The NIGMS National Center for Biomedical Glycomics, formerly the Integrated Resource Center for Biomedical Glycomics, was established in 2004 to develop and implement new technologies to investigate the Glycome of cells and exploit the use of these technologies to develop embryonic stem cell (ESC) markers during their differentiation. ESC and induced pluripotent stem cell (iPSC) differentiation is our platform for glycomic technology development.
Critical components of the National Center for Biomedical Glycomics (NCBG) are its Analytical Service and Training Programs. From 2008 to mid-2012, for example, the Service Program analyzed over 1000 samples for investigators from across the U.S. and the world, from both academia and industry. In addition, as Glycomics becomes recognized as a fundamental element of biomedical research, pharmaceutical and biotechnology companies, as well as diverse academic laboratories send students for our summer workshops, such as the Mass Spectrometry of Glycans and Glycoconjugates. More information regarding our Service and Training is available by clicking on the buttons in the navigation bar.
Overview of the National Center for Biomedical Glycomics:
Over the past ten years, Glycobiology has emerged as field of study that impacts most areas of mainstream biomedical research. Cell-cell recognition and adhesion, the innate immune response, and cell surface receptor signaling are all areas that have been significantly impacted by discoveriesrelating to the structure and function of specific glycans, the proteins and sphingolipids that express them (glycoconjugates), and the family of proteins that recognize and bind them (lectins). As evidence of the impact of Glycobiology, the U.S. National Academy of Sciences has commissioned a report to be issued this summer entitled, "Assessing the importance and impact of Glycomics and Glycosciences." The acceleration of our knowledge of the critical functions of glycans in biomedicine is due mainly to developments in technology; in particular, increased analytical capabilities to determine glycan structures and comprehensive databases of the genes involved in glycan synthesis and expression of these genes. Our Center has participated in the development of some of these advances and we propose to extend and add additional Aims for technology development in this proposal. Glycomics can be defined in several ways, but in terms of our Center, we define Glycomics as the study of glycans, glycoconjugates, the proteins that regulate their expression, and the proteins that recognize them; in particular, we are interested in developing tools to facilitate learning how glycans and glycoconjugates change during early embryonic development and during the onset of disease as a result of mutations that affect glycan expression, thereby illuminating their specific functions.
The great challenge to the Glycobiology research community is to be able to understand the fine details of glycan expression and its regulation in light of the diversity of glycan structures that could be expressed at any particular glycosylation site on a specific protein, for example. This diversity is also driven by the fact that the "template" for glycosylation not only depends on the expression of particular enzymes and transporters responsible for glycan processing expressed in a cell at a given time, but that the ultrastructure of the cell itself can significantly influence the glycosylation process and the final biosynthetic products. Scheme I is modified from Ohtsubo and Marth and Hart and Copeland  and depicts the biosynthesis of cellular glycans as most of them are on glycoconjugates that transverse the ER and Golgi apparatus, destined for the cell surface, secretion, or the lysosome. Three of the Resource Center's Investigators have recently written an article for Nature Reviewsof Cell and Molecular Biologyon the topic of Glycoprotein Biosynthesis . Scheme 2 demonstrates the expression of glycoconjugates on the surfaces of cells (glycocalyx) via an osmium-stained electron micrograph of a lymphocyte, emphasizing the function of cell surface glycans in cell-cell communication, including adhesion (lymphocyte trafficking), innate immunity (recognition by cell surface lectins such as the Siglecs), and modulation of receptor function (regulation of the surface residence time of the EGF receptor). In addition, the spatial information contained in a typical N-linked glycan is illustrated by a model of erythropoietin and its N-glycan structures using NMR spectroscopic tools and modeling software developed by Robert Woods in the NIGMS Resource Center for Glycotechnology.
To develop technologies that will impact the understanding and regulation of the diversity of glycosylation, therefore, we felt it necessary to focus on Glycomics from more of a Systems Biology approach: investigation of glycosylation in a particular biological context, analysis of the fine details of glycan expression on glycoconjugates produced in specific cell types, identification of the glyco-genes that participate in glycan biosynthesis and the biosynthetic pathways that the enzymes and transporters expressed from the glyco-genes constitute. Furthermore, using a bioinformatics approach, the integration and sharing of this structural and biosynthetic knowledge is beginning to illuminate how glycan expression is regulated and coordinated. The biological context of glycosylation we have chosen to utilize for the development of Glycomic technologies is the differentiation of embryonic stem cells in culture. When we began our Center, there was limited evidence that glycan expression changed as embryonic stem cells (ESC) differentiated in vitro, in ways similar to what had been determined using antibody and lectin binding to study mouse embryonic development.
We began in 2003 by concentrating on the in vitro differentiation of mouse ESC and applying lectin fractionation tools to characterize glycan structures by mass spectrometry. In addition, we explored the development of chip-based and qRT-PCR-based assays for glyco-gene transcript expression. Many problems with this approach became apparent. First, although there was a growing database of enzymes that acted on carbohydrates, CAZy, there was no source that compiled comprehensive glycan biosynthetic pathways and certainly none that assigned Open Reading Frame (ORF) sequences to specific biosynthetic steps. Moreover, the carbohydrate structural databases available were rudimentary and poorly curated (this is still a problem that we have made some progress in solving). From a glycan structural analysis perspective, there was no efficient means to prepare glycans from membrane-bound and soluble, cell-associated glycoproteins and determine their structures in a quantitative way. This was possible using fairly complex mixtures of soluble proteins, however, and several laboratories were working to extend these types of analyses to whole cells.
Since that time, our Center and other researchers have made significant progress in solving these problems. As outlined in the accomplishments below and in individual TR&D, DBP and Collaboration descriptions, we have developed a robust qRT-PCR glycotranscriptome program (TR&D3) to quantify glyco-gene transcripts and have also generated detailed glycan biosynthetic pathways with associated ORF from the CAZy database. Together, these advances have allowed us to take significant steps into quantifying changes in the glycotranscriptome as embryonic stem cells differentiate in culture. Moreover, we have developed a robust means to extract total glycoproteins and glycosphingolipids from whole cells (and tissues) and isolate glycans, N-linked and O-linked, for relative quantitative analysis after permethylation using ESI-LTQ-MSn. Glycosphingolipids are permethylated directly and analyzed similarly. We have also been able to integrate transcript changes and glycan changes for the first steps of mouse embryonic stem cell differentiation. Technology advances have also been made in analysis of glycoprotein-derived glycopeptides for glycosylation site analysis on peptides. Our glycobioinformatics program has developed novel ontologies to store this information and portals to communicate our findings with other laboratories and databases around the world. Many of the analyses are becoming automated in terms of data collection and data analysis by the development of new software programs. The study of embryonic stem cell differentiation itself has also made huge strides-we have now transitioned completely to studying human embryonic stem cells (hESC) which require no "feeder" layers to culture and can be grown in chemically defined media. Moreover, we have developed technologies to differentiate these cells down multiple pathways to form the early cellular precursors to various organs; for example, cardiac myocyte precursor cells and pancreatic endodermal cells. The advances in technologies for the glycan and transcript analyses are now being applied to the glycomes of these differentiated cells, as well as to specific cells and glycoproteins via the DBP and Collaborations associated with the Center.
Fig. 1 below depicts a biantennary N-linked glycan (attached to asparagine) using the Consortium for Functional Glycomics convention for representing glycan structures. This code will be used throughout the application. It is also important to note one of the special problems-or opportunities-that has to be considered when dealing with the analysis of glycans. This problem highlights the challenges for using mass spectrometry to perform glycomic analyses compared to proteomic analyses. Not only is there a great diversity of glycan structures that can be expressed on an asparagine or serine/threonine reside, significant numbers of these structures that can be assigned are isobaric, that is, they share the same mass, just as glucose, galactose, and mannose, common constituents of vertebrate glycans, share the same mass, as do N-acetylglucosamine and N-acetylgalactosamine. Fig.2 illustrates several common isobaric glycan structures.
The Biomedical Glycomics Center was originally conceived to focus on developing tools for the analysis of glycoproteins, glycosphingolipids, and the transcripts of the enzymes that synthesize these glycoconjugates using mouse ESC. For the first renewal in 2007, we expanded the scope of the Resource to the study of human ESC and added two investigators. In the present competitive renewal application, we are adding the expertise of an additional investigator, Dr. Richard Steet, who joined the University of Georgia and the CCRC in 2007 and is highly experienced with research that focuses on the family of disorders known as the Congenital Disorders of Glycosylation (CDG). Researchers studying the CDG have quite limited tools to apply to the understanding of these disorders. We now have proofs-of-concept that technologies developed in our Center by TR&D1 and 2 can be successfully applied to the Glycomics of the CDG; consequently we have expanded out technology development significantly in this area.
The structure of the Resource is focused on four TR&D programs plus Analytical Service and Training (please see diagram below).
TR&D1: Stem Cell and Induced Pluripotent Stem Cell (IPSC) Resources
Senior Investigators Dr. Stephen Dalton and Dr. Richard Steet
TR&D2: Glycomics and Glycoproteomics
Senior Investigators Drs. Lance Wells, Michael Tiemeyer, Ron Orlando, and Michael Pierce
TR&D3: Transcriptome and Glycome Regulation
Senior Investigator Dr. Kelley Moremen
Senior Investigator Dr. William York
Analytical Service and Training: Dr. Parastoo Azadi
RECENT KEY ACCOMPLISHMENTS AND PROGRESS
A detailed Progress Report for each TR&D and Service/Training are imbedded in the section for each program.
TR&D1 Stem Cell and IPSC Resources
TR&D2 Glycomics and Glycoproteomics
- Production of hESC and differentiated cell types: Developed technology to grow, characterize, and distribute human embryonic stem cells (hESC) and cells differentiated from hESC without feeder layers and in defined media (without serum). Typically this program distributes at least 107 of each cell type with rigorous quality control based on flow cytometry for analysis by TR&D2 and 3. A complete list of these cell types is found in the TR&D1 section, but they include definitive endoderm, foregut endoderm, pancreatic endoderm, liver, nascent mesoderm, smooth muscle, epicardium, and cardiomyocytes. The technology to differentiate hESC into most of these differentiated cell types was developed first by TR&D1, and these differentiation events have been optimized to drive down the costs of cell production required for large scale Glycomic analyses.
- Generation of neural crest cells and derivative cell types for the first time: TR&D1 accomplished a major breakthrough (Menendez, et al.) by differentiating hESC into neural crest cells and further into cell types derived from the neural crest. Neural crest cells normally arise in very early embryonic development and give rise to a diverse spectrum of cells that include peripheral neurons, those cells that give rise to facial features-chrondrocytes and osteocytes, plus adipocytes and melanocytes. It was impossible to grow these cell types in culture until now; TR&D1 now routinely grows 107 - 108 of these cells for further analyses by the Center. These cells are also available for distribution to other investigators, separate from our DBP and Collaborations.
- Development of technology to investigate the Glycomics of specific types of a family of diseases known as the Congenital Disorders of Glycosylation (CDG): Many of the phenotypes that are manifest from the CDG are found in differentiated cell types such as peripheral neurons and chondrocytes; yet, the only means now available to study the molecular details of these diseases is to use skin fibroblast biopsies that have been placed in various repositories. TR&D1 has developed a successful protocol to reprogram these primary fibroblasts into induced pluripotent stem cells (iPSC), which can be expanded to large numbers, and then further differentiate the iPSC into neural crest cells and their derivatives. TR&D1 has demonstrated proof-of-concept of this protocol by differentiating fibroblasts from a patient with CDG ST3Gal5 into iPSC and further into neural crest cells and peripheral neurons. This differentiation was required, since the causal mutation in this patient is in the glycosyltransferase that synthesizes a particular glycosphingolipid found in these neurons and not present in the fibroblasts. Indeed, the neurons differentiated from the CDG iPSC and neural crest cells showed a defect in synthesizing the glycosphingolipid (a ganglioside). Glycomics experiments show compensatory changes in both other glycosphingolipids and in glycoprotein glycan biosynthesis in the ST3Gal5 null neurons; for example, there is a large increase in sulfated glycans that may compensate for the loss of sialic acid. In this way, the various CDG can serve as a tool to understand the underlying compensation and coordination of glycan biosynthesis that regulates levels of particular glycans. Based on this successful proof-of-concept, initiated by Dr. Richard Steet who is now a Senior Investigator in TR&D1, we have expanded technology development to other CDG. DBP have been initiated with several human genetic disease programs that study these CDG, and they will serve as repositories for the CDG iPSC.
- Validation of glycotranscriptome analysis to identify functional glycan changes during differentiation: Experiments in collaboration with TR&D2 and 3 have shown that glycotranscriptome analysis can yield promising leads to understand the regulation of specific glycosyltransferases and their function. Significant increases in the enzymes that synthesize polysialic acid on glycoproteins were shown to be upregulated in both mouse and human ESC as they differentiated and lost pluripotency. The glycoprotein shown to express polysialic acid during differentiation is N-CAM, the "neural" cell adhesion molecule, but at a much later stage of development. We demonstrated a critical step of differentiation and loss of pluripotency of hESC was the induction of the PST polysialyltransferase. When the expression of PST is blocked by shRNA expression, differentiation is blocked or greatly retarded, showing that PST expression in both mESC and hESC is required for differentiation and loss of pluripotency.
TR&D3 Transcriptome and Glycome Regulation
- Development of methods for released, permethylated glycan analysis preparedfrom cells: Although many research groups have developed methods to analyze complex mixtures of glycans released from glycoproteins, relatively few had attempted analyses to characterize the glycomes of actual cells. Methods were developed to analyze N- and O-linked glycans released from mouse embryonic stem cells and human embryonic stem cells and cells differentiated from both cell types using newly applied mass spectrometry tools.
- Initial development of automated annotation of MS-based data to yield rapid identification of glycan compositions and structural assignments: The experience with analysis of mouse and human cells and differentiated cell types compelled a collaboration with TR&D4 to develop an automated annotation of MS data such that assignment time post MS decreased from months to a few days. Automated annotation required a database of vetted glycan structures; however, it was quickly obvious that the databases of N- and O-linked glycans available around the world were significantly contaminated with incorrect structures. Building a database of glycan structures vetted by Center investigators who were experienced in glycan structural analysis has yielded a unique database resource that allowed initial attempts at automated annotation of MS data to succeed. This approach will be expanded as an Aim in this application for high throughput glycan analysis.
- Development of tools to analyze site-specific glycoprotein glycosylation: As an example of the development of technologies for glycopeptide analysis, a DBP was developed with TR&D2 in collaboration with Kevin Campbell's laboratory to identify the glycosylation site on alpha-dystroglycan that expresses an O-mannosyl-linked glycan that functions to be bound by laminin. Several forms of muscular dystrophy involve mutations in glycosyltransferases that synthesize this glycan (and are classified as CDG). TR&D2 was able to identify the precise site on the N-terminal portion of alpha-dystroglycan that expresses this glycan critical to nerve-muscle adhesion. The analysis involved using MSn approaches to fragment first glycan structure, followed by peptide in order to identify the site expressing the glycan of interest O-linked to a specific serine residue. This methodology has now been extended with the use of ETD approaches and HCD-triggered ETD approaches that allow the glycan moiety to stay attached to the peptide and that search for glycan oxonium ion, respectively.
- Application and extension of the IDAWG technology to look at dynamics and quantify relative changes of particular cellular glycans: We have developed and applied an in cell culture isotope labeling methodology, termed IDAWG for isotopic detection of amino-sugars with glutamine, that allows us to perform relative quantification between samples (Fig. A). We have used this method to look at changes in glycosylation between hES cells and derived cell types such as definitive endoderm (Fig. B). We have extended this method in a pulse-chase experiment to allow us to look at the dynamics of glycans that allows us to evaluate degradation times as well as remodeling rates.
- Development of a robust glyco-gene qRT-PCR platform: A major accomplishment of the TR&D has been generating and vetting of over 2500 primer sets for qRT-PCR of glyco-genes and particular glycoproteins that are involved in expression of glycans in mouse, human, plus a more limited set in Drosophila and Zebrafish. The ability to quantify transcripts of these genes has resulted in many collaborations/DBP, and the present platform allows linear quantitation over seven orders of magnitude.
- Development and annotation of glycan biosynthetic pathways with open reading frames of specific enzymes: Although there are databases, notably CAZy, annotating ORF of glycan metabolizing enzymes, there was no biosynthetic pathway context to place this information, particularly since most databases involving Glycomics are created and curated by non-glycobiologists. TR&D2 has taken the lead in codifying these biosynthetic pathways and vetting the information that are in databases regarding particular ORF catalyzing specific biosynthetic reactions.Moreover, the integration of the glycotranscriptome data with those of TR&D2 concerning glycan expression differences during mouse and human ESC differentiation has provided one of the first pictures of how glycosylation is globally modulated as embryonic stem cells differentiate.
- Visualization of transcript and glycan expression data in the context of biosynthesis: representations have been and are being developed by TR&D4 (Fig. 4) that allow the display of these data including replicate assays. These representations allow a comparison of transcript and glycan prevalence data that implicate particular nodes of specific biosynthetic pathways that require further investigation by collaborators.
- Refinement of the qRT-PCR platform: Improvements have been made in the volume of sample needed for assay (now down to 5 ?l), and the sample handling has been robotically automated. These advances have brought down the cost of analysis significantly and increased through-put. This platform is now transitioned to be offered through our Service program to interested investigators.
Service, Training, and Dissemination
- Development of a bioinformatics infrastructure for Glycomics: Several ontologies have been developed, which include GlycO that includes knowledge about glycan structure (see Fig. below); ReactO, which holds knowledge about glycan biosynthesis and metabolism; and EnzyO, which holds knowledge about enzymes involved in glycan biosynthesis and metabolism. The Qrator tool that we developed evaluates candidate glycan structures for consistency with biosynthetic mechanisms (see Fig. below) and allowed us to populate GlycO with 889 curated N-glycan structures for use in automating MS data analysis.
- Development of GLYDE, GLYcan Data Exchange format: The new GLYDE II format makes an easier to represent and evaluate biosynthetic reactions of glycans. These improvements have allowed collaborative efforts to develop uniform interfaces for data exchange and database querying via the internet using data modules developed by EurocarbDB, Macquarie University (Australia), the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Consortium for Functional Glycomics, Imperial College (London), and the CCRC.
- Development of GlycoVault, a novel database that supports semantic annotation of Glycomics and glycotranscriptomics data: This database is now available through the Glycomics Portal, along with downloadable tools, Web applications and Web services that can be used to process or interpret Glycomics data (click TR&D4 tab).
- Collaborating with TR&D2 and 3 to develop Glycomic analysis tools: Various software has been developed that has proved essential for analyses by TR&D2 and 3. For example, software tools have been developed (named Similan tools since they interface with the commercial software SimGlycan) that allow automated annotation of mass spectral features with structural assignments of permethylated glycans. Software to allow analysis of aminosugars utilizing metabolic isotopic labeling with glutamine (iDAWG) has proved absolutely essential to this analysis. In addition a transcriptomics workflow application has allowed the population of GlycoVault with transcriptomics data that is annotated with robust meta-data, including biological sample name and sets of genes whose transcription levels were determined.
Driving Biomedical Projects (DBP)
- Service: During the period May 2007-May 2012, our Service program directed by Dr. Azadi analyzed 842 samples and completed 192 projects, compared to 340 samples and 86 projects from 2004-2007.The most requested analysis has been the detailed structural analysis of N and O-linked oligosaccharides by ESI-MS/MS using the LTQ-Orbitrap mass spectrometer. The success of our service is reflected in the increased number of publications from the Service laboratory that has mainly been due to transfer of glycomics technologies from the TR&Ds and improved instrumentation in the service laboratory. The total number of analyses from 2007-2012 was 6843. Four of these service projects have been described in an expanded form and the rest are listed in a Table in Appendix 4. Clients are found across the United States and 13 foreign countries (See the map with US & foreign service clients). There is a regular transfer of technology from the TR&D programs to Service. The most recent addition to Service has been glycotranscriptome analysis, which was transferred from TR&D3.
- The Service program and the individual PIs have also been involved in many collaborative projects that have involved discourse with the client and have required some method developments and substantial individual attention such that they were elevated from simple service to a formal Collaboration. We have had 72 collaborative projects from 2007-2012 six of these collaborations have been described in an expanded form and the rest are listed in Appendix 3.
- Training: Dr. Azadi has developed and administered four "Hands-on" lecture and laboratory courses on: Separation and Characterization of Glycoprotein and Glycolipid Oligosaccharides and Techniques for Characterization of Carbohydrate Structure of Polysaccharides, as well as Mass Spectrometry of Glycoproteins and Structural Characterization of Glycosaminoglycans.The courses emphasize hands-on laboratory experiments, but also include a variety of lectures and demonstrations led by faculty and senior personnel at the CCRC. The Separation and Characterization of Glycoprotein and Glycolipid Oligosaccharides and the Mass Spectrometry of Glycoproteins courses specifically use the technologies developed in the Glycomics Resource TR&Ds. The total number of participants in these hands-on courses, from both academia and industry, for the period 2007-2011 was 245. The list of name and affiliations of all course participants is included in Appendix 5.
- Dissemination: The Glycomics Resource disseminates information about the progress in its technological research and development projects through this website, publications in scientific journals, presentations at conferences and symposia, and by incorporation of new advances into its analytical services, the upcoming training courses, and collaborative research projects.Brochures outlining the different services and training we offer are distributed at scientific conferences. Distribution of these brochures has resulted in requests for services and training as well as requests for advice on methods for working with glycoconjugates. Dr. Azadi is a member of the US Pharmacopeia's Biologics & Biotechnology 1 Expert Committee and the Carbohydrate Committee at the Association of Biomolecular Resource Facilities (ABRF), and as a result she receives requests for current methods of analysis through these committees. A major event to introduce many glycoscientists to technology developed in the Center will center around the Warren Workshop IV, which is being held at the CCRC, Aug 2012. This is a biennial workshop that brings together investigators working on the frontiers of glycan characterization in order to advance the field of glycomics, glycolipidomics, and glycoproteomics. The workshop is designed to facilitate interactions and foster meaningful discussions that will encompass the nuts-and-bolts of recent technological advances, and the ins-and-outs of glycobioinformatics. A number of our senior investigators including Drs. Tiemeyer, Wells, York, Azadi, Ranzinger and Orlando are organizers and session chairs at this workshop.
These DBP are distinguished from collaborations in that they require and their success depends on a close interaction between scientists in the TR&D and those scientists separate from the Center who are participating in the DBP. There are presently 23 of these DBP, and most of them involve more than one TR&D. There are seven, additional DBP which we have chosen to expand their descriptions. These DBP represent specific projects that have had or will have an exceptional impact on technology development in the Center. The DBP section of this proposal consists of a listing of the expanded DBP, a listing of the non-expanded DBP with basic information on their Significance, Innovation, and Approach, followed by a complete listing of all DBP.
These projects are listed, with a total of ten Collaborative projects and Service projects described in more detail. There are a total of 73 collaborative projects for all of the Resource from 2007-May 2012. A full listing of Service projects is included in the Appendix.
Impact of the National Center for Biomedical Glycomics
Our Center lists 30 DBP, 73 Collaborative Projects, 192 Service projects, and four Courses/Workshops that have had 245 participants over the last four years. We list 83 publications resulting from research associated with the Center, including the TR&D, DBP, Collaboration, and Service. We have developed technologies in each of our TR&D that have significantly impacted the biomedical research community, and our over-arching goal is to amplify that impact over the next funding period. The technology to reprogram and differentiate CDG and CMD patient fibroblasts (TR&D1) will broadly impact and support the efforts of the biomedical communityby providing an unprecedented opportunity to study glycosylation-related diseases in relevant specialized cell types that represent the sites of pathology in these patients. Advancements in MS-based glycan analysis (TR&D2) will yield a comprehensive picture on the breadth and quantity of glycan structures within these cell types - information that will be leveraged by the biomedical research community to deepen our understanding of the tissue-specific roles of protein- and lipid-bound glycoconjugates. Further development of transcriptome and RNA Seq tools (TR&D3) adds a discovery-based, technology platform for the research community and will point to novel ways in which pluripotent and differentiated cell types regulate their glycosylation. Finally, the development of state-of-the-art bioinformatics tools (TR&D4) will provide the world-wide biomedical research community with a functional and "user-friendly" platform that can be employed to mine, integrate, analyze, and share Glycomics data.
- Ohtsubo, K. and J.D. Marth, Glycosylation in cellular mechanisms of health and disease. Cell, 2006. 126(5): p. 855-67.
- Hart, G.W. and R.J. Copeland, Glycomics hits the big time. Cell, 2010. 143(5): p. 672-6.
- Moremen, K.W., M. Tiemeyer, and A.V. Nairn, Protein glycosylation: diversity, synthesis, and function Nature Reviews of Cell and Molecular Biology, 2012. in press.
- Menendez, L., et al., Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proc Natl Acad Sci U S A, 2011. 108(48): p. 19240-5.
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