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News Article | May 5, 2017
Site: globenewswire.com

At the 2017 Waterfront Conference on May 10, the Waterfront Alliance Previews the Region’s First-Ever Harbor Scorecard How protected are our coastal communities and our infrastructure? Are waterways meeting Clean Water Act standards? Where are opportunities for public access to the water? Turn to the Harbor Scorecard for answers and action, neighborhood by neighborhood NEW YORK, May 05, 2017 (GLOBE NEWSWIRE) -- At this year’s Waterfront Conference, titled “Measuring Our Harbor: Strong, Healthy, and Open,” the Waterfront Alliance will preview the region’s first-ever Harbor Scorecard. Comprehensive and user-friendly, the Scorecard compiles research in public access, ecology, and resiliency for a neighborhood-by-neighborhood evaluation of the waterfronts of New York City and northern New Jersey. Distilling extensive research into an easy-to-use tool, the Harbor Scorecard will be previewed at the Waterfront Alliance’s annual Waterfront Conference on Wednesday, May 10, and rolled out three weeks later in time for the start of hurricane season. The Waterfront Conference takes place aboard the Hornblower Infinity, dockside at Hudson River Park, Pier 40, in the morning, and cruising New York Harbor throughout the afternoon. Featured speakers include Rep. Nydia M. Velázquez, Member of Congress; Lauren Brand, U.S. Department of Transportation, Maritime Administration; Hon. Ras J. Baraka, Mayor, City of Newark; and Alicia Glen, Deputy Mayor for Housing and Economic Development, City of New York, along with dozens of expert panelists and workshop leaders, and hundreds of policy-makers, waterfront advocates, and professionals. Hornblower Cruises & Events is the venue sponsor. For the third year, Arcadis is the premier sponsor of the Waterfront Conference, and for the second year, the sponsor of Arcadis Waterfront Scholars, a program that invites more than 70 undergraduate and graduate students to participate in the conference and engage with professional mentors. On May 10, Arcadis Global Lead for Water Management Piet Dircke will discuss the Harbor Scorecard in a global context using the Arcadis Sustainable Cities Water Index, which assesses the water resources of 50 cities around the world. “New York City is making strides in protecting its coastline, and the Waterfront Conference is an important step in bringing together the best minds to help benchmark our harbors and waterways,” said Mr. Dircke. “Waiting until the next big storm to create safeguards against future disasters is not a sound resiliency strategy.” “As the Waterfront Alliance begins our milestone tenth year of work, we have put together the Harbor Scorecard, an essential tool to take stock of progress along our waterways,” said John Boulé, vice-chair of the Waterfront Alliance and senior vice president at Dewberry, where he is business manager for New York operations. “While we’ve got a lot to be proud of, it’s clear that we need to do better, and the Harbor Scorecard will give citizens and policy-makers alike the information they need to act.” “The Waterfront Alliance cannot solve all of New York City’s waterfront and sea level rise issues, but its Harbor Scorecard will indicate whether we are moving in the right direction, becoming more resilient while at the same time providing more and better access to the waterfront,” said Klaus Jacob of Columbia University’s Lamont-Doherty Earth Observatory. “Right now, New York City is making decisions that will affect clean water investments for the next generation,” said Larry Levine, a senior attorney at Natural Resources Defense Council. “Thanks to the Clean Water Act, our harbor is cleaner than it used to be. But far too often the water is still too polluted to touch. Sewage overflows still foul our waterways after it rains, making them unsafe for eight million New Yorkers to use recreationally. The Harbor Scorecard will provide a call to action for local, state and federal officials, shining a light on where we need to invest in our infrastructure for a cleaner, healthier future.” Learn more about the Waterfront Conference and purchase tickets ($150 regular ticket; $75 government agencies and nonprofits; $50 students). Registration and breakfast is 8am to 8:45am; the boat is dockside at Pier 40, Hudson River Park until 1pm; the afternoon harbor cruise returns at 5pm. Continuing education credits will be offered (AIA CES; with APA AICP CM, and LA CES credits pending). The Waterfront Conference is generously sponsored by: Venue Sponsor: Hornblower Cruises & Events Premier Sponsor and Waterfront Scholars: Arcadis Commander: AECOM, GCA , New York City Economic Development Corporation Supporter: Dewberry, ExxonMobil, GBX Gowanus Bay Terminal, Hudson River Foundation, Newtown Creek Group, Red Hook Container Terminal, Seastreak, Stantec, Studio V, Two Trees, United Metro Energy Champion: Entertainment Cruises, HDR, Industry City, Langan, New York Water Taxi, Park Tower Group, Queens Chamber of Commerce, Sims Metal Management/Sims Municipal Recycling, Friend: ARUP, Ecology and Environment, HATCH, Kyle Conti Construction, M.G. McLaren Engineering Group, Moffatt & Nichol, Mott MacDonald, NY Waterway, Perkins & Will, Scape Studio, Starr Whitehouse Landscape Architects and Planners, Steer Davies & Gleave, Williams, WSP/Parsons Brinckerhoff Continuing Education Partners: AIA New York, APA NY Metro Chapter, ASLA NY The Waterfront Alliance works to protect, transform, and revitalize our harbor and waterfront.


News Article | April 17, 2017
Site: www.acnnewswire.com

Rex International Holding Maps Out Value Creation Strategy for Norway - Technology and infrastructure-led, de-risked exploration approach taken by Lime Petroleum Norway AS - First test production in Edvard Grieg South (Rolvsnes) anticipated in 2019 - Cluster of assets around Edvard Grieg South (Rolvsnes) discovery added to portfolio Rex International Holding Ltd (SGX:5WH,REXI), a new-generation technology driven oil company, has mapped out a value creation strategy for its assets in Norway - particularly in the North Sea - built on the Group's unique technology-led, de-risked exploration approach. Development feasibility studies are being carried out on the Edvard Grieg South (Rolvsnes) ("EGS") discovery in licence PL338C made in December 2015, for which a recent Gaffney, Cline & Associates ("GCA") Qualified Persons Report[1] (dated 10 March 2017) disclosed GCA's independent assessment of gross contingent resources attributable to the PL338C licence, on an unrisked basis, of up to 77.9 million barrels of oil (3C resources: high estimate of potentially recoverable oil from the discovery) and up to 78.7 billion standard cubic feet of natural gas (3C resources: high estimate of potentially recoverable gas from the discovery). Rex's 87.84 per cent subsidiary Lime Petroleum Norway AS ("Lime Norway") holds a 30 per cent stake in the licence. The Group expects test production from EGS to start in 2019. - Infrastructure-led strategy to fast-track value creation - Mr Mans Lidgren, CEO of Rex International Holding, said, "EGS is a prime example of our technology and infrastructure-led strategy in Norway. The EGS discovery is located near to Johan Sverdrup, one of the five largest oil fields on the Norwegian continental shelf with expected resources of between 1.9 to 3.0 billion barrels of oil[2], and adjacent to the producing Edvard Grieg and Ivar Aasen fields with estimated combined reserves of some 400 million barrels of oil[3]. We have used our Rex Virtual Drilling technology to select and build a cluster of investments in this oil prolific area that already has pipeline infrastructure in place, allowing a fast-track path to potential commercialisation and return on investment (ROI) when we make more oil discoveries. To this end, Lime Norway also holds a 20 per cent interest in licence PL815, where the undrilled Goddo prospect is believed to be a geological continuation of the EGS discovery, as well as a 30 per cent stake in licence PL818 comprising the Orkja prospect located within easy tie-back distance to the Ivar Aasen field. Our aim is to prove up these adjacent fields in the mid to long term to grow our pool of resources, on top of achieving production in EGS in the short term." The EGS discovery made in December 2015 is located in water depths of about 100 metres on the prolific Utsira High, and is the second discovery in licence PL338C. The first discovery was made in exploration well 16/1-12 in 2009, which proved a 42-metre oil column in fractured granitic basement. - Rex Virtual Drilling an effective exploration de-risking tool - The EGS discovery well was the first time that the Group's Rex Virtual Drilling ("RVD") technology was applied to an unconventional weathered and fractured basement reservoir. RVD has also proven to be highly accurate in predicting dry wells. Over the past two years, Lime Norway has declined participation in more than 15 licences in Norway after RVD analyses. All the wells that were subsequently drilled in these licences came up dry, saving the Group millions of dollars in futile capital expenditure. Rex's multi-attribute version of the technology, RVD version 3 ("RVDv3"), can identify the location of oil reserves using conventional seismic data, independent of porosity and permeability estimates from conventional geological studies; hence further de-risking the exploration assets. "Risk and commercialisation are the two key factors guiding smart exploration in the region. Exploration and drilling remain hugely expensive and time-consuming, so any technology that can derisk these activities - especially in a low oil-price environment - is incredibly valuable," added Mr Mans Lidgren. - Optimisation of Lime Norway's portfolio - "Exploration in Norway continues to benefit from long-term political stability and an exploration friendly tax structure, which enables accredited pre-qualified petroleum companies such as Lime Norway to be eligible for tax rebates of 78 per cent of their upfront exploration costs," said Mr Mans Lidgren. Lime Norway secured a two-year extension of a credit facility of NOK 400 million (about US$46 million) in December 2016. Over the past months, Lime Norway has reviewed and optimised its portfolio of licences to build an enviable portfolio of exploration licences that showcases its unique approach to exploration and commercialisation. Licences failing to meet prospectivity criteria set by RVD and conventional geological evaluations have been relinquished to reduce future capital expenditure. Today, the Group holds in total interests in six offshore assets in Norwegian waters, spanning the established oil fields of the North Sea and Norwegian Sea, as well as the less-developed Barents Sea. The Group believes these holdings offer a balance between longer term development prospects and more short term assets that are close to existing pipeline infrastructure, resulting in a clear and fast route to potential commercialisation and ROI. Lime Norway is also evaluating several farm-in opportunities that are already oil producing, which would offer financial and tax benefits in addition to reducing overall business risk. Besides the three licences in the North Sea, Lime Norway holds a 20 per cent interest in licence PL841 in the Norwegian Sea, alongside operator Edison Oil (40 per cent), Statoil (20 per cent) and the Norwegian government-owned Petoro (20 per cent). The block had previously been explored in the 1990s. Current technologies, including RVDv3, indicate that a well drilled then, to be a missed discovery. RVDv3 will be used to determine whether commercialisation is now viable. The Group's other licence in the Norwegian Sea is PL762, which covers the entirety of the Vagar prospect, is also currently being evaluated using RVDv3. More than half of the estimated oil and gas resources in Norwegian waters remain to be produced[4]. In addition to developing prospects in established areas, the Group is also active in the further afield Barents Sea South region, where Lime Norway holds a 20 per cent share of the PL850 licence, east of the producing oil field Goliat. Once again, RVDv3 offers the Group the opportunity to invest in drilling in this field only when it is satisfied that commercial returns can be potentially realised for partners and shareholders. Rex's Norway strategy is underpinned not only by its technology, but also by the experience of the specialist Lime Norway team, led by CEO Terje Hagevang. "Our vision is for Lime Norway to be an oil producing company, with oil reserves of more than 100 million barrels of oil and at least 10 concessions prospected with Rex Virtual Drilling. We are working towards these aspirations," said Mr Terje Hagevang. [1]Source: http://rex.listedcompany.com/misc/QPR2016.pdf [2]Source: https://www.statoil.com/en/what-we-do/johan-sverdrup.html [3]Sources: http://bit.ly/2nU4UDC; http://bit.ly/2hnUUtW [4]Source: http://bit.ly/2nUkLlE About Lime Petroleum Norway AS Lime Petroleum Norway AS ("Lime Norway") is a small and fast growing exploration company established in 2012 in Skoyen in Oslo. The Company was pre-qualified in February 2013 and has since built a balanced portfolio of licences in frontier and mature areas. The company uses state-of-the-art data and Rex Virtual Drilling technology, in addition to market seismic attributes, in its exploration efforts. Lime Norway's vision: - To be an oil-producing company; - To have oil reserves of more than 100 million barrels; - To have at least 10 concessions all upgraded with Rex Virtual Drilling; and - To be a listed company, subject to necessary regulatory approvals. About Rex International Holding Rex International Holding was listed on Singapore Exchange Securities Trading Limited's Catalist Board on 31 July 2013. The Company owns a key set of proprietary and innovative exploration technologies, Rex Technologies, originating from the Company's Swedish founders. These include the game-changing Rex Virtual Drilling technology, the world's first direct hydrocarbon detector using seismic data, which literally enables the Group to 'see oil in the ground' by pinpointing the location of oil reservoirs in the sub-surface. Through the exploration accuracy of Rex Technologies which are applicable to both onshore and offshore oil exploration, the oil discovery success rate is significantly increased. The Company also owns the unique Rexonic ultrasound technology which is used for well bore cleaning which allows for significantly increased oil production in wells that have issues with clogging and deposits. Rex International Holding has stakes in exploration assets in the Oman, Norway, the US and Trinidad & Tobago. These offshore and onshore concessions cover an aggregate area of over 19,000 square kilometres in regions known for previous oil and gas discoveries. Located in politically stable countries with well-developed oil and gas infrastructures, Rex International Holding has a portfolio of assets that is geographically diversified and consists of both onshore and offshore concessions. Issued by Rex International Holding Limited Tel: +65 6908 4858 / +65 8518 8945 Mok Lai Siong This press release has been prepared by the Company and its contents have been reviewed by the Company's sponsor, PrimePartners Corporate Finance Pte. Ltd. (the "Sponsor") for compliance with the Singapore Exchange Securities Trading Limited (the "SGX-ST") Listing Manual Section B: Rules of Catalist. The Sponsor has not verified the contents of this press release. The Sponsor has also not drawn on any specific technical expertise in its review of this press release. This press release has not been examined or approved by the SGX-ST. The Sponsor and the SGX-ST assume no responsibility for the contents of this press release, including the accuracy, completeness or correctness of any of the information, statements or opinions made or reports contained in this press release. The contact person for the Sponsor is Ms Gillian Goh, Director, Head of Continuing Sponsorship, at 16 Collyer Quay, #10-00 Income at Raffles, Singapore 049318, telephone +65 6229 8088. Disclaimer This press release may contain projections and forward-looking statements that reflect the Company's current views with respect to future events and financial performance. These views are based on estimates and current assumptions which are subject to business, economic and competitive uncertainties and contingencies as well as various risks and these may change over time and in many cases are outside the control of the Company and its directors. Actual future performance, outcome and results may differ materially from those expressed in forward-looking statements as a result of a number of risks, uncertainties and assumptions. Representative examples of these factors include (without limitation) general industry and economic conditions, interest rate trends, cost of capital and capital availability, availability of real estate properties, competition from other companies and venues for the sale/distribution of goods and services, shifts in customer demands, customers and partners, changes in operating expenses, including employee wages, benefits and training, governmental and public policy changes and the continued availability of financing in the amounts and the terms necessary to support future business. No assurance can be given that future events will occur, that projections will be achieved, or that the Company's assumptions are correct. The Company does not assume any responsibility to amend, modify or revise any forward-looking statements, on the basis of any subsequent developments, information or events, or otherwise. These statements can be recognised by the use of words such as "expects," "plans," "will," "estimates," "projects," or words of similar meaning. Such forward-looking statements are not guarantees of future performance and actual results may differ from those forecast and projected or in the forward-looking statements as a result of various factors and assumptions. Shareholders and investors are cautioned not to place undue reliance on these forward looking statements, which are based on the current view of management of future events.


News Article | May 22, 2017
Site: www.businesswire.com

SOUTH SAN FRANCISCO, Calif.--(BUSINESS WIRE)--Genentech, a member of the Roche Group (SIX: RO, ROG; OTCQX: RHHBY), announced today that the U.S. Food and Drug Administration (FDA) has approved Actemra® (tocilizumab) subcutaneous injection for the treatment of GCA, a chronic and severe autoimmune condition. Actemra is the first therapy approved by the FDA for the treatment of adult patients with GCA. This is the sixth FDA approval for Actemra since the medicine was launched in 2010. “Today’s FDA decision means people living with giant cell arteritis will, for the first time, have an FDA-approved treatment option for this debilitating disease,” said Sandra Horning, M.D., chief medical officer and head of Global Product Development. “With no new treatments in more than 50 years, this approval could be transformational for people with GCA and for their physicians.” The approval is based on the positive outcome of the Phase III GiACTA study evaluating Actemra in patients with GCA. The results showed that Actemra, initially combined with a six-month steroid (glucocorticoid) regimen, more effectively sustained remission through 52 weeks (56 percent in the Actemra weekly group and 53.1 percent in the Actemra bi-weekly group) compared to placebo combined with a 26-week steroid taper (14 percent) and placebo combined with a 52-week steroid taper (17.6 percent).1 GiACTA (NCT01791153) is a Phase III, global, randomized, double-blind, placebo-controlled trial investigating the efficacy and safety of Actemra as a novel treatment for GCA. It is the first successful clinical trial conducted in GCA and the first to use blinded, variable-dose, variable-duration steroid regimens. The multicenter study was conducted in 251 patients across 76 sites in 14 countries. The primary endpoint was evaluated at 52 weeks. GCA, also known as temporal arteritis (TA), affects an estimated 228,000 people over the age of 50 in the U.S., and the disease is two to three times more likely to affect women than men.2,3 GCA is often difficult to diagnose because of the wide and variable spectrum of signs and symptoms. GCA can cause severe headaches, jaw pain and visual symptoms and if left untreated, can lead to blindness, aortic aneurysm or stroke.3 Treatment to date for people with GCA has been limited to high-dose steroids that play a role as an effective ‘emergency’ treatment option to prevent damage such as vision loss.3 Due to the variability of symptoms, complexity of the disease and disease complications, people with GCA are often seen by several physicians including rheumatologists, ophthalmologists and neurologists. Actemra is the first humanized interleukin-6 (IL-6) receptor antagonist approved for the treatment of adult patients with moderately to severely active rheumatoid arthritis (RA) who have used one or more disease-modifying antirheumatic drugs (DMARDs), such as methotrexate (MTX), that did not provide enough relief. The extensive Actemra RA IV clinical development program included five Phase III clinical studies and enrolled more than 4,000 people with RA in 41 countries. The Actemra RA subcutaneous clinical development program included two Phase III clinical studies and enrolled more than 1,800 people with RA in 33 countries. In addition, Actemra is also used as an IV formulation for patients with active polyarticular juvenile idiopathic arthritis (PJIA) or systemic juvenile idiopathic arthritis (SJIA) two years of age and older. Actemra is not approved for subcutaneous use in people with PJIA or SJIA. It is not known if Actemra is safe and effective in children with PJIA or SJIA under two years of age or in children with conditions other than PJIA or SJIA. Actemra is intended for use under the guidance of a healthcare practitioner. Actemra can cause serious side effects. Actemra changes the way a patient’s immune system works. This can make a patient more likely to get infections or make any current infection worse. Some people taking Actemra have died from these infections. Actemra can cause other serious side effects. These include: Patients should not receive Actemra if they are allergic to Actemra or if they have had a bad reaction to Actemra previously. Patients should tell their doctor if they have these or any other side effect that bothers them or does not go away: Patients should tell their doctor if they are planning to become pregnant, are pregnant, plan to breastfeed, or are breastfeeding. The patient and their doctor should decide if the patient will take Actemra or breastfeed. Patients should not do both. If a patient is pregnant and taking Actemra, they should join the pregnancy registry. To learn more, patients should call 1-877-311-8972 or talk to their doctor to register. Patients should tell their doctor right away if they are experiencing any side effects. Report side effects to the FDA at 1-800-FDA-1088 or http://www.FDA.gov/medwatch. Report side effects to Genentech at 1-888-835-2555. Please visit http://www.actemra.com for the full Prescribing Information, including Boxed Warning and Medication Guide, for additional Important Safety Information or call 1-800-ACTEMRA (228-3672). Actemra is part of a co-development agreement with Chugai Pharmaceutical Co. and has been approved in Japan since June 2005. Actemra is approved in the European Union, where it is known as RoActemra, and several other countries, including China, India, Brazil, Switzerland and Australia. Founded more than 40 years ago, Genentech is a leading biotechnology company that discovers, develops, manufactures and commercializes medicines to treat patients with serious or life-threatening medical conditions. The company, a member of the Roche Group, has headquarters in South San Francisco, California. For additional information about the company, please visit http://www.gene.com.


News Article | May 24, 2017
Site: www.nature.com

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.

Loading GCA Co. collaborators
Loading GCA Co. collaborators