 after NC specification induced the expression of cranial NC markers, AP2alpha, MSX1, and DLX1, during NC cell differentiation from human pluripotent stem cells. On the other hand, the proportion of cells expressing p75(NTR) or HNK1 decreased compared with that of cells cultured without BMP4, whereas gene expression analysis demonstrated that the expression levels of cranial NC-associated genes increased in BMP4-treated NC cells. These BMP4-treated NC cells were capable of differentiation into osteocytes and chondrocytes. The results of the present study indicate that BMP4 regulates cranial positioning during NC development.

Abstract 12515: Enhanced Transforming Growth Factor β Signaling Rescues Defective Differentiation of Smooth Muscle Cell in Bicuspid Aortic Valve With Thoracic Aortic Aneurysm

Individuals with bicuspid aortic valves (BAV) are at a high risk to develop a thoracic aortic aneurysms (TAA) than patients with trileaflet aortic valves (TAV), most commonly involving ascending aorta and sparing the descending aorta. Smooth muscle cells (SMCs) in the ascending and descending aorta arise from neural crest and paraxial mesoderm, respectively. We hypothesize the aortopathy associated with BAVs is due to aberrant differentiation of neural crest stem cells (NCSCs) to SMCs. Induced pluripotent stem cell (iPSC) lines were established from a patient with BAV/TAA, and control patients with TAV and a normal aorta. The iPSCs pluripotency was verified by standard stem cell markers and teratoma formation in SCID mice. iPSCs were differentiated into NCSCs and paraxial mesoderm cells (PMCs) separately, and verified by specific neural crest markers and paraxial mesoderm markers. Both NCSCs and PMCs were differentiated into SMCs, NCSC-SMCs and PMC-SMCs, by treating with TGFβ1 and PDGF-bb. With differentiation, both SMCs lines expressed SMC specific markers including MYH11, CNN1, ACTA2, and TAGLN by quantitative RT-PCR and immunohistochemical staining. However, NCSC-derived SMCs from BAV/TAA patient demonstrated significantly decreased expression and protein levels of MYH11 and ACTA2 and impaired contraction by gel contraction assay compared to NCSC-SMCs derived from control. In contrast, the PMC-derived SMCs from BAV/TAAD patient had similar or higher expression and levels of contractile proteins, including MYH11, CNN1, ACTA2, and TAGLN, and contractile response to carbacol treatment when compared with control PMC-SMCs. The defective differentiation of NCSC-SMC from the BAV/TAA patient was coupled with the decreased TGFβ signaling as assessed by decreased gene expression of TGFβ1 and connective tissue growth factor (CTGF) when compared to control cells. Increasing the concentration of TGFβ1, rescued the gene expressions of TGFβ1, CTGF, MYH11 and ACTA2 in the NCSC-SMCs from the BAV/TAA. Taken together, our data demonstrate that defective TGFβ signaling and differentiation of specifically NCSC-derived SMCs contributes to aortopathy in BAV with ascending aortic aneurysm.

Enteric Neural Cells From Hirschsprung Disease Patients Form Ganglia in Autologous Aneuronal Colon.

Background & AimsHirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells.MethodsCells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2′-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed.ResultsENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia.ConclusionsNC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient.

Heterogeneity of neuroblastoma.

Neuroblastoma (NB) is the most common pediatric solid tumor and represents 15% of all childhood cancer deaths. High cellular heterogeneity is a hallmark of NB, which may account for the wide range of clinical presentations and non-uniform response to treatment. Compelling evidence indicates that cancer cells in solid tumors may not necessarily be hierarchically organized, but rather, also following the clonal evolution model in which cancer cells may evolve from one subtype to the others, generating tumors with distinct features. Cancer cells may even transiently acquire stemness properties depending on the tumor context [1–3]. Importantly, these two models are not mutually exclusive in cancers, accounting for the tumor heterogeneity [4]. To date, there is still no defined surface marker available for the identification of tumor initiating cells (TICs) from NB. TICs isolated from the high staged NBs were mainly based on their stem cell characteristics, including sphere-formation capacity and plasticity with respect to differentiation along several lineages [5, 6]. Transcriptome analysis of NB TICs further highlighted the complexity of these cells and suggested that NB TICs may exist as a dynamic and heterogeneous cell population strongly dependent on the particular niche environment [5]. In our recent manuscript by Liu et. al. [7], we made use of various lines of NB-TICs isolated from the primary and bone-marrow metastasized NB, and NB cell lines to generate in vivo models for studying tumor heterogeneity. We discovered that a population of NB cells, which express high level of c-KIT (c-KIThigh), a tyrosine-kinase receptor for stem cell factor (SCF), is de novo generated and dynamically maintained within the tumors to sustain tumor progression. c-KIThigh NB cells possess the stem cell characteristics: they expressed high levels of neural crest and stem cell markers (SLUG, SOX2, NANOG), possess high clonogenic capacity, differentiation plasticity and drug resistance. Even though c-KIT expression is not required for the tumor formation, serial transplantation assays showed that c-KIThigh cells are more aggressive and induce tumors 9-fold more efficiently than c-KIT−/low cells. More importantly, c-KIThigh cells exhibited a long-term in vivo self-renewal capacity to sustain the formation of the secondary and tertiary tumors in mice. All these data support the notion that NB cells change dynamically, they evolve and generate new progenies during tumor progression. The progenies would be more tumorigenic than the parental cells to sustain the tumor progression. Since c-KIT−/low cells can give rise to c-KIThigh cells and vice versa, targeting c-KIT may not be sufficient to effectively eradicate the tumor. Thus, we proposed to target a growth factor, namely Prokineticin, where its receptors are ubiquitously expressed in both c-KIThigh and c-KIT−/low cells and supports the formation and growth of c-KIThigh cells. We showed that knocking down the receptors for Prokineticin provides a more effective way to inhibit the tumor formation than directly targeting c-KIT, by abolishing the de novo generation of c-KIThigh cells. In summary, we have employed NB-TIC lines isolated from primary and bone-marrow metastasized tumors to establish an in vivo cancer model for understanding how NB TICs give rise to heterogeneous derivatives and how these derivatives are organized and influence disease progression or treatment response. We also provided evidence that targeting the growth factor which supports the formation of moving target represents an effective way to eradicate NB. Obviously, NB is highly heterogeneous, comprising cancer cells with very different molecular features. The next important questions are to understand how the clonal composition of a tumor influences its progression and treatment response, how the molecular features determine the behavior of tumor cells, and what are the common features or molecular signatures shared by the most aggressive tumor cells of various tumors. NB-TIC derived tumor model would be a useful platform for understanding tumor formation, progression and tumor heterogeneity. Recent advances in single cell sequencing technology allow a precise examination of the expression profile of the heterogeneous cancer populations at a single cell level. Comparisons of the molecular signatures of the tumor cells derived from various NB-TICs will allow a systematic analysis of heterogeneous populations of a tumor and help identify candidate genes for future drug intervention. Nevertheless, the most formidable challenge of the field is to know which population of cancer cells within the individual tumor we should target: TIC or its most aggressive progenies, in order to translate our knowledge of cancer biology to establish the personalized treatment strategy for the patients.

Neural crest specification by Prohibitin1 depends on transcriptional regulation of prl3 and vangl1.

A complex network of transcription factors regulates specification of neural crest cells at early neurula stage by stabilizing neural crest identity and activating neural crest effector genes so that distinct subpopulations evolve. In this network, c-myc acts on top of the gene hierarchy controlling snail2, AP2 and prohibitin1 (phb1) expression. While snail2 and AP2 are well studied neural crest specifier genes little is known about the role of phb1 in this process. To identify phb1 regulated genes we analyzed the transcriptome of neural crest explants of phb1 morphant Xenopus embryos. Among 147 phb1 regulated genes we identified the membrane-associated protein-tyrosine phosphatase PRP4A3 (prl3) and the atypical cadherin and Wnt-PCP component van gogh like1 (vangl1). Gain of function, loss of function and epistasis experiments allowed us to allocate both genes in the neural crest specification network between phb1 and twist. Interestingly, both, vangl1 and prl3 regulate only a small subset of neural crest marker genes. The identification of two membrane-associated proteins as novel neural crest specifiers indicates that in addition to gene regulation by combinatory effects of transcription factors also post-translational modifications (prl3) and cell-cell adhesion and/or regulation of cell-polarity (vangl1) specify the identity of neural crest cell populations. genesis 53:627-639, 2015. © 2015 Wiley Periodicals, Inc.

Mechanisms of cadmium-caused eye hypoplasia and hypopigmentation in zebrafish embryos

Cadmium-caused head and eye hypoplasia and hypopigmentation has been recognized for a long time, but knowledge of the underlying mechanisms is limited. In this study, we found that high mortality occurred in exposed embryos after 24hpf, when cadmium (Cd) dosage was above 17.8μM. Using high-throughput in situ hybridization screening, we found that genes labelling the neural crest and its derivative pigment cells exhibited obviously reduced expression in Cd-exposed embryos from 24hpf, 2 days earlier than head and eye hypoplasia and hypopigmentation occurred. Moreover, based on expression of crestin, a neural crest marker, we found that embryos before the gastrula stage were more sensitive to cadmium toxicity and that damage caused by Cd on embryogenesis was dosage dependent. In addition, by phenotype observation and detection of neural crest and pigment cell markers, we found that BIO and retinoic acid (RA) could neutralize the toxic effects of Cd on zebrafish embryogenesis. In this study, we first determined that Cd blocked the formation of the neural crest and inhibited specification of pigment cells, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in Cd-exposed embryos. Moreover, we found that compounds BIO or RA could neutralize the toxic effects of Cd.

Abstract 2682: Melanoma phenotype switching to adapt to BRAF inhibition

Abstract Melanoma can adapt to several stress factors, such as TNF treatment, by changing its phenotype from a highly differentiated (MITF and MART-1 positive) to undifferentiated (neural crest markers) states. We studied this phenomenon as a mechanism of adaptive resistance to BRAF inhibition since melanomas with both MITF high and low have been reported to result in BRAF inhibitor resistance. We treated 17 BRAFV600E mutant melanoma cell lines with the BRAF inhibitor vemurafenib for short term (3 days) or long term (17 days), and compared them to no treatment or TNF treated conditions. We profiled them phenotypically for MART1 antigen (differentiated phenotype) and nerve growth factor receptor (NGFR, neural crest phenotype) expression by flow cytometry. Results comparing median fluorescence intensity (MFI) showed a wide spectrum of adaptive responses to short term BRAF inhibition, followed by solidification or further evolution of these changes upon long term exposure and development of drug tolerance. According to each cell line distribution, we calculated a (x,y) value that organized the cell lines and conditions in four quadrants according to MART1 (x axis) or NGFR (y axis) expression. Two double negative (MART1-/NGFR-) cell lines that were unaffected by treatment and stayed in the same quadrant. Seven MART1-/NGFR+ and four MART1+/NGFR- cell lines slightly changed phenotype with vemurafenib exposure but stayed in the same quadrant. Four cell lines with MART1+/NGFR+ phenotype were greatly influenced by treatment, showing wide plasticity with cells shifting towards NGFR+/MART1- and others towards NGFR-/MART1+. Changes in position between baseline and tolerance to vemurafenib after 17days treatment were numerically quantified as an inferred number from plasticity. Cell lines with double positive (MART1+/NGFR+) phenotype had a significant change in plasticity compared to the rest of cell lines in other quadrants (p = 0.0004). This arranges cells in a four-quadrant pattern that could be likened to a U-turn toward de-differentiation. Cells that de-differentiated with BRAF inhibition mimicked the effect induced by TNF. This effect was driven by a decrease in MITF activity confirmed with a tyrosinase promoter-luciferase assay. In conclusion, baseline melanoma differentiation phenotypes guide the phenotype switch response, with those double positive for differentiation and neural crest markers being at a semi-stable state that can be prone to different plastic changes upon BRAF inhibition. Citation Format: Lidia Robert, Jennifer Tsoi, Angel Garica Diaz, Blanca Homet Moreno, Nhat Truong, Thomas Graeber, Antoni Ribas. Melanoma phenotype switching to adapt to BRAF inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2682. doi:10.1158/1538-7445.AM2015-2682

Abstract 1508: UV-induced Wnt7a in the human skin microenvironment specifies the fate of neural crest -like cells via suppression of Notch

Abstract Multipotent stem cells with neural crest-like properties have been identified in the dermis of human skin. These neural crest stem cell (NCSC)-like cells display self-renewal capacity and differentiate into neural crest derivatives including epidermal pigment-producing melanocytes. NCSC-like cells share many properties with aggressive melanoma cells, such as high migratory capabilities and expression of the neural crest markers. However, little is known about which intrinsic or extrinsic signals determine the proliferation or differentiation of these neural crest-like stem cells. Here, we show that in NCSC-like cells, Notch signaling is highly activated, similar to melanoma cells. Inhibition of Notch signaling reduced proliferation of NCSC-like cells, induced cell death, and down-regulated non-canonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells. In three-dimensional skin reconstructs, canonical Wnt signaling promoted the differentiation of NCSC-like cells into melanocytes. This differentiation was triggered by the endogenous Notch inhibitor Numb, which is up-regulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes. Together, these data reveal a novel crosstalk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells. Citation Format: Mizuho Fukunaga-Kalabis, Denitsa Hristova, Joshua Wang, Ling Li, Markus Heppt, Zhi Wei, Alexandra Gyurdieva, Marie Webster, Masahiro Oka, Ashani Weeraratna, Meenhard Herlyn. UV-induced Wnt7a in the human skin microenvironment specifies the fate of neural crest -like cells via suppression of Notch. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1508. doi:10.1158/1538-7445.AM2015-1508

Abstract A13: MSX1-induced neural crest-like reprograming promotes melanoma progression

Abstract Melanoma cells share many biological properties with neural crest cells. Here, we show that the homeodomain transcription factor Msh homeobox 1 (MSX1), which is essential for neural crest specification, reprograms melanocytes towards a neural crest precursor-like state. MSX1-reprogrammed melanocytes express the neural crest marker p75 and are able to differentiate into neuronal and mesenchymal lineages. Mechanistically, MSX1 suppresses the proximal promoter of microphthalmia-associated transcription factor (MITF), the master transcriptional regulator of melanogenesis. MSX1 expression is also significantly correlated with melanoma progression. MSX1 prompts melanoma cell motility and depletion of MSX1 significantly inhibits melanoma metastasis. These results demonstrate that not only can neural crest-like reprogramming in melanocytes be achieved by a single factor, but also that similar dedifferentiation is a critical process for melanoma progression. Citation Format: Markus V. Heppt, Wang X. Joshua, Denitsa M. Hristova, Zhi Wei, Martin Irmler, Carola Berking, Robert Besch, Johannes Beckers, Frank J. Rauscher, III, David E. Fisher, Meenhard Herlyn, Mizuho Fukunaga-Kalabis. MSX1-induced neural crest-like reprograming promotes melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A13.

Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection

The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells. Therefore, they can be potential cell sources for tooth regeneration. However, these previous methods still have problems, such as usage of other cell types, heterogeneity of differentiated cells, and tumorigenicity. In the present study, a novel method to differentiate iPS cells into odontoblast-like cells without tumorigenicity using gene transfection was established. It is an important advance in the establishment of efficient methods to generate homogeneous functional odontogenic cells derived from iPS cells.

UV-Induced Wnt7a in the Human Skin Microenvironment Specifies the Fate of Neural Crest–Like Cells via Suppression of Notch

Multipotent stem cells with neural crest-like properties have been identified in the dermis of human skin. These neural crest stem cell (NCSC)-like cells display self-renewal capacity and differentiate into neural crest derivatives, including epidermal pigment-producing melanocytes. NCSC-like cells share many properties with aggressive melanoma cells, such as high migratory capabilities and expression of the neural crest markers. However, little is known about which intrinsic or extrinsic signals determine the proliferation or differentiation of these neural crest-like stem cells. Here, we show that in NCSC-like cells, Notch signaling is highly activated, similar to melanoma cells. Inhibition of Notch signaling reduced proliferation of NCSC-like cells, induced cell death, and down-regulated non-canonical Wnt5a, suggesting that the Notch pathway contributes to the maintenance and motility of these stem cells. In three-dimensional skin reconstructs, canonical Wnt signaling promoted the differentiation of NCSC-like cells into melanocytes. This differentiation was triggered by the endogenous Notch inhibitor Numb, which is up-regulated in the stem cells by Wnt7a derived from UV-irradiated keratinocytes. Together, these data reveal a crosstalk between the two conserved developmental pathways in postnatal human skin, and highlight the role of the skin microenvironment in specifying the fate of stem cells.

Abstract 679: Modeling Thoracic Aortic Aneurysm of Bicuspid Aortic Valve Patients with Induced Pluripotent Stem Cells Reveals Distinct Phenotypes of Smooth Muscle Cells from Different LIneages

Ascending aortic aneurysm occurs in 35%-68% patients with bicuspid aortic valves (BAV), which is more frequently than it does in patients with normal trileaflet aortic valves (TAV). In contrast to recurrent aneurysms of the ascending aorta, descending aortic aneurysms is very rare in patients with BAV. Vascular Smooth Muscle Cells (SMCs) make up the media of the aortic walls, and their contractile properties help sustain vessel tone. Interestingly, ascending aorta is populated by SMCs arising from neural crest and descending aorta by SMCs from paraxial mesoderm. An intriguing hypothesis is that the distinct susceptibility of aneurysm in the ascending and descending aorta in patients with BAV is due in part to disparities in SMC embryonic origins. To prove our hypothesis, we used an induced Pluripotent Stem Cells (iPS) model system to generate SMCs from neural crest and paraxial mesoderm. Firstly, iPSCs were established from a patient with BAV and ascending thoracic aortic aneurysm (BAV/TAA) and two control patients with normal aorta and TAV. The iPSCs pluripotency was verified by standard stem cell markers and teratoma formation. Neural crest stem cells (NCSCs) were differentiated from iPSCs and expressed neural crest markers, including P75, HNK1, AP2 and Sox9. In parallel, paraxial mesoderm cells (PMCs) were differentiated from iPSCs and expressed specific markers, including TCF15, TBX6, Meox1 and Pax1. The SMCs differentiated from both lineages had expression of SMC markers including MYH11, CNN1, ACTA2, SM22α. Interestingly, SMCs derived from BAV-NCSCs had significantly decreased in MYH11 and ACTA2 gene expression and impaired contractile property by gel contraction assay compared to SMCs derived from control-NCSCs. However, SMCs from BAV-PMCs had similar level of expression of MYH11 and ACTA2, and strong contractile activity with Carbacol treatment compared to SMCs derived from control PMCs. In conclusion, the iPS model system revealed decreased expression of contractile proteins and impairment of contractility in SMCs derived from NCSCs in BAV patient, but not in the SMCs derived from paraxial mesoderm cells. The high susceptibility of aneurysm in the ascending aorta in patients with BAV might be partly due to embryonic origin of the SMCs.

Sox9 regulates development of neural crest and otic placode in a time- and dose-dependent fashion

Neural crest and placodes share a number of important features, pointing to a possible common evolutionary origin. They both arise from the neural plate border, which is the boundary between the non-neural ectoderm and neural plate. The transcription factor Sox9 has been implicated in neural crest and otic placode induction in several species. To investigate the differential regulation of neural crest and otic placode induction by Sox9, a gain of function assay was performed using a hormone-inducible version of the Sox9 construct at different doses and time periods. Sox9 was expressed in both neural crest and otic placode cell populations in the same stage embryos by in situ hybridization. Using a gain of function approach, increased expression of neural crest marker (Snail2) and otic placode marker (Pax8) in Sox9-overexpressed embryos was observed. Higher dose of Sox9 reduced or eliminated both neural crest and placode cells in the embryos. Interestingly, otic placodes cells were more strongly affected as compared to neural crest cells. So, optimal dosage and timing of Sox9 expression are important for the development of the neural crest and otic placode. The development of the neural crest and otic placode are affected by Sox9 in a time- and dose-dependent manner.