Latest Articles Include:
- Conflicts and collaborations
- Nat Biotech 28(11):1133 (2010)
Nature Biotechnology | Editorial Conflicts and collaborations Journal name:Nature BiotechnologyVolume: 28 ,Page:1133Year published:(2010)DOI:doi:10.1038/nbt1110-1133Published online05 November 2010 Claims of conflicts of interest concerning authorship of a scientific paper highlight the difficulties facing regulators participating in collaborations with industry. View full text Additional data - Novartis eyes oral MS drug as potential blockbuster
- Nat Biotech 28(11):1135-1136 (2010)
The first oral medication to treat multiple sclerosis (MS) has been given a regulatory green light after getting an overwhelmingly positive recommendation by an advisory panel three months earlier. In September, the US Food & Drug Administration (FDA) approved Novartis's Gilenya (fingolimod, also called FTY720) as a first-line treatment for people with the relapsing form of MS. - Bristol-Myers Squibb reaps biologics in ZymoGenetics windfall
- Nat Biotech 28(11):1137-1138 (2010)
Following a $885 million buyout announced on 7 October, Bristol-Myers Squibb (BMS) of New York now has added Seattle-based ZymoGenetics to its biologics portfolio. Some critics have chided ZymoGenetics' board of directors for selling the company too cheaply to the pharmaceutical giant, which last year also acquired antibody pioneer Medarex and its rich pipeline of antibody products. - Shire's replacement enzymes validate gene activation
- Nat Biotech 28(11):1139-1140 (2010)
Genzyme's manufacturing strife and the urgent search for alternatives to meet patients' needs have propelled Shire and its gene-activation technology to the fore. When Genzyme's Allston Landing facility was shut down in 2009 after the discovery of viral contamination, the US Food and Drug Administration (FDA) requested Chineham, UK–based Shire's help in maintaining enzyme supplies for Fabry and Gaucher patients, prompting the company to accelerate its manufacturing timeline for VPRIV (velaglucerase alfa, glucocerebrosidase) by 18 months. - Pharmacogenomics row
- Nat Biotech 28(11):1139 (2010)
A new US government–sponsored report on three pharma-cogenetic tests for targeted cancer treatments has confirmed the usefulness of KRAS testing but raised doubts about two other widely adopted tests. The Agency for HealthCare Research and Quality (AHCRQ) commissioned the report at the request of the Centers for Medicare and Medicaid Services (CMS) to help set clinical guidelines and reimbursement policies. - Joint inspections still cool
- Nat Biotech 28(11):1140 (2010)
Regulatory agencies on both sides of the Atlantic, the European Medicines Agency (EMA) and US Food and Drug Administration (FDA), are urging companies to apply to its joint good manufacturing practice (GMP) inspections because, since its launch in August 2009, the program has had a slow uptake. The regulators aim to increase the number of sites inspected and avoid duplication. - Stimulus trickle
- Nat Biotech 28(11):1140 (2010)
Private biotech companies have received only a small fraction of the $10 billion from the American Recovery and Reinvestment Act (ARRA) of 2009 funds intended for biomedical research. In fiscal year 2010 the National Health Institute awarded $196 million dollars of stimulus funding to for-profit organizations, representing 4. - Transgenic salmon inches toward finish line
- Nat Biotech 28(11):1141-1142 (2010)
A fast-growing Atlantic salmon developed by AquaBounty Technologies is poised to become the first transgenic animal to enter the food chain. After a ten-year wait, officials at the US Food and Drug Administration (FDA) reviewed the transgenic fish owned by the Waltham, Massachusetts company. - CIRM spurs translation
- Nat Biotech 28(11):1141 (2010)
As the first US Food and Drug Administration–approved clinical trial of human embryonic stem (hES) cells gets underway, the California Institute of Regenerative Medicine in San Francisco (CIRM) is pushing forward with a second round of translational grants. The first round of Disease Team Research Awards, expected recipients to have an approvable investigational new drug (IND) application ready to file within four years. - Irish bait
- Nat Biotech 28(11):1141 (2010)
The Irish government expects to lure venture capital (VC) firms to its shores with a 500 ($693) million fund to boost investment in local startups. Innovation Fund Ireland will focus on biotech, information technology, medical devices and cleantech. - Sanofi/Genzyme hostile
- Nat Biotech 28(11):1142 (2010)
Its efforts to acquire Genzyme rebuffed in August, Sanofi-aventis has begun a hostile tender offer for the Cambridge, Massachusetts, biotech, for the $69 per share ($18.5 billion) it originally offered. - Adverse-events fraud trial
- Nat Biotech 28(11):1142 (2010)
A company's failure to disclose nonstatistically adverse clinical data does not constitute fraud argues BayBio, the San Francisco–based biotech company association, in an amicus brief submitted to the US Supreme Court. In Matrixx Initiatives, Inc. et al. v. James Siracusano et al - Newsmaker: Anaphore
- Nat Biotech 28(11):1143 (2010)
Nature Biotechnology | News Newsmaker: Anaphore * Jennifer Rohn1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume: 28 ,Page:1143Year published:(2010)DOI:doi:10.1038/nbt1110-1143Published online05 November 2010 This protein engineering firm claims its therapies, modeled on the naturally secreted human serum protein tetranectin, could compete with antibodies. View full text Additional data Affiliations * London * Jennifer Rohn - Biotech rallies in Q3
- Nat Biotech 28(11):1144 (2010)
Biotech stocks stormed back with the rest of the markets last quarter. Although funding for public biotechs was $9. - When patients march in
- Nat Biotech 28(11):1145-1148 (2010)
Nature Biotechnology | News | News Feature When patients march in * Virginia Hughes1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume: 28 ,Pages:1145–1148Year published:(2010)DOI:doi:10.1038/nbt1110-1145Published online05 November 2010 Impatient with the slow pace of clinical research, families of individuals suffering from untreatable diseases are taking matters into their own hands—with some success. Virginia Hughes reports. View full text Additional data Affiliations * Brooklyn, New York * Virginia Hughes - Making the leap into entrepreneurship
- Nat Biotech 28(11):1149-1151 (2010)
- Grant management skills are critical for young scientists
- Nat Biotech 28(11):1152-1153 (2010)
In a commentary last April, Singhvi & Sachdev1 communicate several skills fundamental for career development of young scientists. Although this article was based upon views from a limited pool of post-docs and faculty at Rockefeller University, New York, it offers a valuable analysis of the universal training challenges faced in preparing scientists to embark upon a successful career, and I sincerely hope it will inspire similar interactive discussion at all research training institutions. - Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins
- Nat Biotech 28(11):1153-1156 (2010)
Nature Biotechnology | Opinion and Comment | Correspondence Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins * Carlos J Bosques1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brian E Collins1 Search for this author in: * NPG journals * PubMed * Google Scholar * James W Meador III1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hetal Sarvaiya1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer L Murphy1 Search for this author in: * NPG journals * PubMed * Google Scholar * Guy DelloRusso1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dorota A Bulik1 Search for this author in: * NPG journals * PubMed * Google Scholar * I-Hsuan Hsu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nathaniel Washburn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra F Sipsey1 Search for this author in: * NPG journals * PubMed * Google Scholar * James R Myette1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rahul Raman2 Search for this author in: * NPG journals * PubMed * Google Scholar * Zachary Shriver2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ram Sasisekharan2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ganesh Venkataraman1ganesh@momentapharma.com Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1153–1156Year published:(2010)DOI:doi:10.1038/nbt1110-1153Published online05 November 2010 To the Editor: Chinese hamster ovary (CHO) cells are widely used for the manufacture of biotherapeutics, in part because of their ability to produce proteins with desirable properties, including 'human-like' glycosylation profiles. For biotherapeutics production, control of glycosylation is critical because it has a profound effect on protein function, including half-life and efficacy. Additionally, specific glycan structures may adversely affect their safety profile. For example, the terminal galactose-α-1,3-galactose (α-Gal) antigen can react with circulating anti α-Gal antibodies present in most individuals1. It is now understood that murine cell lines, such as SP2 or NSO, typical manufacturing cell lines for biotherapeutics, contain the necessary biosynthetic machinery to produce proteins containing α-Gal epitopes2, 3, 4. Furthermore, the majority of adverse clinical events associated with an induced IgE-mediated anaphylaxis response in patients treated with the commercial antibody! Erbitux (cetuximab) manufactured in a murine myeloma cell line have been attributed to the presence of the α-Gal moiety4. Even so, it is generally accepted that CHO cells lack the biosynthetic machinery to synthesize glycoproteins with α-Gal antigens5. Contrary to this assumption, we report here the identification of the CHO ortholog of N-acetyllactosaminide 3-α-galactosyltransferase-1, which is responsible for the synthesis of the α-Gal epitope. We find that the enzyme product of this CHO gene is active and that glycosylated protein products produced in CHO contain the signature α-Gal antigen because of the action of this enzyme. Furthermore, characterizing the commercial therapeutic protein abatacept (Orencia) manufactured in CHO cell lines, we also identified the presence of α-Gal. Finally, we find that the presence of the α-Gal epitope likely arises during clonal selection because different subclonal populations from the same parental cell line differ in their e! xpression of this gene. Although the specific levels of α-Gal! required to trigger anaphylaxis reactions are not known and are likely product specific, the fact that humans contain high levels of circulating anti-α-Gal antibodies suggests that minimizing (or at least controlling) the levels of these epitopes during biotherapeutics development may be beneficial to patients. Furthermore, the approaches described here to monitor α-Gal levels may prove useful in industry for the surveillance and control of α-Gal levels during protein manufacture. View full text Figures at a glance * Figure 1: Structural complex of CHO Ggta1 exons 8–9 gene product with UDP-2F-Gal donor and LacNAc acceptor substrate. () Shown in stereo view is the cartoon representation of the enzyme active site with the side chains of the key residues labeled using the single amino acid code and the numbering based on the bovine α3GalT crystal structure (PDB: 1G93). The UDP-2F-Gal is shown in stick representation in green and the type II LacNAc acceptor substrate is shown in stick representation in yellow. The location of the Mn2+ cation (in purple) is obtained from the bovine α3GalT crystal structure template used to construct the homology model. () Sequence alignment of the various α3GalT with the highly conserved critical active site residues highlighted in gray. * Figure 2: MS/MS fragmentation analysis of N-glycan species observed in abatacept. (–) Comparison of the MS/MS fragmentation profile between an N-glycan species with a neutral mass of 2,070 Da observed in Orencia () and the fragmentation profile of an isobaric hybrid species () and an isobaric α-Gal–containing species () from our N-glycan fragmentation database. Nomenclature for glycan structures: blue squares, GlcNAc; red triangles, fucose; green circles, mannose; yellow circles, galactose. The additional galactose is depicted in the lower branch to simplify the fragmentation nomenclature. Fragmentation nomenclature is based on ref. 10. Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Momenta Pharmaceuticals, Cambridge, Massachusetts, USA. * Carlos J Bosques, * Brian E Collins, * James W Meador III, * Hetal Sarvaiya, * Jennifer L Murphy, * Guy DelloRusso, * Dorota A Bulik, * I-Hsuan Hsu, * Nathaniel Washburn, * Sandra F Sipsey, * James R Myette & * Ganesh Venkataraman * Harvard-MIT Division of Health Sciences and Technology, Koch Institute for Integrative Cancer Research, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Rahul Raman, * Zachary Shriver & * Ram Sasisekharan Competing financial interests Momenta Pharmaceuticals is a biotechnology company specializing in the detailed characterization and engineering of complex drugs including glycoprotein therapeutics. Momenta currently does not have marketed protein products but has products in development (http://www.momentapharma.com/). Contributing authors from Momenta (all except R.R., Z.S. & R.S.) hold stocks and stock options in the company. Corresponding author Correspondence to: * Ganesh Venkataraman (ganesh@momentapharma.com) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (800K) Supplementary Figs. 1–5, Supplementary Table 1 and Supplementary Methods Additional data - A policy approach to the development of molecular diagnostic tests
- Nat Biotech 28(11):1157-1159 (2010)
Nature Biotechnology | Opinion and Comment | Commentary A policy approach to the development of molecular diagnostic tests * Kevin A Schulman1kevin.schulman@duke.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Sean R Tunis2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1157–1159Year published:(2010)DOI:doi:10.1038/nbt1110-1157Published online05 November 2010 Efficiently generating evidence of clinical utility is a major challenge for ensuring clinical adoption of valuable diagnostics. A new approach to reimbursement in the United States offers a balance between evidence and incentives for molecular diagnostic tests. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Kevin A. Schulman is at the Duke Clinical Research Institute, Duke University School of Medicine and the Health Sector Management Program, The Fuqua School of Business, Duke University, Durham, North Carolina, USA. * Sean R. Tunis is at the Center for Medical Technology Policy, Baltimore, Maryland, USA. Competing financial interests K.A.S. received research support from Actelion Pharmaceuticals, Allergan, Amgen, Astellas Pharma, Bristol-Myers Squibb, The Duke Endowment, Genentech, Inspire Pharmaceuticals, Johnson & Johnson, Kureha Corporation, LifeMasters Supported SelfCare, Medtronic, Merck & Co., Nabi Biopharmaceuticals, National Patient Advocate Foundation, North Carolina Biotechnology Center, NovaCardia, Novartis, OSI Eyetech, Pfizer, Sanofi-aventis, Scios, Tengion, Theravance, Thomson Healthcare and Vertex Pharmaceuticals; personal income for consulting from McKinsey & Company and the National Pharmaceutical Council; had equity in Alnylam Pharmaceuticals; had equity in and served on the board of directors of Cancer Consultants, Inc; and had equity in and serving on the executive board of Faculty Connection. K.A.S. has made available online a detailed listing of financial disclosures (http://www.dcri.duke.edu/research/coi.jsp). S.R.T. is an employee of the Center for Medical Technology Policy (CMPT)! , a nonprofit policy and research center that works on issues of comparative effectiveness research and coverage with evidence development. CMPT receives funding from a range of health plans, life sciences companies, foundations and government grants. Corresponding author Correspondence to: * Kevin A Schulman (kevin.schulman@duke.edu) Additional data - What is the value of oncology medicines?
- Nat Biotech 28(11):1160-1163 (2010)
Nature Biotechnology | Opinion and Comment | Commentary What is the value of oncology medicines? * Joshua Cohen1 Search for this author in: * NPG journals * PubMed * Google Scholar * William Looney2 Search for this author in: * NPG journals * PubMed * Google Scholar * AffiliationsJournal name:Nature BiotechnologyVolume: 28 ,Pages:1160–1163Year published:(2010)DOI:doi:10.1038/nbt1110-1160Published online05 November 2010 Coverage with evidence development (CED), rather than quality-adjusted-life-year (QALY) thresholds, offers the best way forward in balancing evidence-based policy for new oncology products with the needs of developers, payers, physicians and patients. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Joshua Cohen is at Tufts University School of Medicine, Center for the Study of Drug Development, Boston, Massachusetts, USA * William Looney is at Pfizer, New York, New York, USA. Competing financial interests The authors declare no competing financial interests. Additional data - What's fueling the biotech engine—2009–2010
- Nat Biotech 28(11):1165-1171 (2010)
Nature Biotechnology | Feature What's fueling the biotech engine—2009–2010 * Saurabh Aggarwal1saurabhaggarwal@gmail.com Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature BiotechnologyVolume: 28 ,Pages:1165–1171Year published:(2010)DOI:doi:10.1038/nbt1110-1165Published online05 November 2010 Last year, the biologics sector managed single-digit growth in the United States, driven mainly by products indicated for oncology, diabetes and autoimmune disorders. Lurking on the horizon, though, are challenges, such as pricing, competition and follow-on molecules. View full text Figures at a glance * Figure 1: Growth trends in the US biotech market for biologic drugs (2005–2009). () Total sales and growth rate trends. () Quarterly sales growth for biologic drugs (2005–Q2, 2010). * Figure 2: Top companies that comprise the majority of sales of biologic drugs in 2009. The pie chart shows the fraction of total biotech sales of the top 17 companies. The table shows the annual growth rates of the top ten companies. Red boxes indicate companies that had biologics sales growth >15%. For the purpose of this analysis, Rituxan US sales have been split equally between Genentech and Biogen Idec; Erbitux US sales were split 40/60 between Imclone and BMS. * Figure 3: Top nine categories of biologic drugs in terms of US sales in 2009. The pie chart shows US sales of these drug categories. The table shows the growth rates of the categories between 2008 and 2009. The red boxes indicate the major categories showing the fastest growth rate during that period. For therapeutic enzymes, their manufacturers do not break out the US sales, so their sales were estimated assuming 20–30% of worldwide sales were generated in the United States. * Figure 4: Trends in US sales of mAbs. () 2009 sales for US markets for mAbs ($ billions). 'Other' includes all mAbs with sales <$300 million per year. () Trends in US sales show Remicade leading, Humira rising and Herceptin lagging. * Figure 5: Trends in US sales of growth factors. () 2009 sales in US market for growth factors ($ billions). () Trends in US sales show Neulasta and Epogen leading with Aranesp lagging. * Figure 6: Trends in US sales of recombinant hormones. () 2009 sales in US market for recombinant hormones ($ billions). () Trends in US sales show Lantus to be the leader, Novolog a rising star and Levemir new on the block. 'Other' includes all hormones with sales of <$200 million per year. * Figure 7: US sales of cytokines and therapeutic enzymes ($ billions). () Cytokines sales, showing the top four brands. () Therapeutic enzyme sales, showing the top three brands. 'Other' includes all drugs with sales of <$200 million per year. * Figure 8: US sales of recombinant vaccines, blood factors and anticoagulants in ($ billions). () Recombinant vaccines, dominated by one brand. () Blood factors dominated by two brands. () Recombinant anticoagulants market, which is dominated by two brands. 'Other' includes all drugs with sales of <$50 million per year. Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Saurabh Aggarwal is a healthcare consultant based in New York. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Saurabh Aggarwal (saurabhaggarwal@gmail.com) Additional data - A shadow falls over gene patents in the United States and Europe
- Nat Biotech 28(11):1172-1173 (2010)
Nature Biotechnology | Feature | Patents A shadow falls over gene patents in the United States and Europe * Gareth Morgan1lisa.haile@dlapiper.com Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa A. Haile1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1172–1173Year published:(2010)DOI:doi:10.1038/nbt1110-1172Published online05 November 2010 Will a pair of court decisions that restrict the protection offered to DNA-based claims reduce financial incentives and thus chill investment? View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Gareth Morgan and Lisa A. Haile are at DLA Piper LLP, London, UK. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gareth Morgan (gareth.morgan@dlapiper.com; lisa.haile@dlapiper.com) Additional data - Recent patent applications in biological imaging
- Nat Biotech 28(11):1174 (2010)
Table 1 - Plant natural products from cultured multipotent cells
- Nat Biotech 28(11):1175-1176 (2010)
Cultured cambial meristematic cells could enable large-scale production of certain natural products. - Making antibodies from scratch
- Nat Biotech 28(11):1176-1178 (2010)
Synthesis and screening of a small library of antibody fragments yields promising hits. - Toward global RNA structure analysis
- Nat Biotech 28(11):1178-1179 (2010)
Deep sequencing provides a first view of the RNA structures in a eukaryotic transcriptome. - Research highlights
- Nat Biotech 28(11):1180 (2010)
- In silico research in the era of cloud computing
- Nat Biotech 28(11):1181-1185 (2010)
Nature Biotechnology | Computational Biology | Commentary In silico research in the era of cloud computing * Joel T Dudley1 Search for this author in: * NPG journals * PubMed * Google Scholar * Atul J Butte1abutte@stanford.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1181–1185Year published:(2010)DOI:doi:10.1038/nbt1110-1181Published online05 November 2010 Snapshots of computer systems that are stored and shared 'in the cloud' could make computational analyses more reproducible. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Joel T. Dudley and Atul J. Butte are in the Division of Systems Medicine, Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA, and at the Lucile Packard Children's Hospital, Palo Alto, California, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Atul J Butte (abutte@stanford.edu) Additional data - Directed differentiation of human embryonic stem cells toward chondrocytes
- Nat Biotech 28(11):1187-1194 (2010)
Nature Biotechnology | Research | Article Directed differentiation of human embryonic stem cells toward chondrocytes * Rachel A Oldershaw1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Melissa A Baxter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Emma T Lowe1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicola Bates1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa M Grady1 Search for this author in: * NPG journals * PubMed * Google Scholar * Francesca Soncin3 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel R Brison1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Timothy E Hardingham1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Susan J Kimber1sue.kimber@manchester.ac.uk Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1187–1194Year published:(2010)DOI:doi:10.1038/nbt.1683Received25 March 2010Accepted23 August 2010Published online22 October 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95–97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replace! ment therapies for cartilage repair. View full text Figures at a glance * Figure 1: Schematic of directed differentiation protocol in three stages. In stage 1, pluripotent hESCs are directed toward a primitive streak–mesendoderm population; in stage 2, differentiation proceeds to a mesoderm population; and in stage 3, toward chondrocytes. As some genes are expressed in different cell lineages and at different stages, the developmental status of each cell population was characterized by expression of panels of marker genes including SOX2, which is expressed by both pluripotent hESCs and cells derived from the neurectoderm germ layer, CDH1, expressed on pluripotent and mesendoderm cells and CXCR4, used to identify cell lineages from both the endoderm and mesodermal-derived hemangioblast. * Figure 2: Morphology of hESC cultures (HUES1) at different stages of the protocol. (,) Pluripotent hESCs on a mouse embryonic fibroblast (MEF) feeder layer. Cell cultures were heterogeneous with hESCs forming individual, tightly packed colonies. (,) Pluripotent hESCs cultured on a fibronectin matrix in a defined medium. hESCs appeared as a homogeneous 2D monolayer, with individual cells appearing larger than those in colonies maintained on feeder layers. The cells had characteristic hESC morphology, with a high nucleus-to-cytoplasm ratio and prominent nucleoli. FF-hESC; feeder-free hESC. (,) At the end of stage 1, cells were ~80% confluent and still retained morphological features of pluripotent hESCs. (,) At stage 2, cell cultures were densely packed, with phase-bright cell clusters distributed throughout the culture (circled). These clusters formed 3D nodules with cells clustered into a 'rosette-like' morphology (circled). (,) During stage 3 (day 12), the flatter cells surrounding the 3D cell aggregates began to detach, leaving behind the 3D cell aggrega! tes (circled). Cultures are on fibronectin-gelatin substrate and were imaged immediately before passaging onto gelatin as detailed in Table 1. (,) By end of stage 3 (day 14), intermediate cells between the cell aggregates had also become detached. All scale bars, 100 μm. * Figure 3: Gene expression analysis of hESCs at different stages of the protocol. Pluripotent hESCs (HUES1; gray bars) and differentiating cultures at the end of stage 1 (day 4), stage 2 (day 9) and stage 3 (day 14) (black bars) were analyzed for gene expression, as denoted in Figure 1. (–) Genes associated with hESC pluripotency. (–) Genes expressed by cell lineages from the primitive streak–mesendoderm. (–) Genes expressed by mesodermal cell types. (–) Genes expressed by endoderm cell types. (–) Genes expressed by neurectodermal cell types (also in ). (–) Genes expressed by chondrocytes. hESCs transiently expressed genes associated with a primitive streak–mesendoderm phenotype, before expression of genes associated with mesodermal cell lineages. Embryoid body–derived spontaneous differentiation cultures (SDC; hatched bars) taken at day 14 were used as a positive control to confirm the specificity of the primers used and to verify the pluripotent phenotype of the original hESC cultures. Gene expression was normalized to GAPDH. Values re! present means ± s.e.m. (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 4: Sulfated glycosaminoglycan accumulation during directed differentiation of hESCs (HUES1) to chondrocytes. (,) Cell clusters that formed throughout stage 2 cultures stained discretely with safranin O at day 9, indicating the accumulation of sGAG. (,) At stage 3, independent cell aggregates were larger than the cell clusters in stage 2 and at day 14 showed markedly stronger staining for safranin O. The frequency of cell clusters staining for safranin O in stage 2 cultures and cell aggregates in stage 3 cultures was determined as described in Online Methods. Values represent means ± s.e.m. (n = 4). (e–h) To demonstrate the specificity of safranin O staining for sGAG, some cultures were treated with chondroitinase ABC before staining. Control stage 3 cultures before () and after () staining with safranin O. Stage 3 cultures treated with chondroitinase ABC before () and after () staining with safranin O. All scale bars, 100 μm. () Quantification of sGAG production per cell at stage 2 and 3. Values represent means ± s.e.m. (n = 4). *P < 0.05. * Figure 5: Immunofluorescence of SOX9 and collagen II. Proteins were indirectly labeled with secondary Alexa Fluor 488 antibodies (green channel) and cell nuclei labeled with DAPI (blue channel). (–) Expression of chondrogenic transcription factor SOX9 was low in pluripotent hESC cultures (HUES1). (–) At the end of the differentiation protocol (day 14), SOX9 was highly expressed and SOX9 protein was localized within the nuclei of aggregated cells. (–) Phase images and immunofluorescence of collagen II deposited within the matrix of cell aggregates at the end of the differentiation protocol. Scale bar, 100 μm. * Figure 6: Flow cytometry analyses of HUES1-derived cells at the end of stage 3 (day 14). (–) Cells were analyzed for expression of the chondrocyte transcription factor SOX9 (,), the cell surface receptor CD44 (,) and the adult stem cell surface antigen CD105 (,). Immunological control () for SOX-9. The proportion of SOX9-expressing cells () was 74.8% ± 3.1. Immunological control () for CD44. The proportion of CD44-expressing cells () was 34.1%. ± 2.1. Immunological control () for CD105. The proportion of CD105-expressing cells () was 12.7% ± 2.6. Values represent means ± s.e.m. (n = 4) FL-2 indicates exclusion gate for flow cytometry analysis. Author information * Abstract * Author information * Supplementary information Affiliations * North West Embryonic Stem Cell Centre, Faculty of Life Sciences, Core Technology Facility, University of Manchester, Manchester, UK. * Rachel A Oldershaw, * Melissa A Baxter, * Emma T Lowe, * Nicola Bates, * Lisa M Grady, * Daniel R Brison, * Timothy E Hardingham & * Susan J Kimber * Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, UK. * Emma T Lowe & * Timothy E Hardingham * Faculty of Medical and Human Sciences, Core Technology Facility, University of Manchester, Manchester, UK. * Francesca Soncin * Department of Reproductive Medicine, Saint Mary's Hospital for Women and Children, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK. * Daniel R Brison * Present address: North East England Stem Cell Institute, Institute of Cellular Medicine, Newcastle University, International Centre for Life Bioscience Centre, Newcastle-Upon-Tyne, UK. * Rachel A Oldershaw Contributions R.A.O., D.R.B., T.E.H. and S.J.K. were responsible for study concept and design, analysis and interpretation of data and preparation of the manuscript. R.A.O., M.A.B., E.T.L., N.B., F.S. and L.M.G. were responsible for the acquisition of data. Competing financial interests R.A.O., D.R.B., T.E.H. and S.J.K. are named inventors on UK Intellectual Property Office patent applications GB 1012495.6 (filed 26 July 2010) and GB 1012559.9 (filed 27 July 2010). Corresponding author Correspondence to: * Susan J Kimber (sue.kimber@manchester.ac.uk) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Tables 1,2 and Supplementary Figs. 1–8 Additional data - Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization
- Nat Biotech 28(11):1195-1202 (2010)
Nature Biotechnology | Research | Article Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization * Hongyuan Mao1 Search for this author in: * NPG journals * PubMed * Google Scholar * James J Graziano1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tyson M A Chase1 Search for this author in: * NPG journals * PubMed * Google Scholar * Cornelia A Bentley1 Search for this author in: * NPG journals * PubMed * Google Scholar * Omar A Bazirgan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Neil P Reddy1 Search for this author in: * NPG journals * PubMed * Google Scholar * Byeong Doo Song1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Vaughn V Smider1, 2vvsmider@scripps.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1195–1202Year published:(2010)DOI:doi:10.1038/nbt.1694Received24 June 2010Accepted27 September 2010Published online24 October 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ~10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity inform! ation at the first step of the discovery process, and could enable novel antibody discovery in functional screens. View full text Figures at a glance * Figure 1: A spatially addressed antibody library for discovery and optimization. () Schematic of spatially addressed combinatorial library design and generation. () Schematic of screening and optimization. * Figure 2: Screening and identifying hits using ECL detection. () Antigen map of the ten-spot 96-well plate used in the screen. () Examples of four hits identified from ECL detection: F0001 against DLL4, F0003 against ERBB2/Fc, F0032 against EPOR/Fc and F0079 against HGFR/Fc. Quantified ECL signals are listed in Supplementary Table 1. () Titration of F0002 confirms its binding affinity and specificity to DLL4. Serial dilutions of F0002 were tested (ECL image is inset), and the resulting signal for each of the ten spots is graphed as a function of F0002 concentration. * Figure 3: Affinity maturation of F0002 binding to DLL4. () SAR analysis of F0002. The ECL image of F0002 (well C9) and a closely related 'non-hit' (well C8) from the initial screen is shown. Below is the sequence alignment of their heavy chain CDR3s (residues 96 to 112). Both Fabs share the same light chain and a similar heavy chain, the only differences are six amino acids within the CDR3 of their heavy chains. () ELISA titration of F0002 and its affinity-matured mutants binding to DLL4. Error bars, s.d. (n = 3). (). ECL titration of F0002 and affinity-matured mutants binding to DLL4. * Figure 4: Epitope mapping the anti-DLL4 Fabs binding sites on the DLL4 extracellular domain. () ECL-based competition binding assay. Competition between the binding of Ru-labeled M0008 and increased concentrations of either unlabeled M0022 or M0031 to DLL4, quantified by ECL. Error bars, s.d. (n = 2). () Mapping of DLL4 binding region of M0026 and M0035 using an unreduced western blot. A series of DLL4 extracellular domain truncations are depicted in the cartoon diagram, where the DSL domain is highlighted as the open rectangular box; and the EGF-like domains 1 to 8 are highlighted as the shaded rectangular boxes labeled as 1, 2, etc. Anti-DLL4 binding observed in the westerns is marked as +, and no binding is marked as −. * Figure 5: Assays of anti-DLL4 antibodies in cell-based assays. (–) FACS profiles of M0026, M0035 Fabs and NOTCH1-Fc binding to CHO-DLL4 cells. M0026 and M0035 binding was detected using phycoerythrin-labeled anti-human kappa IgGs (PE-α-kappa), phycoerythrin-labeled anti-human lambda IgG (PE-α-lambda), respectively. Biotinylated NOTCH1-Fc binding to CHO-DLL4 was detected with phycoerythrin-labeled streptavidin (PE-strep). () Anti-DLL4 Fab competition of NOTCH1/Fc binding to CHO-DLL4 cells using FACS. () Inhibition of NOTCH1 luciferase reporter by anti-DLL4 antibodies. CHO-DLL4 activation of NOTCH1 reporter in T98G cells is inhibited by both M0026 and M0035 Fabs and IgGs in a dose-dependent manner. A control nonbinding Fab (F1001) has no effect on NOTCH1 reporter levels. Shown is background-subtracted counts per second (CPS) with error bars, s. e. (n = 4). Author information * Abstract * Author information * Supplementary information Affiliations * Fabrus LLC, La Jolla, California, USA. * Hongyuan Mao, * James J Graziano, * Tyson M A Chase, * Cornelia A Bentley, * Omar A Bazirgan, * Neil P Reddy, * Byeong Doo Song & * Vaughn V Smider * The Scripps Research Institute, La Jolla, California, USA. * Vaughn V Smider * Present address: Scripps Korea Antibody Institute, Chuncheon-si, Korea. * Byeong Doo Song Contributions H.M. designed and constructed the plasmid vectors for heavy and light chain library; performed Fab library generation; designed and executed affinity maturations, including alanine-scanning mutagenesis, NNK mutagenesis and cassette mutagenesis, of F0001 and F0002; performed a small subset of library screening using ECL; conducted ECL based epitope mapping competition assay. H.M. also designed the cloning strategy for constructing the VH3-23 library with all possible germline D-J combinations, together with T.M.A.C., J.J.G., C.A.B., O.A.B. and N.P.R. made the VH3-23 library. J.J.G. performed the automated expression and purification on Piccolo. J.J.G. and V.V.S. designed the software for generating the V(D)J recombinant sequences and selecting the representative sequences for gene synthesis. T.M.A.C. performed the majority of the library screening using ECL and performed ELISA on the affinity matured Fabs with DLL4. C.A.B. performed the Fab binding assays on CHO-DLL4 cells us! ing FACS and executed inhibition assays of NOTCH1-DLL4 interaction using ELISA and FACS. C.A.B. and O.A.B. designed and performed the Luciferase reporter assays on inhibition of NOTCH1-DLL4 interaction. N.P.R. made all DLL4 extracellular domain constructs and executed the epitope mapping with western blots, generated CHO-DLL4 cell line and helped H.M. for Fab library transformation. B.D.S. also supervised the construction of DLL4 extracellular domains. O.A.B. made the NOTCH1 reporter plasmid (p6xCBF), which was modified from an earlier reporter plasmid (p4XCBF) made by B.D.S. O.A.B. also constructed the full length IgG eukaryotic expression vectors and expressed and purified the IgGs. V.V.S. conceptualized the spatially addressed antibody library and oversaw the concept development at Fabrus. V.V.S. and H.M. wrote the manuscript. All authors discussed and commented on the manuscript. Competing financial interests The authors own equity interests in Fabrus LLC. Corresponding author Correspondence to: * Vaughn V Smider (vvsmider@scripps.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Tables 1–4 and Supplementary Figs. 1–3 Additional data - Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization
- Nat Biotech 28(11):1203-1207 (2010)
Nature Biotechnology | Research | Letter Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization * Tomoyuki Igawa1igawatmy@chugai-pharm.co.jp Search for this author in: * NPG journals * PubMed * Google Scholar * Shinya Ishii1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tatsuhiko Tachibana1 Search for this author in: * NPG journals * PubMed * Google Scholar * Atsuhiko Maeda1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshinobu Higuchi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shin Shimaoka1 Search for this author in: * NPG journals * PubMed * Google Scholar * Chifumi Moriyama1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tomoyuki Watanabe1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ryoko Takubo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshiaki Doi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tetsuya Wakabayashi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Akira Hayasaka1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shoujiro Kadono1 Search for this author in: * NPG journals * PubMed * Google Scholar * Takuya Miyazaki1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kenta Haraya1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yasuo Sekimori1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tetsuo Kojima1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshiaki Nabuchi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshinori Aso1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshiki Kawabe1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kunihiro Hattori1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1203–1207Year published:(2010)DOI:doi:10.1038/nbt.1691Received31 August 2010Accepted23 September 2010Published online17 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra)1, an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R2 suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the intera! ctions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses. View full text Figures at a glance * Figure 1: Surface plasmon resonance (SPR) sensorgrams of tocilizumab (TCZ), two variants with pH-dependent binding to hsIL-6R (PH1, PH2), TCZ and PH2 with increased affinity to FcRn (TCZ-FcRn, PH2-FcRn) and an affinity matured variant with increased affinity to FcRn (AM-FcRn). (–) SPR measurements of hsIL-6R association (3 min) at pH 7.4 and dissociation (30 min) at pH 7.4 (pH 7.4–pH7.4; green), and hsIL-6R association at pH 7.4 (3 min) and dissociation at pH 6.0 (30 min) (pH 7.4–pH 6.0; red) are shown for TCZ (), PH1 (), PH2 (), TCZ-FcRn (), PH2-FcRn () and AM-FcRn (). * Figure 2: In vivo characterization of pH-dependent binding variants in mice. (,) In vivo study of TCZ, PH1 and PH2 in hIL-6R transgenic mice. TCZ, PH1 and PH2 were administered intravenously at single doses of 25 mg/kg. 4 μg/kg of hIL-6 was intraperitoneally administered at 48 h, 72 h and 96 h for TCZ, PH1, PH2 and hIL-6 control (IL-6 + vehicle) group. Vehicle group was injected with buffer, instead of antibody and hIL-6 (vehicle + vehicle). The concentration of SAA was measured as a marker of neutralization of hIL-6R21. Time profiles of plasma antibody concentration () and plasma SAA concentration () are shown. Each data point represents the mean ± s.d. for antibody concentration and mean ± s.e.m. for SAA concentration (n = 3–4 each). (,) In vivo study of TCZ and PH2 in normal mice. hsIL-6R, TCZ + hsIL-6R and PH2 + hsIL-6R were intravenously administered at single doses of 50 μg/kg for hsIL-6R and 1 mg/kg for antibody. Time profiles of plasma antibody concentration () and plasma hsIL-6R concentration () are shown. Each data point represents th! e mean ± s.d. (n = 3 each). * Figure 3: In vivo characterization of pH-dependent binding variants in cynomolgus monkeys. (,) In vivo study of TCZ, TCZ-FcRn, PH2-FcRn and AM-FcRn in cynomolgus monkeys. TCZ, TCZ-FcRn, PH2-FcRn and AM-FcRn were intravenously administered at single doses of 1 mg/kg. TCZ-FcRn, PH2-FcRn and AM-FcRn were engineered to improve binding to FcRn. 5 μg/kg of cynomolgus IL-6 was subcutaneously administered daily from day 3 to day 10 for TCZ, from day 3 to day 11 for TCZ-FcRn and from day 6 to day 18 for PH2-FcRn and AM-FcRn. The concentration of CRP was measured as a marker of neutralization of IL-6R. Time profiles of plasma antibody concentration () and plasma CRP concentration () are shown. Each data point represents the mean ± s.d. for antibody concentration and mean ± s.e.m. for CRP concentration (n = 3–4 each). (,) In vivo study of TCZ and PH2-FcRn in cynomolgus monkeys. TCZ, PH2-FcRn and vehicle were subcutaneously administered at single doses of 2 mg/kg. 5 μg/kg of cynomolgus IL-6 was subcutaneously administered every other day from day 5 to day 27. CRP concen! tration was measured as a marker of neutralization of IL-6R. Time profiles of plasma antibody concentration () and plasma CRP concentration () are shown. Each data point represents the mean ± s.d. for antibody concentration and mean ± s.e.m. for CRP concentration (n = 2–5 each). Time points when cynomolgus monkey anti-TCZ antibodies or anti-PH2-FcRn antibodies were detected in the plasma were not used for calculating mean concentration. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Entrez Nucleotide * AB591055 * AB591062 Author information * Accession codes * Author information * Supplementary information Affiliations * Chugai Pharmaceutical Co. Ltd., Fuji-Gotemba Research Laboratories, Shizuoka, Japan. * Tomoyuki Igawa, * Shinya Ishii, * Tatsuhiko Tachibana, * Atsuhiko Maeda, * Yoshinobu Higuchi, * Shin Shimaoka, * Chifumi Moriyama, * Tomoyuki Watanabe, * Ryoko Takubo, * Yoshiaki Doi, * Tetsuya Wakabayashi, * Akira Hayasaka, * Shoujiro Kadono, * Takuya Miyazaki, * Kenta Haraya, * Yasuo Sekimori, * Tetsuo Kojima, * Yoshiaki Nabuchi, * Yoshinori Aso, * Yoshiki Kawabe & * Kunihiro Hattori Contributions T.I. led the overall pH-dependent binding antibody program, designed experiments, generated tocilizumab variants and wrote the manuscript. S.I. and A.M. generated tocilizumab variants. T.T., R.T., Y.H. and K. Haraya performed in vivo studies. S.S. led the anti-IL-6R antibody program. C.M. and A.H. performed affinity analysis of tocilizumab variants. T. Watanabe performed in vitro studies of tocilizumab variants. Y.D. and T. Wakabayashi performed purification of tocilizumab variants. S.K. and T.M. provided structural information for designing tocilizumab variants. Y.S., T.K., Y.N., Y.A., Y.K., and K. Hattori provided direction and guidance for the various functional areas. Competing financial interests The authors are employees of Chugai Pharmaceutical Co. Ltd. Corresponding author Correspondence to: * Tomoyuki Igawa (igawatmy@chugai-pharm.co.jp) Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (620K) Supplementary Tables 1–6 and Supplementary Figs. 1–4 Additional data - Programmable in situ amplification for multiplexed imaging of mRNA expression
- Nat Biotech 28(11):1208-1212 (2010)
Nature Biotechnology | Research | Letter Programmable in situ amplification for multiplexed imaging of mRNA expression * Harry M T Choi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Joann Y Chang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Le A Trinh2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer E Padilla1 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott E Fraser1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Niles A Pierce1, 3niles@caltech.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1208–1212Year published:(2010)DOI:doi:10.1038/nbt.1692Received28 June 2010Accepted24 September 2010Published online31 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In situ hybridization methods enable the mapping of mRNA expression within intact biological samples1, 2. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos3, 4, 5, 6, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR)7. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed w! hole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization. View full text Figures at a glance * Figure 1: Multiplexed in situ hybridization using fluorescent HCR in situ amplification. () HCR mechanism. Metastable fluorescent RNA hairpins self-assemble into fluorescent amplification polymers upon detection of a specific RNA initiator. Initiator I nucleates with hairpin H1 via base pairing to single-stranded toehold '1', mediating a branch migration30 that opens the hairpin to form complex I·H1 containing single-stranded segment '3*-2*'. This complex nucleates with hairpin H2 by means of base pairing to toehold '3', mediating a branch migration that opens the hairpin to form complex I·H1·H2 containing single-stranded segment '2*-1*'. Thus, the initiator sequence is regenerated, providing the basis for a chain reaction of alternating H1 and H2 polymerization steps. Red stars denote fluorophores. () Validation in a test tube. Agarose gel demonstrating orthogonal amplification in a reaction volume containing four HCR amplifiers and zebrafish total RNA. Minimal leakage from metastable states is observed in the absence of initiators. () Detection stage. Probe! sets are hybridized to mRNA targets and then unused probes are washed from the sample. () Amplification stage. Initiators trigger self-assembly of tethered fluorescent amplification polymers and then unused hairpins are washed from the sample. () Experimental timeline. The same two-stage protocol is used independent of the number of target mRNAs. For multiplexed experiments (three-color example depicted), probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal HCR amplification cascades labeled by spectrally distinct fluorophores. * Figure 2: Validation of fluorescent HCR in situ amplification in fixed whole-mount zebrafish embryos. (–) The target is the transgenic transcript Tg(flk1:egfp), expressed below the notochord and between the somites (see the expression atlas of Fig. 3a). Embryo morphology is depicted by autofluorescence in the gray channel. Probe set: 1 RNA probe. Fluorescent staining (green channel) using in situ HCR in Target+ () and Target− () embryos compared to (green channel) autofluorescence in the absence of probes and hairpins (). No amplification in the absence of probes () or of one hairpin species (,). Modification of hairpin stem sequences (H1′, H2′) disrupts (,) and restores () toehold-mediated branch migration, confirming that staining arises from triggered polymerization rather than from random aggregation of hairpins. Typical for zebrafish, the yolk sack (bottom left of each panel) often exhibits autofluorescence. (–) Characterizing the signal-to-background ratio for fluorescent HCR in situ amplification. The target is a muscle gene transcript (desm) expressed in th! e somites. Embryo morphology is depicted by autofluorescence in the gray channel. Pixel intensity histograms are calculated using the green channel. WT embryos. Probe set: three RNA probes, except panel . () Sample penetration with in situ HCR: probes and hairpins penetrate the sample before executing triggered self-assembly of tethered amplification polymers in situ. () Sample penetration with ex situ HCR: probes trigger self-assembly of amplification polymers before penetrating the sample. () Background and signal contributions. Histograms of pixel intensity are plotted for a rectangle partially within the expression region and partially outside the expression region (e.g., ,). Background arises from three sources: autofluorescence (AF; buffer only), nonspecific amplification (NSA; hairpins only); nonspecific detection (NSD; in situ HCR amplification after detection of absent target Tg(flk1:egfp)). NSD studies use a probe set of three RNA probes targeting transgenic trans! cript Tg(flk1:egfp), which is absent from the WT embryo. () Mu! ltiple probes per mRNA target. Comparison of autofluorescence and in situ HCR using probe sets with 1, 3 or 9 RNA probes (compare curves of the same color). The microscope photomultiplier tube gain was decreased as the size of the probe set increased to avoid saturating pixels in the images using in situ HCR amplification (this accounts for the reduction in AF intensity as the size of the probe set increases). Embryos fixed at 25 h.p.f. Scale bar, 50 μm. * Figure 3: Multiplexed imaging in fixed whole-mount and cross-sectioned zebrafish embryos. () Expression atlas for five target mRNAs (lateral view: Tg(flk1:egfp), tpm3, elavl3, ntla, sox10). () mRNA expression imaged using confocal microscopy at four planes within an embryo. This multiplexed experiment is performed using the same two-stage protocol that is used for single-color experiments (summarized in Fig. 1c–e). Detection is performed using five probe sets carrying orthogonal initiators. The probe sets have different numbers of RNA probes (10,7,18,30,20) based on the strength of expression of each mRNA target and the strength of the autofluorescence in each channel. Amplification is performed using five orthogonal HCR amplifiers carrying spectrally distinct fluorophores. () Expression atlas for five target mRNAs (anterior view). () mRNA expression imaged within a 200-μm zebrafish section using confocal microscopy. Vibratome sectioning was performed after HCR in situ amplification and post-fixation. See also the image stacks of Supplementary Movies 1 and 2. ! Embryos fixed at 27 h.p.f. Scale bars, 50 μm. * Figure 4: Sharp signal localization and co-localization in fixed whole-mount zebrafish embryos. Redundant two-color mapping of one target mRNA expressed predominantly in the somites (desm; two probe sets, two HCR amplifiers, channels 1 and 2) simultaneous with redundant two-color mapping of a second target mRNA expressed predominantly in the interstices of somites (Tg(flk1:egfp): two probe sets, two HCR amplifiers, channels 3 and 4). () Sharp co-localization of desm signal (Pearson correlation coefficient, r = 0.93). () Sharp co-localization of Tg(flk1:egfp) signal (Pearson correlation coefficient, r = 0.97). () Sharp signal localization within the two interleaved expression regions. The interstice between somites is only the width of a single stretched cell. Embryos fixed at 27 h.p.f. Scale bars, 10 μm. Author information * Author information * Supplementary information Affiliations * Department of Bioengineering, California Institute of Technology, Pasadena, California, USA. * Harry M T Choi, * Joann Y Chang, * Jennifer E Padilla, * Scott E Fraser & * Niles A Pierce * Department of Biology, California Institute of Technology, Pasadena, California, USA. * Le A Trinh & * Scott E Fraser * Department of Applied & Computational Mathematics, California Institute of Technology, Pasadena, California, USA. * Niles A Pierce Contributions S.E.F. and N.A.P. conceived the application of HCR to multiplexed bioimaging; H.M.T.C., J.Y.C., J.E.P. and N.A.P. engineered HCR hairpins for use in stringent hybridization buffers; H.M.T.C. and N.A.P. designed the experiments; H.M.T.C. performed the experiments; L.A.T. selected targets, provided technical guidance and performed the control experiments using traditional in situ hybridization; H.M.T.C., L.A.T., S.E.F. and N.A.P. analyzed the data; H.M.T.C. and N.A.P. wrote the manuscript; and all authors edited the manuscript. Competing financial interests The authors declare competing financial interests in the form of US patents and pending US and EU patents. Corresponding author Correspondence to: * Niles A Pierce (niles@caltech.edu) Supplementary information * Author information * Supplementary information Movies * Supplementary Movie 1 (8M) Image stack for Figure 3b * Supplementary Movie 2 (4M) Image stack for Figure 3d PDF files * Supplementary Text and Figures (23M) Supplementary Notes Additional data - Cultured cambial meristematic cells as a source of plant natural products
- Nat Biotech 28(11):1213-1217 (2010)
Nature Biotechnology | Research | Letter Cultured cambial meristematic cells as a source of plant natural products * Eun-Kyong Lee1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Young-Woo Jin1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Joong Hyun Park1 Search for this author in: * NPG journals * PubMed * Google Scholar * Young Mi Yoo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sun Mi Hong1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rabia Amir2 Search for this author in: * NPG journals * PubMed * Google Scholar * Zejun Yan2 Search for this author in: * NPG journals * PubMed * Google Scholar * Eunjung Kwon2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Alistair Elfick3 Search for this author in: * NPG journals * PubMed * Google Scholar * Simon Tomlinson4 Search for this author in: * NPG journals * PubMed * Google Scholar * Florian Halbritter4 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Waibel2 Search for this author in: * NPG journals * PubMed * Google Scholar * Byung-Wook Yun2 Search for this author in: * NPG journals * PubMed * Google Scholar * Gary J Loake2gloake@ed.ac.uk Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature BiotechnologyVolume: 28 ,Pages:1213–1217Year published:(2010)DOI:doi:10.1038/nbt.1693Received24 August 2010Accepted27 September 2010Published online24 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A plethora of important, chemically diverse natural products are derived from plants1. In principle, plant cell culture offers an attractive option for producing many of these compounds2, 3. However, it is often not commercially viable because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale3. To bypass the dedifferentiation step, we isolated and cultured innately undifferentiated cambial meristematic cells (CMCs). Using a combination of deep sequencing technologies, we identified marker genes and transcriptional programs consistent with a stem cell identity. This notion was further supported by the morphology of CMCs, their hypersensitivity to γ-irradiation and radiomimetic drugs and their ability to differentiate at high frequency. Suspension culture of CMCs derived from Taxus cuspidata, the source of the key anticancer drug, paclitaxel (Taxol)2, 3, circumvented obstacles routinely associated with the commercial growth o! f DDCs. These cells may provide a cost-effective and environmentally friendly platform for sustainable production of a variety of important plant natural products. View full text Figures at a glance * Figure 1: Isolation and culture of T. cuspidata CMCs. () Schematic cross-section illustrating the location of cambium cells within a typical twig. Reproduced with permission from reference 12. () Preparation of T. cuspidata explant by peeling off cambium, phloem, cortex and epidermal cells from the xylem. Cell types are indicated by the following colored arrows: yellow, pith; white, xylem; green, cambium; red, phloem; blue, cortex; and turquoise, epidermis. Scale bar, 0.5 mm. () Natural split of CMCs from DDCs induced from phloem, cortex and epidermal cells. The top layer is composed of CMCs whereas the bottom layer consists of DDCs. Scale bar, 1 mm. () CMCs proliferated from the cambium. Scale bar, 1 mm. () DDCs induced from the tissue containing phloem, cortex and epidermal cells. Scale bar, 1 mm. () DDCs induced from the cut edge of a needle explant. Scale bar, 0.5 mm. () DDCs induced from the cut edge of an embryo explant. Scale bar, 0.5 mm. () Micrographs of DDCs and a CMC. CMCs are significantly smaller and possess charac! teristic numerous, small vacuole-like structures. The black arrow indicates a vacuole-like structure. Scale bars, 20 μm. () Single CMC stained with neutral red, which marks the presence of vacuoles. Two of many stained vacuoles are denoted by black arrows. Scale bar, 10 μm. () Needle-derived DDC stained with neutral red. The single large vacuole present in this cell is marked by a black arrow. Scale bar, 10 μm. () Conditional differentiation of T. cuspidata CMCs to tracheary elements, at the times indicated, after addition of differentiation media. Scale bar, 25 μm. () Time-course of differentiation of different T. cuspidata cell lines over time into tracheary elements. () Quantification of cell death in T. cuspidata cells after exposure to increasing levels of ionizing radiation. () Levels of cell death in T. cuspidata cells after exposure to the radiomimetic drug, Zeocin (phleomycin). Experiments were repeated at least twice with similar results. Data points represent! the mean of three samples ± s.d. * Figure 2: Characterization of CMCs from T. cuspidata, including transcriptome profiling using digital gene expression tags. () Scatter plot showing differentially expressed genes (DEGs) (blue and red) in CMCs and non-DEGs (black). The deployment of further filtering approaches identified more robust DEGs (red), whereas other DEGs (blue) were filtered out. FDR ≤ 0.05; n = 1,229. () Analysis of the expression of contig 01805 and contig 10710. () Relative percentage of GO groups within CMC DEGs. () Growth of CMCs and DDCs derived from needles or embryos on solid growth media from an initial 3 g f.c.w. 95% confidence limits are too small to be visible on this scale. () Bar graph reporting the extent of cell aggregation in DDC and CMC suspension cultures. () Paclitaxel production by 3-month-old DDCs and CMCs 10 d after elicitation, following batch culture in a flask format. Error bars represent 95% confidence limits. These experiments were repeated three times with similar results. * Figure 3: Growth and natural product biosynthesis of CMC suspensions. () Growth of CMCs and needle-derived DDCs in a 10 liter stirred tank bioreactor. () Growth of given cell suspension cultures in a 3 liter air-lift bioreactor format determined as d.c.w. At each passage, 14 d after inoculation, suspension cells were transferred to additional 3 liter air-lift bioreactors, as required. () Growth of needle- and embryo-derived DDCs and CMC suspension cultures in a 20 liter air-lift bioreactor, determined as d.c.w. accumulation following a single passage. () Total paclitaxel production following elicitation of the indicated 6-month-old repeatedly subcultured cell suspensions, after batch culture in a 3 liter air-lift bioreactor. () Intracellular and extracellular paclitaxel yield from the indicated batch cultured suspension cells grown in a 3 liter air-lift bioreactor. () Percentage of paclitaxel released into the production medium after batch culture of the given cell suspensions in a 3 liter air-lift bioreactor. () Synthesis of baccatin III and ! 10-deacetylbaccatin III in CMCs relative to needle-derived DDCs. () Magnitude of paclitaxel biosynthesis following elicitation of 28-month-old CMCs in a 20 liter air-lift bioreactor. Needle- and embryo-derived DDC suspensions did not routinely grow in this size bioreactor. () Intracellular and extracellular paclitaxel yield after 45 d of perfusion of cultured needle- and embryo-derived DDCs and CMCs in a 3 liter air-lift bioreactor. () Percentage of paclitaxel released into the production medium after perfusion culture of the given cell suspensions as indicated in . () Synthesis of taxamairin A and C in CMCs and needle-derived DDCs after batch culture in a 3 liter air-lift bioreactor. () Synthesis of ginsenosides in P. ginseng CMC and pith-derived DDC suspension cells after batch culture in a 3 liter air-lift bioreactor. Error bars represent 95% confidence limits. These experiments were repeated twice with similar results. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Sequence Read Archive * ERP000352 Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Eun-Kyong Lee & * Young-Woo Jin Affiliations * Unhwa Corp., Wooah-Dong, Dukjin-gu, Jeonju, South Korea. * Eun-Kyong Lee, * Young-Woo Jin, * Joong Hyun Park, * Young Mi Yoo & * Sun Mi Hong * Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, King's Buildings, Edinburgh, UK. * Rabia Amir, * Zejun Yan, * Eunjung Kwon, * Thomas Waibel, * Byung-Wook Yun & * Gary J Loake * School of Engineering, University of Edinburgh, King's Buildings, Edinburgh, UK. * Eunjung Kwon & * Alistair Elfick * Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, King's Buildings, Edinburgh, UK. * Simon Tomlinson & * Florian Halbritter Contributions E.-K.L., Y.-W.J., J.H.P., T.W. and B.-W.Y. performed experiments. R.A., E.K., S.T. and F.H. contributed to bioinformatic and statistical analysis. Z.Y., Y.M.Y. and S.M.H. carried out experiments. A.E. co-supervised E.K. E.-K.L., Y.-W.J. and G.J.L. formulated experiments. G.J.L. and E.-K.L. wrote the paper. All authors discussed results and commented on the manuscript. Competing financial interests E.K.L. and Y.W.J. hold stock in Unhwa Corp. Corresponding author Correspondence to: * Gary J Loake (gloake@ed.ac.uk) Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Tables 1–7 and Supplementary Figs. 1–17 * Supplementary Data Set 1 (27M) Assembled T. cuspidata transcriptome. * Supplementary Data Set 2 (2M) BLAST analysis of T. cuspidata contigs. * Supplementary Data Set 3 (1M) Digital gene expression tag profiling of CMCs. * Supplementary Data Set 4 (108K) Differentially expressed contigs between T. cuspidata CMCs and DDCs. * Supplementary Fig. 7 (2M) Comparison of the cultural properties of the given cell lines in a 3 L air-lift bioreactor. Additional data - Third-quarter biotech job picture
- Nat Biotech 28(11):1218 (2010)
Biotech and pharmaceutical positions on the three representative job databases tracked by Nature Biotechnology (Tables 1 and 2) were mixed in the third quarter of 2010. Compared with the second quarter (Nat. Biotechnol. 28, 875, 2010 - People
- Nat Biotech 28(11):1220 (2010)
Privately held NKT Therapeutics (Waltham, MA, USA) has announced the appointment of Barbara Finck (below, right) to the position of chief medical officer. She was most recently senior vice president of R&D and chief medical officer at Osprey Pharmaceuticals.
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