News Article | May 9, 2017
On May 5, Global Container Terminals (GCT) Canada welcomed Antwerpen Express, the largest container vessel to call the country at GCT Deltaport, located in the Port of Vancouver. Owned by German shipping company Hapag-Lloyd, the 13,200 TEU ship has been deployed in THE Alliance’s Transpacific mainline West Coast PN3 service. The 142,295 gross ton boxship was built by South Korean Hyundai Heavy Industries (HHI) shipyard in 2013. GCT Deltaport is Antwerpen Express’s first-port-of-call directly from the Far East (Hong Kong-Yantian-Ningbo-Shanghai-Pusan-Vancouver). Known as Canada’s flagship container terminal, GCT Deltaport handles large transpacific containerships and features a fleet of post-Panamax cranes.
News Article | May 11, 2017
VANCOUVER, BRITISH COLUMBIA--(Marketwired - May 11, 2017) - GVIC Communications Corp. ("GVIC" or the "Company") (TSX:GCT) reported cash flow, earnings and revenue for the period ended March 31, 2017. The following results are presented on an adjusted basis(1) to include the Company's share of its joint venture operations on a proportionate basis, because this is the basis on which management bases its operating decisions and performance. For a reconciliation to the results in accordance with International Financial Reporting Standards ("IFRS"), refer to the "Reconciliation of IFRS to Adjusted Results" as presented below and in Management's Discussion & Analysis ("MD&A"). GVIC's results for the first quarter were soft. Adjusted(1) consolidated EBITDA, including the Company's share of its joint venture interests, decreased to $7.4 million for the period ended March 31, 2017 compared to $8.4 million for the same period in the prior year. Adjusted consolidated revenue was $55.4 million for the quarter compared to $58.5 million for the same period in the prior year. The Company's commodity information operations continued to face adverse conditions in the quarter mainly due to the depressed energy, metals and agricultural prices. Adjusted revenues declined by 9.6% to $14.6 million while adjusted EBITDA declined by 16.2% to $4.2 million. The environmental, property and financial information operations experienced another strong quarter with solid revenue growth in all operations. While the Company continues to invest in these businesses, adjusted EBITDA for these operations experienced strong growth. Adjusted revenues grew by 13.6% to $6.9 million while adjusted EBITDA grew by 12.3% to $1.7 million. The challenged Prairies' economy weighed on the community media operations. While certain community media operations outside of the Prairies experienced EBITDA growth, overall the remaining operations had a difficult quarter. Adjusted revenues declined by 6.5% to $33.8 million while adjusted EBITDA declined by 8.5% to $3.3 million. GVIC operates as an information and marketing solutions company pursuing growth in sectors where the provision of essential information and related services provides high customer value. The Company's "go to market" strategy is being pursued through two operational areas: Through its brands and operations, GVIC serves clients in three segments: At March 31, 2017, senior debt was $40.6 million. During the quarter, the Company made net repayments of $3.1 million of senior debt, some of which was sourced from the sale of non-core real estate assets. On an adjusted basis, GVIC's consolidated debt net of cash outstanding before deferred financing charges was 2.63x trailing 12-months EBITDA as at March 31, 2017. The following table is a reconciliation of the IFRS results to the adjusted results (which include the Company's proportionate share of its joint venture operations). Refer to the MD&A for further discussion and analysis of these results: The qualitative discussion of the results for the period ended March 31, 2017 in this Press Release is relevant and applicable for the adjusted results and the IFRS results. Near-term uncertainty and market risk continues, especially given the ongoing impact of weak energy and commodity market conditions on the Western Canadian economy. The Company's agriculture, energy, mining and community media operations are continuing to be negatively impacted by these market conditions. In light of these conditions, the Company will continue to evaluate cost reduction initiatives while simultaneously improving the revenue generating operations of the businesses. The Company remains confident in the longer term outlook for the energy and mining sectors and a rebound in the mining industry appears to be underway. The Company continues to invest in its environmental, property and commodity information operations and expects continued strong growth. Additional investment is planned for businesses such as ERIS and REW.ca that are experiencing high growth and address large potential markets. Within agricultural information, a number of operations including WIN, the agricultural exhibitions and AgDealer are performing well and are expected to expand. The Company also continues to invest in and improve the value of its energy and mining database and subscription offerings, positioning itself for when the cyclical downturn reverses. Given the varied outlook, management plans to continue the progress of the last few years in strengthening the Company's financial position by further reducing debt. A strengthened balance sheet will mitigate risk while allowing the ongoing and planned operational and capital investments. These investments are necessary to continue the strong growth in a number of the Company's businesses that are creating real shareholder value. Shares in GVIC are traded on the Toronto Stock Exchange under the symbol GCT. About the Company: GVIC Communications Corp. is an information & marketing solutions company pursuing growth in sectors where the provision of essential information and related services provides high customer utility and value. GVIC's strategy is implemented through two operational areas: content and marketing solutions; and data, analytics and intelligence. To supplement the interim consolidated financial statements presented in accordance with International Financial Reporting Standards, GVIC uses certain non-IFRS measures that may be different from the performance measures used by other companies. These non-IFRS measures include cash flow from operations (before changes in non-cash operating accounts and non-recurring items), net income attributable to common shareholders before non-recurring items, net income from continuing operation attributable to common shareholders before non-recurring items, earnings before interest, taxes, depreciation and amortization (EBITDA) and all 'adjusted' measures which are not alternatives to IFRS financial measures. Management focuses on operating cash flow per share as the primary measure of operating profitability, free cash flow and value. EBITDA per share is also an important measure as the Company has low ongoing capital expenditures and depreciation and amortization largely relates to acquisition goodwill and copyrights and does not represent a corresponding sustaining capital expense. These non-IFRS measures do not have any standardized meanings prescribed by IFRS and accordingly they are unlikely to be comparable to similar measures presented by other issuers. The adjusted consolidated financial results have been adjusted to include the Company's share of revenue, expenses, assets and liabilities from its joint venture operations on a proportionate accounting basis as this is the basis on which management bases its operating decisions and performance evaluation. IFRS does not allow for the inclusion of the joint ventures on a proportionate basis. These results include additional non-IFRS measures such as EBITDA, cash flow from operations and net income attributable to common shareholders before non-recurring items. The adjusted results are not generally accepted measures of financial performance under IFRS. The Company's method of calculating these financial performance measures may differ from other companies and accordingly, they may not be comparable to measures used by other companies. Refer to the MD&A for a reconciliation of these non-IFRS measures and adjusted results. This news release contains forward-looking statements that relate to, among other things, the Company's objectives, goals, strategies, intentions, plans, beliefs, expectations and estimates. These forward-looking statements include, among other things, statements relating to our expectations regarding revenues, expenses, cash flows, future profitability and the effect of our strategic initiatives and restructuring, including our expectations to grow our business information operations, and to reduce debt levels. These forward-looking statements are based on certain assumptions, including continued economic growth and recovery and the realization of cost savings in a timely manner and in the expected amounts, which are subject to risks, uncertainties and other factors which may cause results, performance or achievements of the Company to be materially different from any future results, performance or achievements expressed or implied by such forward-looking statements, and undue reliance should not be placed on such statements. Important factors that could cause actual results to differ materially from these expectations include failure to implement or achieve the intended results from our strategic initiatives, the failure to reduce debt and the other risk factors listed in our Annual Information Form under the heading "Risk Factors" and in our interim MD&A under the heading "Business Environment and Risks", many of which are out of our control. These other risk factors include, but are not limited to, the ability of the Company to sell advertising and subscriptions related to its publications, foreign exchange rate fluctuations, the seasonal and cyclical nature of the agricultural and energy industry, discontinuation of the Department of Canadian Heritage's Canada Periodical Fund's Aid to Publishers, general market conditions in both Canada and the United States, changes in the prices of purchased supplies including newsprint, the effects of competition in the Company's markets, dependence on key personnel, integration of newly acquired businesses, technological changes, tax risk, financing risk and debt service risk. The forward-looking statements made in this news release relate only to events or information as of the date on which the statements are made. Except as required by law, the Company undertakes no obligation to update or revise publicly any forward-looking statements, whether as a result of new information, future events or otherwise, after the date on which the statements are made or to reflect the occurrence of unanticipated events.
News Article | May 23, 2017
Poster presentations also will highlight preclinical data examining the role of DS-8201 in combination with immunotherapy, the study design of the phase 1 study of U3-1402 in patients with HER3-expressing metastatic breast cancer and patient-reported outcomes in a phase 1 study of pexidartinib in tenosynovial giant cell tumor (TGCT), which includes pigmented villonodular synovitis (PVNS) and giant cell tumor of the tendon sheath (GCT-TS). "These presentations continue to underscore the promise of DS-8201 and U3-1402, which employ our unique ADC technology, to potentially change the standard of care in both HER2-expressing and HER3-expressing metastatic breast cancer," said Antoine Yver, MD, MSc, Executive Vice President and Global Head, Oncology Research and Development, Daiichi Sankyo. "In the context of recent advances made in the field of molecularly targeted new science for breast cancer, our own research further contributes to providing advanced or metastatic breast cancer with precision medicine options." DS-8201, U3-1402, pexidartinib and tivantinib have not been approved for any indication in any country. About DS-8201 and U3-1402 Antibody drug conjugates (ADCs) are a type of targeted cancer medicine that deliver cytotoxic chemotherapy ("payload") directly to cancer cells via a linker attached to a monoclonal antibody that binds to a specific target expressed on cancer cells. DS-8201 and U3-1402 are ADCs that use Daiichi Sankyo's proprietary payload and linker-payload technology, which has broad application across multiple types of cancer, and are designed to deliver enhanced cancer cell destruction with less systemic exposure to the cytotoxic payload. DS-8201 is an investigational ADC currently in phase 1 clinical development for HER2-positive advanced or metastatic breast cancer or gastric cancer, HER2-low-expressing breast cancer and other HER2-expressing solid cancers. The U.S. Food and Drug Administration (FDA) granted Fast Track designation to DS-8201 for the treatment of HER2-positive unresectable and/or metastatic breast cancer in patients who have progressed after prior treatment with HER2-targeted therapies including ado-trastuzumab emtansine (T-DM1). U3-1402 is an investigational and potential first-in-class ADC currently in phase 1 clinical development for HER3-expressing metastatic or unresectable breast cancer. About Pexidartinib Pexidartinib is an investigational, novel, oral small molecule that potently inhibits CSF-1R (colony stimulating factor-1 receptor), which is a primary growth driver of abnormal cells in the synovium that cause TGCT. Pexidartinib was discovered by Plexxikon Inc., the small molecule structure-guided R&D center of Daiichi Sankyo. Pexidartinib has been granted Breakthrough Therapy Designation for the treatment of TGCT and Orphan Drug Designation for PVNS/GCT-TS by the U.S. Food and Drug Administration (FDA). Pexidartinib also has received Orphan Designation from the European Commission for the treatment of TGCT. Pexidartinib is also being evaluated in several additional potential clinical indications, including glioblastoma, melanoma, ovarian, colorectal and pancreatic cancer. It is also being investigated in combination with anti-PD-1 immunotherapy, pembrolizumab, for multiple tumor types, including melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, ovarian cancer and gastrointestinal stromal tumors. About Daiichi Sankyo Cancer Enterprise The vision of Daiichi Sankyo Cancer Enterprise is to leverage our world-class, innovative science and push beyond traditional thinking in order to create meaningful treatments for patients with cancer. We are dedicated to transforming science into value for patients, and this sense of obligation informs everything we do. Anchored by our Antibody Drug Conjugate (ADC) and Acute Myeloid Leukemia (AML) Franchises, our cancer pipeline includes more than 20 small molecules, monoclonal antibodies and ADCs stemming from our powerful research engines: our two laboratories for biologic/immuno-oncology and small molecules in Japan, and Plexxikon Inc., our small molecule structure-guided R&D center in Berkeley, CA. Compounds in development include: quizartinib, an oral FLT3 inhibitor, for newly-diagnosed and relapsed/refractory AML with FLT3-ITD mutations; DS-8201, an ADC for HER2-expressing breast cancer, gastric cancer and other HER2-expressing solid tumors; and pexidartinib, an oral CSF-1R inhibitor, for tenosynovial giant cell tumor (TGCT), which is also being explored in a range of solid tumors in combination with the anti-PD1 immunotherapy pembrolizumab. For more information, please visit: www.DSCancerEnterprise.com. About Daiichi Sankyo Daiichi Sankyo Group is dedicated to the creation and supply of innovative pharmaceutical products to address diversified, unmet medical needs of patients in both mature and emerging markets. With over 100 years of scientific expertise and a presence in more than 20 countries, Daiichi Sankyo and its 16,000 employees around the world draw upon a rich legacy of innovation and a robust pipeline of promising new medicines to help people. In addition to a strong portfolio of medicines for hypertension and thrombotic disorders, under the Group's 2025 Vision to become a "Global Pharma Innovator with a Competitive Advantage in Oncology," Daiichi Sankyo research and development is primarily focused on bringing forth novel therapies in oncology, including immuno-oncology, with additional focus on new horizon areas, such as pain management, neurodegenerative diseases, heart and kidney diseases, and other rare diseases. For more information, please visit: www.daiichisankyo.com. Daiichi Sankyo, Inc., headquartered in Parsippany, New Jersey, is a member of the Daiichi Sankyo Group. For more information on Daiichi Sankyo, Inc., please visit: www.dsi.com. To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/daiichi-sankyo-cancer-enterprise-pipeline-data-showing-swift-progress-in-precision-medicine-for-breast-cancer-to-be-presented-at-2017-american-society-of-clinical-oncology-asco-annual-meeting-300461748.html
News Article | May 24, 2017
A step-by step protocol describing the HSPC conversion of human PSCs can be found at Protocol Exchange48. All experiments were performed with H9 hESC (NIHhESC-10-0062), PB34 iPS49, MSC-iPS150, 1045-iPSC, and 1157-iPSC established by the hES Core Facility at Boston Children’s Hospital. Human ESCs and iPSCs were maintained on hESC-qualified Matrigel (BD) in mTeSR1 media (Stem Cell Technologies) or mouse embryonic fibroblasts (GlobalStem) feeders in DMEM/F12 + 20% KnockOutSerum Replacement (Invitrogen), 1 mM l-glutamine, 1 mM NEAA, 0.1 mM β-mercaptoethanol, and 10 ng ml−1 bFGF on 10 cm gelatinized culture dishes. Medium was changed daily, and cells were passaged 1:4 onto fresh feeders every 7 days using standard clump passaging with dispase. Morphology of PSCs was checked by microscopy daily. As a quality control, only dishes with more than 70% of typical PSC colonies were processed for embryoid body formation. Cell lines were tested for mycoplasma routinely. Embryoid body differentiation was performed as previously described19. Briefly, hPSC colonies were dissociated with 0.05% trypsin for 5 min at 37 °C, pipetted thoroughly with p1000 to form small aggregates, washed twice with PBS + 2% FBS, and resuspended in StemPro-34 (Invitrogen, 10639-011) supplemented with l-glutamine (2 mM), penicillin/streptomycin (10 ng ml−1), ascorbic acid (1 mM), human holo-Transferrin (150 μg ml−1, Sigma T0665), monothioglycerol (MTG, 0.4 mM) (referred to as ‘supplemented StemPro-34’), BMP4 (10 ng ml−1), and Y-27632 (10 μM). Cells were then seeded into non-adherent spheroid formation 10 cm plates (Ezsphere, Asahi Glass; well size diameter 400–500 μm, depth 100–200 μm; number of wells 14,000 per dish) at a density of 5 million per dish. Twenty-four hours later, bFGF (5 ng ml−1) and BMP4 (10 ng ml−1) were added to the medium. On day 2, the developing embryoid bodies were collected and resuspended in supplemented StemPro-34 with SB431542 (6 μM), CHIR99021 (3 μM), bFGF (5 ng ml−1), and BMP4 (10 ng ml−1). The formation of embryoid bodies was checked by microscopy on day 4 and the decision was made to continue embryoid body formation on the basis of the size and morphology of aggregations (quality control; >100 μM, compaction-like tight contact of cells). On day 4, medium was replaced by supplemented StemPro-34 with VEGF (15 ng ml−1) and bFGF (10 ng ml−1). At day 6, medium was replaced by supplemented StemPro-34 with bFGF (10 ng ml−1), VEGF (15 ng ml−1), interleukin (IL)-6 (10 ng ml−1), IGF-1 (25 ng ml−1), IL-11 (5 ng ml−1), and SCF (50 ng ml−1). Cultures were maintained in a 5% CO /5% O /90% N environment. All recombinant factors were human and purchased from Peprotech. To avoid potential damage resulting from hydrodynamic pressure and contamination through fluorescence-activated cell sorting (FACS), for functional assay, isolation of haemogenic endothelium was performed by magnetic cell isolation. Freshly dissociated embryoid body cells (at the day 8 time point) by 0.05% trypsin were filtered through a 70 μm filter and stained with CD34 microbeads (Miltenyi) for 30 min at 4 °C. CD34+ cells were isolated with LS columns (Miltenyi). Around 0.3 × 105 to 1.0 × 105 cells were obtained per 10 cm dish of embryoid body formation. A sample from each batch was analysed by FACS to validate its purity of haemogenic endothelium with the panel CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD), and 4′,6-diamidino-2-phenylindole (DAPI). For expression profiling by microarray and qRT–PCR, isolation of haemogenic endothelium was performed by FACS. Dissociated embryoid bodies (at the day 8 time point) were resuspended at 1 × 106 to 3 × 106 per 100 μl of staining buffer (PBS + 2% FBS). Cells were stained with a 1:50 dilution of CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), CD43 PE (1G10; BD), and DAPI for 30 min at 4 °C in the dark. All FACS sorting was performed on a BD FACS Aria II cell sorter using an 85 μm nozzle to avoid potential damage to haemogenic endothelium. All the samples used for microarray analysis were FACS-sorted. Haemogenic endothelium panel: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), and CD43 PE (1G10; BD). Fetal-liver HSCs were purchased from StemCell Technologies and stained with HSC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34 PE-Cy7 (8G12; BD), and CD45 PE (HI30; BD). Between 10,000 and 50,000 cells were sorted for each cell type with two or three biological replicates. An RNAeasy Microkit (Qiagen) was used to collect and prepare total RNA for microarray analysis. The Ovation Picokit (Nugen) was used for preamplification, where required. Gene expression profiling was performed on Affymetrix 430 2.0 gene chips according to standard protocol. Microarray data were analysed according to standard protocol using R/Bioconductor. Embryoid bodies were dissociated on day 8 by digestion with 0.05% trypsin for 5 min at 37 °C, pipetted thoroughly with p1000 to generate a single-cell suspension and washed with PBS + 2%FBS. Dissociated embryoid bodies were immediately processed for isolation of haemogenic endothelium. Cells were resuspended in 1mL PBS+2%FBS and incubated with human CD34 MicroBead kit for 1 h (Miltenyl Biotec, 130-046-702). After incubation, cells were washed with PBS+2%FBS and isolated by magnetic cell isolation using LS columns (Miltenyl Biotec, 130-042-401). Sorted CD34+ cells were resuspended in supplemented StemPro-34 medium, containing Y-27632 (10 μM), TPO (30 ng ml−1), IL-3 (10 ng ml−1), SCF (50 ng ml−1), IL-6 (10 ng ml−1), IL-11 (5 ng ml−1), IGF-1 (25 ng ml−1), VEGF (5 ng ml−1), bFGF (5 ng ml−1), BMP4 (10 ng ml−1), and FLT3 (10 ng ml−1) as reported20 and seeded at a density of 25 × 103 to 50 × 103 cells per well onto thin-layer Matrigel-coated 24-well plates. All recombinant factors were human and most were purchased from Peprotech. Plasmids for the transcription factor library were obtained as Gateway plasmids (Harvard Plasmid Service; GeneCopoeia). Open reading frames were cloned into lentiviral vectors using LR Clonase (Invitrogen). Two vectors were used, pSMAL-GFP (constitutive) and pINDUCER-21 (ORF-EG)51. pINDUCER21 (ORF-EG) was a gift from S. Elledge and T. Westbrook (Addgene plasmid 46948). Lentiviral particles were produced by transfecting 293T-17 cells (ATCC) with the second-generation packaging plasmids (pMD2.G and psPAX2 from Addgene). Virus were harvested 36 and 60 h after transfection and concentrated by ultracentrifugation at 23,000 r.p.m. for 2 h 15 min at 4 °C. Viruses were reconstituted with 50 μl of EHT culture medium. Constructs were titred by serial dilution on 293T cells using GFP as an indicator. Polycistronic vectors were made as follows: LCOR-P2A-HOXA9-T2A-HOXA5 and RUNX1-P2A-ERG DNA fragments were synthesized and cloned into pENTR-D/TOPO cloning vector by GenScript, then Gateway-recombined with pINDUCER-21 (ORF-EG). At day 3 of EHT culture, haemogenic endothelium cells were beginning to produce potentially haematopoietic ‘round’ cells; the occurrence of this phenomenon was used as quality control of haemogenic endothelium induction and transition to haematopoietic cells for each batch of experiments. The infection medium was EHT culture medium supplemented with Polybrene (8 μg ml−1, Sigma). Lentiviral infections were performed in a total volume of 250 μl (24-well plate). The multiplicity of infection for the factors was as follows: Library 3.0 for each, ERG 5.0, HOXA5 5.0, HOXA9 5.0, HOXA10 5.0, LCOR 5.0, RUNX1 5.0, SPI1 5.0, LCOR–HOXA9–HOXA5 2.0, and RUNX1–ERG 2.0. Haemogenic endothelium was vulnerable to damage during spinoculation, thus infections were performed static for 12 h, then 250 μl of fresh EHT medium was supplemented to dilute Polybrene. Parallel wells were cultured for an additional 3 days to measure infection efficiency by the percentage of GFP+ DAPI cells by FACS, achieving 30–70% of infection efficiency. Followed by lentiviral gene transfer, cells were maintained for 5 days in EHT culture medium supplemented with doxycycline (2 μg ml−1, Sigma) to induce transgene expression in vitro. Fifty thousand cells were plated into 3 ml complete methylcellulose (H4434; StemCell Technologies). Additional cytokines added were 10 ng ml−1 FLT3, 10 ng ml−1 IL6, and 50 ng ml−1 TPO (R&D Systems). The mixture was distributed into two 60 mm dishes and maintained in a humidified chamber at 37 °C for 14 days. Colonies were scored manually or using a BD Pathway 855 fluorescent imager. At 14 days, granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colonies were picked up by P20 pipette. Between 10 and 20 GEMM colonies were picked with 2 or 3 biological replicates. A QIAamp DNA Micro Kit (Qiagen) was used to collect and prepare total genomic DNA for PCR detection of transgenes. Nested PCR reaction was as follows: first round with LNCX forward primer (5′-AGC TCG TTT AGT GAA CCG TCA GAT C-3′) and EGFP N reverse primer (5′-CGT CGC CGT CCA GCT CGA CCA G-3′), 95 °C 5 min, 36 cycles of (95 °C for 30 s, 60 °C for 30 s, 72 °C for 5 min), 72 °C for 5 min, 4 °C hold; second round with forward primer for each gene and HA reverse primer (5′-TCT GGG ACG TCG TAT GGG TA-3′), 95 °C 5 min, 36 cycles of (95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min, 4 °C hold. Twelve hours after lentiviral gene transfer, cells were recovered by dispase for 5 min at 37 °C, and washed by PBS three times to ensure no carry-over of virus. Cells were resuspended at 0.3 × 105 to 3.0 × 105 cells per 25 μl buffer (PBS + 2% FBS from StemCell Technologies) and kept on ice until injection. Thirty thousand to 3.0 × 105 cells were intrafemorally injected in to NOD/LtSz-scidIL2Rgnull (NSG) mice and treated with doxycycline as described below (see section on ‘Mouse transplantation’). Up to 100 μl peripheral blood was collected every 2–4 weeks, to 14 weeks. Mice were euthanized and bone marrow and thymus removed at 8–14 weeks. For transgene detection in engrafted cells, each lineage of cells was FACS-sorted from bone marrow. Myeloid cells: CD33 APC (P67.6; BD), CD45 PE-Cy5 (J33; Coulter). B cells: CD19 PE (HIB19; BD), CD45 PE-Cy5 (J33; Coulter). T cells: CD3 PE-Cy7 (SK7; BD), CD45 PE-Cy5 (J33; Coulter). Between 10,000 and 50,000 cells were isolated with 2 or 3 biological replicates for multiple cell lines (iPSCs and ESCs). The QIAamp DNA Micro kit (Qiagen) was used to collect and prepare total genomic DNA for PCR detection of transgenes. Nested PCR reaction was performed similarly to the in vitro screening described in the above section. NOD/LtSz-scidIL2Rgnull (NSG) mice (The Jackson Laboratory) were bred and housed at the Boston Children’s Hospital animal care facility. Animal experiments were performed in accordance with institutional guidelines approved by Boston Children’s Hospital Animal Care Committee. Intrafemoral transplantations were conducted with 6- to 10-week-old female mice irradiated (250 rad) 12 h before transplantation. Before transplantation, mice were temporarily sedated with isoflurane. A 26-half-gauge needle was used to drill the femur and a 0.3 × 105 to 3.0 × 105 range of cells was transplanted in a 25 μl volume using a 28.5-gauge insulin needle. Sulfatrim was administered in drinking water to prevent infections after irradiation. Doxycycline Rodent Diet (Envigo-Teklad Diets; 625 p.p.m.) and doxycycline (1.0 mg ml−1) were added to the drinking water to maintain transgene expression in vivo for 2 weeks (ref. 2). Secondary transplantation was performed with 1,000–3,000 human CD34+ cells (isolated from bone marrow by magnetic cell isolation with CD34 microbeads) at 8 weeks. Isolated cells were resuspended at 1,000–3,000 cells per 25 μl buffer (PBS + 2% FBS from StemCell Technologies) and kept on ice until injection. Cells were intrafemorally injected in to NSG mice. Sorted CD34+CD43+CD45+ (25,000 cells) or CD34+CD43−CD45− (25,000 cells) HE-7TF cells were either intrafemorally or intravenously injected. For non-irradiated c-Kit-deficient immune-deficient recipients, the NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ model was used (The Jackson Laboratory). Investigators were blinded for the analysis of mice. The experiments were not randomized. No statistical methods were used to predetermine sample size. For this analysis, multi-lineage engraftment was defined as chimaerism of human CD45+ cells in bone marrow encompassing four distinct lineages (myeloid, erythroid, B- and T-lymphoid, each comprising more than 1% of engrafted human CD45+ cells) in this study. Cells grown in EHT culture or harvested animal tissues were stained with the following antibody panels. Haemogenic endothelium panel: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), and CD43 PE (1G10; BD). HSPC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34 PE-Cy7, and CD45 PE (HI30; BD). Lineage panel: CD235a/glycophorin (GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter), CD33 APC (P67.6; BD), CD19 PE (HIB19; BD), IgM BV510 (G20-127; BD), CD4 PE-Cy5 (13B8.2; Coulter), CD3 PE-Cy7 (SK7; BD), CD8 V450 (RPA-T8; BD), TCRαβ BV510 (T10B9; BD), TCRγδ APC (B1; BD), CD45 PE-Cy5 (J33; Coulter), CD15 APC (HI98; BD), and CD31/PECAM PE (WM59; BD). All stains were performed with fewer than 1 × 106 cells per 100 μl staining buffer (PBS + 2% FBS) with 1:100 dilution of each antibody, for 30 min at 4 °C in the dark. Compensation was performed by automated compensation with anti-mouse Igk negative beads (BD) and cord blood MNC stained with individual antibodies. All acquisitions were performed on a BD Fortessa cytometer. For detection of engraftment, human cord-blood-engrafted mouse marrow was used as a control to set gating; sorting was performed on a BD FACS Aria II cell sorter. Five thousand to 10,000 FACS-sorted erythroid cells (CD235a/glycophorin (GLY)-A PE-Cy7 or FITC (11E4B-7-6; Coulter)), plasmacytoid lymphocytes (CD19 PE (HIB19; BD), IgM BV510 (G20-127; BD), CD38 PE-Cy5 (LS198-4-3; Clontech)), neutrophils (CD15 APC (HI98; BD), CD31/PECAM PE (WM59; BD) and CD45 PE-Cy5 (J33; Coulter)) were cytospun onto slides (500 r.p.m. for 10 min), air dried, and stained with May-Grunwald and Giemsa stains (both from Sigma), washed with water, air dried, and mounted, followed by examination by light microscopy. RNA extraction was performed using an RNAeasy Microkit (Qiagen). Reverse transcription was performed using Superscript III (>5,000 cells) or VILO reagent (<5,000 cells) (Invitrogen). Quantitative PCR was performed in triplicate with SYBR Green (Applied Biosystems). Transcript abundance was calculated using the standard curve method. Primers used for globin genes were as follows52: huHbB F (5′-CTG AGG AGA AGT CTG CCG TTA-3′), huHbB R (5′-AGC ATC AGG AGT GGA CAG AT-3′), huHbG F (5′-TGG ATG ATC TCA AGG GCA C-3′), huHbG R (5′-TCA GTG GTA TCT GGA GGA CA-3′), huHbE F (5′-GCA AGA AGG TGC TGA CTT CC-3′), and huHbE R (5′-ACC ATC ACG TTA CCC AGG AG-3′). FACS-isolated neutrophils (CD15+PECAM+CD45+) and T cells (CD3+CD45+) were cultured in IMDM + 10%FBS overnight in 96-well plates (flat-bottom), seeding 5,000–20,000 cells per well obtained from mice engrafted over 10% in primary recipients, or pooled mice engrafted less than 5% in primary recipients. Then supernatant was taken and analysed by MPO- or IFN-γ-ELISA –Ready-SET-Go! Kit (eBioscience) according to the manufacturer’s protocol. The amount of IFN-γ was normalized per 1,000 cells. PMA (20 ng ml−1) and ionomycin (1 μg ml−1) were added to either neutrophils or T cells, then cells were cultured overnight (6–18 h). Human Ig production was measured from 50 μl of serum from NSG mice at 8 weeks (IgM) and 14 weeks after engraftment (IgG). Immunization of mice was done with OVA (F5503, Sigma) with Freund’s complete adjuvant (F5881, Sigma), followed by booster doses of Freund’s incomplete adjuvant (F5506, Sigma) according to the manufacturer’s instructions. Six to 14 weeks after engraftment, mice were injected with antigen OVA (0.1%) emulsified in complete adjuvant subcutaneously at two sites on the back, injecting 100 μl at each site. A booster injection of antigen OVA (0.1%) emulsified in incomplete adjuvant was administered 14 days after immunization. The booster was given as a single subcutaneous injection with 100 μl at one site on the back. A serum sample was isolated from mice 7 days after the first booster dose, and human ova-specific antibody concentration was tested with an ovalbumin-specific IgG, OVA sIgG, ELISA Kit (Mybiosource, MB S700766) for human Ova-specific IgG and a Human Anti-Ovalbumin (Gal d 2) IgM ELISA Kit (Alpha Diagnostic, 670-145-OVM) to detect human Ova-specific IgG and IgM, respectively. The technical replicates were done with three measurements of the same experimental setup. Human CD3+ T cells were FACS-isolated from thymus of engrafted NSG mice. Purified DNA was subjected to next-generation sequencing of CDR3 using immunoSEQ (Adaptive Biotechnology, Seattle, Washington, USA) and analysed with immunoSEQ Analyzer software (Adaptive Biotechnology). Aliquots (250 ng) of genomic DNA from human CD45+ bone marrow cells (CD45 PE-Cy5 (J33; Coulter)) from engrafted NSG mice and original PSCs (two biological replicates) were digested with either Nsp1 or Sty1. A universal adaptor oligonucleotide was then ligated to the digested DNAs. The ligated DNAs were diluted with water and three 10 μl aliquots from each well of the Sty 1 plate and four 10 μl aliquots from each well of the Nsp 1 plate were transferred to fresh 96-well plates. PCR master mix was added to each well and the reactions cycled as follows: 94 °C for 3 min; 30 cycles of 94 °C for 30 s, 60 °C for 45 s, 68 °C for 15 s; 68 °C for 7 min; 4 °C hold. After PCR, the seven reactions for each sample were combined and purified by precipitation from 2-propanol/7.5 M ammonium acetate. The ultraviolet absorbance of the purified PCR products was measured to ensure a yield ≥4 μg μl−1. Forty-five microlitres (≥180 μg) of each PCR product were fragmented with DNase 1 so the largest fragments were <185 base pairs. The fragmented PCR products were then end-labelled with a biotinylated nucleotide using terminal deoxynucleotidyl transferase. For hybridization, the end-labelled PCR products were combined with hybridization cocktail, denatured at 95 °C for 10 min and incubated at 49 °C. Two hundred microlitres of each mixture was loaded on a GeneChip and hybridized overnight at 50 °C and 60 r.p.m. After 16–18 h of hybridization, the chips were washed and stained using the GenomeWideSNP6_450 fluidics protocol with the appropriate buffers and stains. After washing and staining, the GeneChips were scanned on a GeneChip Scanner 3000 using AGCC software. Genotype calls (chp files) were generated in Affymetrix Genotyping Console using the default parameters. The resulting chp files were analysed for familial relationships using the identity by state algorithm implemented in Partek Genomics Suite. Engrafted human CD34+CD38−CD45+ HSCs were isolated from bone marrow from either iPS-derived haemogenic endothelium- or cord-blood-injected NSG mice, then RNA was purified with an RNeasy Micro kit (Qiagen). Quality control of RNA was done by Bioanalyzer and qubit analysis. Samples that passed quality control were converted into libraries and sequenced by a Nextseq PE75 kit. Raw reads were aligned to the human genome/transcriptome using TopHat2 software53. Gene expression levels and reads per kilobase per million (RPKM) values were estimated using a htseq-count tool54 and the edgeR package55. For a legitimate transcriptome-wide comparison, we retrieved raw RNA-seq data of two published from the Gene Expression Omnibus database (Long non-coding RNA profiling of human lymphoid progenitors reveals transcriptional divergence of B-cell and T-cell lineages, accession number GSE69239; Distinct routes of lineage development reshape the human blood hierarchy across ontogeny, accession number GSE76234) and calculated RPKM values using a same analysis pipeline. Engrafted human CD34+CD38−CD45+ HSCs were isolated from bone marrow from either iPS-derived haemogenic endothelium- or cord-blood-injected NSG mice, then processed for in-droplet barcoding according to a previous report29. The library was QCed with Bioanalyzer and sequenced by Nextseq PE 75 kit. The t-SNE algorithm was used to visualize transcriptome similarities and population heterogeneity of cord blood HSCs and iPSC-derived HSCs. The t-SNE algorithm performs a dimensionality reduction of multidimensional single-cell RNA-seq data into a low-dimensional space, preserving pairwise distances between data points as well as possible, allowing a global visualization of subpopulation structure and cell–cell similarities. We used the R package tsne in our analyses. The t-SNE map was initialized with point-to-point distances computed by classical multidimensional scaling, and the R plot function was used to visualize t-SNE maps annotated by cord blood or iPSC-derived HSCs. Plots showing t-SNE maps coloured by expression of selected genes were created using the ggplot2 package. For subpopulation identification, we used the top 500 genes with the highest variance to elucidate global differences among single cells. To assess transcriptome similarities in terms of induction of haematopoietic genes in iPSC-derived HSCs, we used 62 haematopoietic genes for t-SNE analysis in Supplementary Table 2. Gene set enrichment analysis was performed with the desktop client version (javaGSEA, http://software.broadinstitute.org/gsea/downloads.jsp) with default parameters. RPKM values from the 7F-HSPC were obtained from the RNA-seq (described previously). These values were normalized to a terminally differentiated cell and the normalized values were used to rank the most differentially expressed genes. These differentially expressed genes were used to run gene set enrichment analysis with gene sets obtained from mSigDB (KEGG, Hallmark, immunological, transcription factors, and chemical and genetic perturbations gene sets were used). In addition, gene sets specific to progenitors, cord blood, or fetal-liver HSC were obtained from previous reports16, 56. FDR < 0.25 with P < 0.05 was considered significant. CD33+ myeloid cells, CD19+ B cells, and CD3+ T cells were isolated from bone marrow from haemogenic-endothelium-injected NSG mice. Genomic DNA was purified with a QIAamp DNA Micro kit (Qiagen). Ligation-mediated PCR-based detection of lentiviral integration sites was done with a Lenti-X Integration Site Analysis Kit (Clontech) according to the manufacturer’s instructions. Sequencing-based detection (integration sequencing) was done as previously described57. RNA-seq from this study have been deposited in the Gene Expression Omnibus under accession number GSE85112. We retrieved raw RNA-seq data of two published from the Gene Expression Omnibus database (Long non-coding RNA profiling of human lymphoid progenitors reveals transcriptional divergence of B-cell and T-cell lineages, accession number GSE69239; Distinct routes of lineage development reshape the human blood hierarchy across ontogeny, accession number GSE76234). The data are all in the paper, or are available from the corresponding author upon reasonable request if not.
News Article | May 8, 2017
DELTA, BC--(Marketwired - May 08, 2017) - On Friday, GCT Canada welcomed the largest container vessel to call the country at GCT Deltaport, located in the Port of Vancouver. The Hapag-Lloyd 13,200 TEU "Antwerpen Express" has been deployed as part of THE Alliance's Transpacific mainline West Coast PN3 service. The Hamburg class vessel is the first of a progressive ship upsizing for the service. Not only will this new fleet of vessels offer more slots for importers and exporters to grow in the market, but it will do so with an improved environmental footprint. GCT Deltaport hosts the largest ships to call the Port of Vancouver. The facility has recently completed major construction on its $280 million Intermodal Yard Reconfiguration project that increases rail capacity by over 50% within the existing footprint, enabling even better handling of big ship surge volumes. GCT Deltaport is the Antwerpen Express's first-port-of-call directly from the Far East (Hong Kong-Yantian-Ningbo-Shanghai-Pusan-Vancouver). "Our dedicated PN3 shuttle service is a competitive East West product with short transit times," stated Wolfgang Schoch, Senior Vice President of Hapag-Lloyd (Canada). "Working together with our supply chain partners, through this facility our cargo reaches more than 20 destinations across Canada and the US Midwest." Collaboration amongst CN, Canadian Pacific (CP), and GCT Canada has improved rail transit times. "CP is proud of its relationship with GCT Canada and Hapag-Lloyd, and looks forward to unlocking future international intermodal growth as a result of the improvements at GCT Deltaport," said John Brooks, CP's Senior Vice-President and Chief Marketing Officer. "CP's customers enjoy the fastest service from Vancouver to Minneapolis, Chicago and beyond and in close collaboration with our supply chain partners, we will continue to leverage our competitive advantage." "CN's vast network connects the Canadian West Coast Gateway to the largest number of destinations across Canada and the United States and is big ship and big alliance ready," said JJ Ruest, Executive Vice President and Chief Marketing Officer at CN. "We welcome THE Alliance's new import service to Vancouver. Our service level agreements with ports and partners across North America have created the fastest and most reliable supply chain from Asia to our customers' front doors. We provide truck and rail options to efficiently move freight from the port and GCT facilities to final destinations from Toronto to Detroit to Chicago to Memphis and beyond." "GCT Deltaport is purpose-built to handle rail cargo seamlessly," said Eric Waltz, President of GCT Canada. "With the terminal's process innovation, efficient equipment and design, beneficial cargo owners calling GCT Deltaport will experience the lowest rail dwells in the industry." To learn about the GCT Deltaport project, visit www.globalterminalscanada.com/#c3. About GCT Global Container Terminals Inc. Headquartered in Vancouver, BC, GCT Global Container Terminals Inc. operates four Green Marine certified terminals in two principal North American ports. Through GCT USA on the East Coast, the company operates two award-winning facilities: GCT New York on Staten Island, NY and GCT Bayonne in Bayonne, NJ. On the West Coast, GCT Canada operates two gateway terminals: GCT Vanterm and GCT Deltaport in Vancouver and Delta, BC. Visit www.globalterminals.com or follow us @BigShipReady to find out more about GCT. About Hapag-Lloyd With a fleet of 166 modern container ships and a total transport capacity of 963,000 TEU, Hapag-Lloyd is one of the world's leading liner shipping companies. The Company has around 9,400 employees at 366 sites in 121 countries. Hapag-Lloyd has a container capacity of 1.6 million TEU -- including one of the largest and most modern fleets of reefer containers. A total of 128 liner services worldwide ensure fast, reliable connections between all the continents. Hapag-Lloyd is one of the leading operators in the Transatlantic, Latin America and Intra-America trades. About CN Rail CN is a true backbone of the economy that transports more than C$250 billion worth of goods annually for a wide range of business sectors, ranging from resource products to manufactured products to consumer goods, across a rail network of approximately 20,000 route-miles spanning Canada and mid-America. CN -- Canadian National Railway Company, along with its operating railway subsidiaries -- serves the cities and ports of Vancouver, Prince Rupert, B.C., Montreal, Halifax, New Orleans, and Mobile, Ala., and the metropolitan areas of Toronto, Edmonton, Winnipeg, Calgary, Chicago, Memphis, Detroit, Duluth, Minn./Superior, Wis., and Jackson, Miss., with connections to all points in North America. For more information about CN, visit the Company's website at www.cn.ca. About Canadian Pacific Canadian Pacific (TSX: CP)( : CP) is a transcontinental railway in Canada and the United States with direct links to eight major ports, including Vancouver and Montreal, providing North American customers a competitive rail service with access to key markets in every corner of the globe. CP is growing with its customers, offering a suite of freight transportation services, logistics solutions and supply chain expertise. Visit www.cpr.ca to see the rail advantages of Canadian Pacific.