text:
Route 66 to Vegas
September 27, 2007 in Arizona/Utah
We're back in civilization again and it seems a bit surreal. How could my vacation be over already, so soon, as if it never happened? I find it hard to believe that I'm sitting in a cubicle again and wonder why time passes so quickly when you're happy, but so agonizingly slow when you're waiting for your computer to be fixed or a shipment of purchases to arrive. At any rate, I must come to acceptance that, here I am at work, and all the anticipation of our vacation has now turned to wonderful memories and digital images.
I suppose it's time I summarize the rest of our vacation. I wanted to write more from Vegas, but the MGM Grand feels like they don't milk you for enough money with the hotel expense, they also charge $12/day for wireless Internet access. There was no way I was paying that, and besides pecking anymore on a tiny keyboard with a little wand was more than I could bear.
Would you be surprised to hear that our car broke down on the way to Vegas or that we got pulled over for a traffic offense and spent the night in jail? If not, then you haven't been paying attention to how our luck runs, but actually, none of those things happened!
Despite our car maintenance light giving us an ominous warning, we decided to push our luck to its limits and take the long road to Vegas via a sight-seeing tour along Route 66. Years ago, National Geographic did an article on Route 66 and ever since I've wanted to see it for myself. We were there, so we thought, "may as well." Plus, there was an actual ghost town on the map, just north of Oatman, AZ. How could we miss that? After all, how often do you get to see a ghost town?
So, we set off along a very long, deserted road through desert and mountains and saw very little of anything, except the occasional lot of trailers and dirt roads. The ghost town turned out to be an abandoned gold mine. We didn't see any other signs of previous life, no houses or stores, nothing that would suggest that humans had ever spent any time inhabiting this land. We didn't even realize we had passed the so called ghost "town" until we arrived in Oatman, a two-block long tourist spot (population 128) that looked like it could have been the original town center. We were surprised to see a number of cars parked there and people milling about. The lonely drive suggested that we were the only people to have ever passed that way.
The best thing about Oatman was the free Internet access. When I got out of the car to take pictures, I noticed one of the houses at the top of a very big hill had several large satellite dishes and I figured one must be for Internet. I pulled out the Pocket PC and was able to connect right away. How odd. The whole trip was that way–Internet access in the most unlikely of places and yet unavailable in the many of the more populated areas.
The rest of Route 66 was a lot of nothing. Desert as far as the eye could see, spotted with tiny little communities and a pocket of houses in the middle of nothingness that left us wondering why, of all the places on earth, the residents chose that spot. When we got to Needles, it seemed like a veritable city in comparison. According to Zip Skinny, the population is 5471. It was huge. There was an entire subdivision and a school. Passing through Needles reminded me of the Three Dog Night's, "Never Been to Spain." Since it was notable enough for them to sing about, here's what they had to say:
"…Well I never been to England
But I kinda like the Beatles
Well, I headed for Las Vegas
Only made it out to Needles
It must be real it
Oh, feels so good"
I'm not sure what feels so good, and the song makes even less sense to me than ever, if that's possible. Did people of the seventies have any standards for lyrics?
When we eventually got to Vegas, it was even more jarring than it's ever been. What a stark contrast to the many impoverished areas we passed through. Where the median household income is $20,000 or less in the rural areas we passed through, you wonder what possible opportunities one could have for education or advancement, or a way out. The Indian Reservations, in particular, seem as hopeless as any place I've ever seen. I mean, rural is wonderful if you love isolation, but if you're trying to give your children options, it's hard to see how it's possible in the middle of such nothingness. I feel like I should defend myself now…as an environmentalist, I see nothing wrong with nothingness. There is great value to me in undeveloped spaces. I think it's admirable to be able to survive in such an inhospitable place where water and rain are scarce, and I understand why people stay for emotional reasons, to remain connected and attached to one's roots. It's just that once you're there, it doesn't seem like there are a lot of options for leaving and I wonder how many stay by choice.
One of the biggest towns we passed through, Cameron, (before reaching the Grand Canyon – not on Route 66) had a number of Native Americans in the restaurant where we breakfasted. The population of Cameron is 2,125, 94% Native American. The median household income is $20,625 and 25% of the residents have a high school education. For many hours along our drives I found myself wondering what kind of business I could open there, if I were that sort of capable person, to create jobs. I wondered how I could bring education and resources, open a library perhaps, if they would want it. It's nearly impossible to pass through such places and not want to do something to help. It gives you a whole new perspective on poverty and the contrast between inner city poverty and rural poverty. They strike me now as two very different things.
That's about all there is to say about Route 66. The interesting part must be the other direction, to the east of Kingman.
In contrast, when we arrived in Vegas, it was utterly shocking the amount of wealth dripping out of the many towering hotels and casinos. The last time I was there was in July of 2000. At the time, the new Aladdin was almost ready to open, after the previous incarnation had been imploded and a new building erected in its place. Sunday, after we arrived in Vegas, we were walking down the Strip and I wondered, "where did the Aladdin go?" That one is gone too. The new building was remodeled to make way for a bigger and better casino, Planet Hollywood. All along the Strip, there are enormous buildings created in such lavishness, air-conditioned to 60 degrees, ornate and energy-demanding displays and lights…All in the middle of the desert, a place not meant to support the number of people who live and vacation there. I couldn't help myself thinking, "what if just one of these casino owners donated a few million dollars to build some libraries, maybe with computers with Internet access, for the rural towns all around Vegas? what would life be like for those residents?" When is it ever enough money? When do people ever have enough? I know, I know, I think too much. I've heard it forever.
So, that said, we arrived happily, uneventfully in Vegas…which I'll describe further in the next post.
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Zip-Pak is the global leader in resealable solutions for the flexible packaging industry. We operate in more than 52 countries around the world with a workforce of more than 270 dedicated flexible packaging professionals. These talented individuals adhere to the highest professional standards and focus their efforts to meeting and exceeding the needs of every Zip-Pak client. Combining this commitment to client success with our technical innovation and unmatched network of resources enables us to confidently and consistently deliver "uniquely flexible" solutions to our customers.
The Operations Manager provides leadership in both day-to-day and long term aspects required to oversee all operation's functions for Zip-Pak's Ottawa, IL facility. This includes: production, process engineering, quality, and maintenance. In addition, this position manages the relationships between the plant, sales, vendors and all activities related to operations. This position provides strategic leadership and coordination on manufacturing projects in support of the Global Director of Operations.
Principal Accountabilities: include the following. Other duties may be assigned.
Continue improvements in safety record and increase ownership of the safety program to the operator level. Focus to be placed on improving tracking and correcting of hazards / near misses reducing overall hazard opportunities in the facility (Sharps / Slips / Soft Tissue).
Develop, implement and oversee manufacturing plans and strategies including capacity planning regarding equipment, training, staffing, raw materials, packaging, systems, and general operations improvements.
Ensure compliance with Company standards for cost control, waste reduction, quality, OSHA, safety, AIB/SQF/HACCP and complete on-time delivery at the lowest possible cost.
Reviews production and operating reports and directs the resolution of operational, manufacturing, and maintenance problems to ensure minimum costs and prevent operational delays.
Monitor, report, and initiate action on results of efficiencies, quality, safety, delivery and costs.
Identify and implement cost savings initiatives.
Promote and implement 80/20 simplification principles.
Oversee fair and consistent administration of policies, work rules, disciplinary actions, promotions, performance reviews, pay increases, skill development, and job performance counseling for reports.
Manage all personnel issues within the facility; including such functions as staffing, compensation, behavioral issues, termination, transfer and promotions. Manage, coach, and develop talent.
Strive to maintain a non-union work environment.
Evaluate new product concepts for manufacturing capabilities.
Identifies, communicates and drives implementation of capital investments and improvement projects.
Balance work capacity across the plant.
Create strong relationships and work closely with all ZP operations in sharing knowledge, resources, technology, cost savings ideas, and product / process improvements.
Bachelor's degree from an accredited four-year college or university required.


text:
"Ask Phil"
Phil Abbott Co-founded Radical in 1997 and was instrumental in the design and production of the Radical model range. Phil's latest focus has been Developing a new race car company called Revolution Race cars that is now producing the A-One model. Phil has a tremendous passion about Race cars and the Racing community, and he has agreed to share his 40+ years of experience with the Radical Sportscar Registry members.
Submit a Question for Phil to Answer
Ask Phil #12 – Hi Phil, my car is an early Radical SR8 (2005) which runs the "closed" MBE ECU that can't be programmed. Is it possible to buy a similar "Open" MBE ECU. I want to do a few performance upgrades to the cylinder heads and was worried about the effects since the ECU can't be programmed Could you also recommend the best options for an upgrades and power gains? Regards Josh Broome (@jwb)
Ask Phil #11 – Hi Phil! I'm curious as to the background of the SR5. The car seems like a great idea to me. A lower stressed engine that would be great for track days where the owner isn't as concerned about getting every last bit out of power to weight. However, it seems it never really took off. I'd like to know what were the thoughts behind the SR5 and why you think it never caught on? The car was also available as a roller and able to accommodate a Sadev which I found to be something Radical doesn't typically do and was wondering if they were trying to tap a new market with this approach? My interest comes from owning SR8 Chassis 42 as a roller and looking to swap in a Honda K24 and a Sadev transaxle. Thanks! Joe (@JoeP)
Ask Phil #10 – Hi Phil, I have the 2013 Radical SR8 VIN 207. Using a new PC Laptop, the Radical ECU cable, and downloaded Life ECU 2.15.40, but when I connect to the SR8 (Life PTMon), it does not find the device. Any suggestions and assistance is appreciated. Vafa Fouroohi
Ask Phil Question # 9 – Hi Phil, I have bought a Prosport 1300 rms 215(one of the last ones?). I believe it was one of several cars sold to Mantorp park driving school in Sweden. I will drive it for the first time this year. Would it be advisable to test it with neutral wing and the front canards off to learn the cars balance with less downforce? I have never driven anything this fast, or mid engined on track before. Regards, Jonas Maurstad (@maurstad) from Norway
Question 8: Hi Phil! Whatever happened to that SR3 turbo project you guys had going on in early 2000's? I know one of these cars was used the first time a Radical broke the Nürburgring track record, but there is very little information currently available about these cars. Thanks, Robert L. (@lunatic)
Question 7: Hi Phil, I own the prototype SR3 SL 'Daisy' as it was known. It is now black rather than the white with the multi coloured stripes. There must be a few tales about this car….one pic I have noticed, is the registration plate looks to have been swapped for a photo shoot / test. The first white SL has the reg AE60 BFA in some shots, which is the reg of my car. Cheers Gareth (@gareth747474)
Question 6: Hi Phil, recently one of the Registry members purchase an RXC body off of Racecars direct and I was wondering if you remember anything about this car? According to the chassis number it appears to be the third RXC built. Also, there are two unique rectangular inlets on the front of the car. Do you remember this car?
Question 5: Hi Phil! Thanks for creating such amazing and affordable race cars! I have an early 2004 SR3 Chassis 165. It has the 1500 long stroke with the Keihin Carburetors and MBE ignition. I've wondered why Radical chose Carburetion for the early cars since the original Hayabusa engine came fuel injected. I did see an option for fuel injection on the Radical promo brochure for SR3's of that era but I believe that it was a Jenvey system and not a modified Suzuki F.I. System. Most of the Gen 1 SR3's I see have the carbs and not the Jenvey system. Crit
Question 4 – "I'm interested in how the SR8 was developed. I've got a couple of them (#42 and #87) and they came with such different equipment! #42 came with an AiM dash and a Quaife QBE58G gearbox, and the #87 came with a Stack dash and a Hewland JFR200 gearbox. Later came the QBE72G gearbox. Can you share any stories or history about how you worked with those different suppliers as the SR8 evolved from 2005 to 2012 (when it all seemed to come together to a single supplier set). Thanks! John (@parsonsj)
Question 3: "I'm interested in the relationship with Quaife, such as, how did the decision (and design/development) to make the GDR (Gear Driven) differential come about? It's a neat piece." (from Russ M)
Question 2: Prosport Suspension tuning (from Neil C) – "I have a 2001 Prosport. I made some adjustable pushrods for the front suspension to give me the ability to better corner weight the car. I proceeded to corner weight the car with both the NIK and rear sway bars detached and found when it was dialed in, I had no travel to the suspension left. Seems the front suspension has a very narrow field of motion. I ended up replacing the adjustable push rods with the stock pushrods. Am I missing the concept of the car/suspension? Is it best just to leave the car alone?"
Question #1: Phil, many people dream of building their own race car or starting a car company to build a special car that meets their needs… But, you actually did it! What elements drove the creation of Radical Sportscars?

redpajama_set_name: RedPajamaCommonCrawl


This family had two separate sessions to take advantage of both the morning light and the sunset in the evening. They did the session at a combination of a private home and down at the beach at the Fairmont Orchid Resort.
During their portrait session they did the separate family groupings as well as a large group portrait.


Poster for LEGO Marvel Super Heroes.
Make way Marvel zombies, there's more heroes -- and villains -- coming to the upcoming LEGO Marvel Super Heroes video game.
At Saturday's Marvel video game panel, the publisher announced the addition of several new playable characters to the October 22 game. Joining this franchise are the heroes Power Man, Iron Fist, Havok, Polaris, Moon Knight, She-Hulk, Ant-Man and Black Bolt, as well as villains such as Taskmaster, Absorbing Man, Nightmare, Pyro, Shocker and Electro (both the comic version and the Amazing Spider-Man 2 movie rendition). And if that wasn't enough, they're also adding Aunt May.
This comes in addition to the previously announced Asgard DLC Pack whichi includes the Asgardian heavyweights of Malekith, Kurse, Sif, Volstagg, Odin, Hogun, Fandral and Earth's Jane Foster. And a third group of characters will be available to those that pre-order the game. This super pack includes Dark Phoenix, Thanos, A-Bomb, Beta Ray Bill, Falcon, Winter Solder, as well as twelve new vehicles including the Spider Mobile.
Marvel also revealed some of the costume variants available for Spider-Man, including his current Superior Spider-Man garb as well as his all-white Future Foundation suit.


We were discussing the concept of laminar and turbulent flow, Reynolds experiment, frictional loss in pipes, derivation of expression for loss of head due to friction in pipes, co-efficient of friction in terms of shear stress, basics of shear stress in turbulent flow, minor head losses in pipe flow, basic concept and working of syphon and also the concept of hydraulic gradient and total energy line, in the subject of fluid mechanics, in our recent posts.
Now we will go ahead to see the, flow through pipes in series and parallel, in the subject of fluid mechanics, with the help of this post.
When pipes of different lengths and different diameters are connected end to end to form a pipe line, such arrangement or connection of pipes will be considered as pipes in series or compound pipes.
Important Point: Discharge passing through each pipe will be same.
Following figure, displayed here, indicates the arrangement of connection of three pipes in series.
We must note it here that difference in liquid surface level will be equal to the sum of total head loss in the pipes.
If we neglect the minor head losses, we will have following equation for total head loss as mentioned here.
Let us consider that co-efficient of friction i.e. f is same for all three pipes and therefore we can write the equation for head loss as mentioned here.
Further we will go ahead to find out the basic concept, Flow through pipes in parallel, in the subject of fluid mechanics, with the help of our next post.


Visit the Gold Coast and 32Red's top online casino - awarded Casino of the Year since 2003 by the Casino watchdog Casinomeister - and try your hand at this fun and rewarding slot machine.
This is a great game in terms of game-play, looks and versatility. It's vibrant colours enhance what is already a superb, simple game to enjoy. Plus it's basic, and truly classic, 3-reel setup is improved by the addition of 5 paylines. Meaning there are more chances to win, if you're a little more adventurous.
The key to the game is filling the paylines with the Gold Coast symbols. Three of them and you could win 6000 coins! However, as you would expect from a 32Red machine, there a host of other features that will net you some serious coin too.


Being an ABAP HR Developer, i'm concerned by SuccessFactors.
What sap education course can i follow to start training in that environment ?
What are the technicals skills needed to be able to work in that environment ?
Would any developer be able to program there ?


Select from one of four color options for the perfect match. Then add the wedding date for a personalized touch in the bottom right corner. These are great in the living room on the course or on the bed for those chilly nights. Take one outdoors and see just how warm the premium sherpa keeps you.


"VidaBox LLC", a company reknowned for innovative media center PCs have just announced the world's first combined Dual HD-DVD *and* Blu-Ray media center systems. No problems with varying formats here! The Dual HD "LUX" and "MAX" systems also feature HDCP-compliant outputs, dual analog / HDTV tuners and much more.
For full details, have a read of the supporting press release on VidaBox's website.
"The Dual HD LUX™ & MAX™ systems are perfect for those who can appreciate convenience, high-fidelity audio, and high-quality video performance in a single, easy-to-use package," says Steven Cheung, co-founder of VidaBox. "High definition is the new video standard, and many customers want the ability to play both Blu-Ray and HD-DVD discs. Combining that ability with the attractive, rich feature set of media center allows everyone to conveniently use the VidaBox as the center of all their digital entertainment needs. There's no need to buy two separate high definition disc players, nor is there a need to use a PVR/TiVo®, stereo, DVD player, etc. when everything - TV, movies, music, pictures, and more - can be easily accessed from a single remote at the touch of a button."
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Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells
Ryo Kurita,
Affiliation Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
Noriko Suda,
Kazuhiro Sudo,
Kenichi Miharada,
Takashi Hiroyama,
Hiroyuki Miyoshi,
Affiliation Subteam for Manipulation of Cell Fate, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
Kenzaburo Tani,
Affiliation Department of Molecular Genetics, Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan
Yukio Nakamura
* E-mail: yukionak@brc.riken.jp
Ryo Kurita Noriko Suda ... Yukio Nakamura
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Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.
Citation: Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, Miyoshi H, et al. (2013) Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells. PLoS ONE 8(3): e59890. https://doi.org/10.1371/journal.pone.0059890
Editor: Dimas Tadeu Covas, University of Sao Paulo - USP, Brazil
Received: November 13, 2012; Accepted: February 19, 2013; Published: March 22, 2013
Copyright: © 2013 Kurita et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the project for realization of regenerative medicine in the Ministry of Education, Culture, Sports, Science, and Technology in Japan (MEXT) and other grants from the MEXT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The transfusion of RBCs is a standard clinical therapy. Currently, the supply of RBCs for transfusion is dependent on donation of blood by large numbers of volunteers. This system has two important shortcomings, namely, shortages of volunteers and contamination of donated blood by microorganisms. One promising way around these problems might be to produce RBCs in vitro [1], [2], [3] from hematopoietic stem/progenitor cells [4], [5], embryonic stem (ES) cells [6], or induced pluripotent stem (iPS) cells [7].
Recently, we developed a new approach in the mouse for producing RBCs in vitro [8]. Using mouse ES cells, we successfully established immortalized erythroid progenitor cell lines, which we termed mouse ES cell-derived erythroid progenitor (MEDEP) cell lines, and confirmed that these cell lines could produce mature RBCs in vitro [8]. The logical next step was to create immortalized human erythroid progenitor cell lines that could provide a convenient and reliable ex vivo source for RBC production. These cell lines could also be of value for a range of basic science investigations, for example, into erythroid differentiation and enucleation. The present study shows the feasibility of establishing immortalized human erythroid progenitor cell lines and demonstrates that enucleated RBCs can be induced to differentiate in these cell lines.
Human iPS cell lines (HiPS-RIKEN-3A and HiPS-RIKEN-4A) and the OP9 cell line were obtained from the Cell Engineering Division of RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). iPS cells were maintained in an undifferentiated state in the presence of a feeder cell line, SNL76/7, as described previously [9]. The SNL76/7 feeder cell line was obtained from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK) and cultured in DMEM (Sigma, St. Louis, MO, USA) supplemented with 7.5% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA).
Establishment of Human iPS Cell Lines Expressing TAL1
The internal ribosomal entry site (IRES)-puromycin resistant gene (Puror) cassette was amplified by polymerase chain reaction (PCR) using pIRESpuro3 plasmid DNA (TAKARA BIO, Otsu, Shiga, Japan) with the following primers: 5′-tga tcc tct aga ctg gaa tta att cgc tgt ctg cga-3′ (sense) and 5′-gtg ggg gtt aac tca ggc acc ggg ctt gcg ggt ca-3′ (anti-sense). After confirmation of the DNA sequence, the IRES-Puror cassette was cloned into the CSII-EF-RfA lentiviral vector plasmid (Figure S1), which contains the human EF-1α promoter and the Gateway system (Invitrogen), to produce CSII-EF-RfA-IRES-Puror. TAL1 cDNA and the recombination sequences for the Gateway system (Invitrogen) were amplified by reverse transcription-PCR (RT-PCR) using human fetal liver total RNA purchased from TAKARA BIO with the following primers: 5′-ggg gac aag ttt gta caa aaa agc agg ctt cac cat gac cga gcg gcc gcc gag cga-3′ (sense) and 5′-ggg gac cac ttt gta caa gaa agc tgg gtc tca ccg agg gcc ggc tcc atc ggc-3′ (anti-sense). The RT-PCR amplification product (1060 bp) was subcloned into the pDONR222 vector (Invitrogen) and verified by DNA sequencing. The TAL1 cDNA was then transferred to the CSII-EF-RfA-IRES-Puror plasmid using Gateway LR clonase (Invitrogen) to produce CSII-EF-TAL1-IRES-Puror. The vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped lentiviral vector preparation was performed as described previously [10] with the exception that Fugene HD (Roche, Mannheim, Germany) was used for transfection instead of polyethyleneimine. Human iPS cell lines were transduced with CSII-EF-TAL1-IRES-Puror lentiviral vector in the presence of polybrene (8 µg/ml; Sigma) and iPS cells expressing TAL1 were selected using puromycin (2 µg/ml; InvivoGen, San Diego, CA, USA).
Construction of Lentiviral Vector Plasmid for Expression of Human Papilloma Virus Type 16-E6/E7 Using the Tetracycline-inducible System
The CSIV-TRE-RfA-UbC-KT lentiviral vector plasmid (Figure S2) contains the humanized Kusabira-Orange 1 (hKO1) fluorescent protein gene [11] and the reverse tetracycline (Tet)-controlled transcriptional transactivator (TAKARA BIO) linked by the Thosea asigna virus 2A peptide sequence under the control of human ubiquitin C promoter; this plasmid is designed to express cDNA under the control of the Tet-responsive promoter (TAKARA BIO). The human papilloma virus 16 (HPV16)-E6/E7 genomic DNA subcloned into pENTR201 (Invitrogen) was a kind gift of Dr. Kiyono (Division of Virology, Molecular Oncology Group, National Cancer Center Research Institute, Japan). The HPV16-E6/E7 genomic DNA was transferred to the CSIV-TRE-RfA-UbC-KT plasmid using Gateway LR clonase to produce CSIV-TRE-HPV16-E6/E7-UbC-KT. Lentiviral vector preparation was performed as described above.
Specific Factors
The following factors were used in this study: human stem cell factor (SCF; R&D systems, Minneapolis, MN, USA), human erythropoietin (EPO; Kirin Brewery, Tokyo, Japan), human FLT3 ligand (FLT3-L; R&D systems), dexamethasone (DEX; Sigma), human vascular endothelial growth factor (VEGF; R&D systems), human insulin-like growth factor-II (IGF-II; R&D systems), and human thrombopoietin (TPO; R&D systems).
Establishment of Immortalized Erythroid Progenitor Cell Lines from Human iPS Cells Expressing TAL1
The protocol schedule is summarized in Table 1 and a graphical depiction of the process is shown in Figure 1. To induce hematopoietic cells from HiPS-RIKEN-3A-TAL1 and HiPS-RIKEN-4A-TAL1 cells, cells were cultured on OP9 feeder cells in basal differentiation medium composed of IMDM (Sigma) supplemented with 15% fetal bovine serum (FBS; Invitrogen), ITS (10 µg/ml human insulin, 5.5 µg/ml human transferrin, and 5 ng/ml sodium selenite; Sigma), 50 mg/mL ascorbic acid (Sigma), 0.45 mM α-monothioglycerol (Sigma), and PSQ (100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine; Invitrogen) in the presence of VEGF (20 ng/ml) and IGF-II (200 ng/ml). From day 10, the cells were cultured on OP9 cells in the presence of SCF (50 ng/ml), EPO (3 IU/ml) and DEX (10−6 M). On day 16, the HPV16-E6/E7 expression system was introduced into the cells by lentiviral transduction. Four days later, the cells were cultured on OP9 cells in the presence of DOX (1 µg/ml), SCF (50 ng/ml), EPO (3 IU/ml) and DEX (10−6 M). The medium was usually changed twice per week, except when cell numbers were low. Around three months after initiation of culture, the cells were able to proliferate without feeder cells. After this time point, we maintained the cells in a serum-free medium, StemSpan SFEM® medium (StemCell Technologies, Vancouver, BC, Canada), in the presence of specific factors.
PowerPoint slide
Figure 1. Schematic outline of the procedures for establishing immortalized human erythroid progenitor cell lines from iPS cells and from hematopoietic stem/progenitor cells in umbilical cord blood.
https://doi.org/10.1371/journal.pone.0059890.g001
Table 1. An outline of the culture schedule used to establish immortalized cell lines from iPS cells.
https://doi.org/10.1371/journal.pone.0059890.t001
Establishment of Immortalized Erythroid Progenitor Cell Lines from Human CD34-positive Hematopoietic Stem/progenitor Cells
The protocol schedule is summarized in Table 2 and a graphical depiction of the process is shown in Figure 1. CD34-positive hematopoietic stem/progenitor cells derived from umbilical cord blood were obtained from the Stem Cell Resource Network in Japan (Banks at Miyagi, Tokyo, Kanagawa, Aichi, and Hyogo) through the RIKEN BioResource Center. CD34-positive cells (1×105 cells) were cultured in a serum-free medium, StemSpan SFEM® medium (StemCell Technologies), in the presence of SCF (50 ng/ml), TPO (50 ng/ml) and FLT3-L (50 ng/ml). On day 1 of culture, the HPV16-E6/E7 expression system was introduced into the cells by lentiviral transduction. Four days after introduction of the HPV16-E6/E7 expression system, the cells were cultured in the presence of doxycycline (DOX, 1 µg/ml; TAKARA BIO), SCF (50 ng/ml), EPO (3 IU/ml) and DEX (10−6 M). DOX was used as a substitute for Tet, and the presence of DOX induced the expression of HPV16-E6/E7. The medium was usually changed twice per week, except when cell numbers were low.
Table 2. An outline of the culture schedule used to establish immortalized cell lines from hematopoietic stem/progenitor cells.
Induction of Differentiation of Immortalized Erythroid Progenitor Cell Lines
The immortalized erythroid progenitor cell lines were induced to differentiate into more mature erythroid cells by culture in erythroid differentiation medium; IMDM (Sigma) containing 10% human AB serum (Kohjin Bio, Saitama, Japan or TAKARA BIO), α-tocopherol (20 ng/ml; Sigma), linoleic acid (4 ng/ml; Sigma), cholesterol (200 ng/ml; Sigma), sodium selenite (2 ng/ml; Sigma), holo-transferrin (200 µg/ml; Sigma), human insulin (10 µg/ml; Sigma), ethanolamine (10 µM; Sigma), 2-ME (0.1 mM; Sigma), D-mannitol (14.57 mg/ml; Sigma), mifepristone (an antagonist of glucocorticoid receptor, 1 µM; Sigma) and EPO (5 IU/ml).
A sample of cells (<1×106) was stained with monoclonal antibodies (MoAbs) in 100 µl of staining medium (phosphate buffered saline [PBS; Sigma] containing 2% FBS [Invitrogen] and 0.25% sodium azide [Sigma]) for 30 min on ice. The cells were washed twice with the staining medium, and analyzed using a FACS Calibur (BD Biosciences, San Jose, CA, USA). MoAbs against human antigens were conjugated with fluorescein isothiocyanate (FITC), Alexa Fluor® 488 (Alexa488) or allophycocyanin (APC). FITC-conjugated CD34 (cat. #555821), CD36 (cat. #555454), CD41a (cat. #555466), and CD71 (cat. #555536), Alexa488-conjugated CD11b (cat. #557701) and APC-conjugated CD33 (cat. #551378), CD45 (cat. #555485), c-KIT (cat. #550412), glycophorin A (GPA) (cat. #551336) and HLA-ABC (cat. #555555) were purchased from BD Biosciences. FITC-conjugated mouse IgG1 (cat. #555748), IgM (cat. #555583), APC-conjugated mouse IgG1 (cat. #555751) and IgG2b (cat. #555745) were also purchased from BD Biosciences and used as isotype controls. Cell viability was monitored by propidium iodide (PI; Sigma) staining. Flow cytometry data was analyzed using CellQuest analysis software (BD Biosciences). In this analysis, PI-positive cells were excluded as being dead and PI-negative cells were analyzed as being viable.
Functional Analysis of Hemoglobin
Following induction of differentiation of immortalized erythroid progenitor cells (5×107 cells) for 4 to 6 days, the cells were collected, washed with PBS, and subjected to analysis. Oxygen equilibrium curves were determined using a Hemox-Analyzer Model B (TSC Scientific, New Hope, PA). The gas phase gradients were obtained using nitrogen and room air, and the curves were run in both directions. Human peripheral blood cells and umbilical cord blood cells were used as the controls.
Quantitative Reverse Transcription-polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from cells using ISOGEN™ reagent (Wako, Osaka, Japan). Reverse transcription was carried out using 4.5 µg total RNA and the SuperScript®III First Strand Synthesis System (Invitrogen) in a 20 µl reaction volume. After reverse transcription, 180 µl of water was added to the reaction mixture and a 1 µl aliquot was used for each PCR. PCR was carried out using FastStart TaqMan® Probe Master and Universal ProbeLibrary Probes (Roche, Mannheim, Germany). PCR products were monitored by FAM (CarboxyFluorescein) dye fluorescence using a Thermal Cycler Dice® Real Time System (Takara Bio). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification was used as the internal control.
Primers and Probes
The following primers and probes were used in this study: human GATA1, sense primer 5′-cac tga gct tgc cac atc c-3′ and antisense primer 5′-atg gag cct ctg ggg att a-3′ (Probe #26); human GATA2, sense primer 5′-aag gct cgt tcc tgt tca ga-3′ and antisense primer 5′-ggc att gca cag gta gtg g-3′ (Probe #70); human GFI1B, sense primer 5′-cct ctt gtg ccc agc act-3′ and antisense primer 5′-cgt gag ggg tgg aga aga c-3′ (Probe #41); human EKLF, sense primer 5′-aca cca aga gct ccc acc t-3′ and antisense primer 5′-gta gtg gcg ggt cag ctc-3′ (Probe #7); human EPO receptor (EPOR), sense primer 5′-ttg gag gac ttg gtg tgt ttc-3′ and antisense primer 5′-agc ttc cat ggc tca tcc t-3′ (Probe #69); human α-globin, sense primer 5′-gac ccg gtc aac ttc aag c-3′ and antisense primer 5′-aga agc cag gaa ctt gtc ca-3′ (Probe #10); human β-globin, sense primer 5′-gca cgt gga tcc tga gaa ct-3′ and antisense primer 5′-cac tgg tgg ggt gaa ttc tt-3′ (Probe #61); human γ-globin, sense primer 5′-tgg atc ctg aga act tca agc-3′ and antisense primer 5′-gcc act gca gtc acc atc t-3′ (Probe #72); human c-MYB, sense primer 5′-agc aag gtg cat gat cgt c-3′ and antisense primer 5′-gat cac acc atg atg aag aat cag-3′ (Probe #37); human SOX6, sense primer 5′-gct tct gga ctc agc cct tt-3′ and antisense primer 5′-gga gtt gat ggc atc ttt gc-3′ (Probe #67); human BCL11A, sense primer 5′-ccc aaa cag gaa cac ata gca-3′ and antisense primer 5′-gag ctc cat gtg cag aac g-3′ (Probe #52); human TAL1 (to detect endogenous TAL1 gene), sense primer 5′-tgt gtg aga gac ggt gtc ttg-3′ and antisense primer 5′-caa ggc tgc aga cag caa-3′ (Probe #19); human Band 3, sense primer 5′-tct tca gga acg tgg agc tt-3′ and antisense primer 5′-cct cat caa agg ttg cct tg-3′ (Probe #89); human Band 4.1, sense primer 5′-cca cac tga gac caa gac ca-3′ and antisense primer 5′-cca agt ctc cac tgt tgt cgt-3′ (Probe #44); human Ankyrin-1, sense primer 5′-gag cac gag gag gtg act gt-3′ and antisense primer 5′-gtg tcg agg tgt gat cct tg-3′ (Probe #65); human α-Spectrin, sense primer 5′-gct ttg aaa ggg acc tcg ta-3′ and antisense primer 5′-ctc tgc tgt ctc ccc cag t-3′ (Probe #14); human GAPDH, sense primer 5′-agc cac atc gct cag aca c-3′ and antisense primer 5′-gcc caa tac gac caa atc c-3′ (Probe #60).
Morphological Analysis and Cell Staining
Cell morphologies were assessed using smears prepared on microscope slides or attached to microscope slides using a Cytospin 3 (Thermo Electron Corporation, Waltham, MA, USA). The cells were stained with Diff-Quik (Sysmex International, Kobe, Japan) and analyzed by microscopy.
Cell size and cell viability were measured using an automated cell counter, ViCell™ (Beckman Coulter, Fullerton, CA, USA).
Supravital staining was performed by incubating the cells in 0.3% new methylene blue solution (Muto Pure Chemicals, Tokyo, Japan) at room temperature for 20 min.
Immunostaining with GPA antibody was performed as reported previously [12]. Cells were spun onto microscope slides, washed with PBS(−) (PBS free of Ca and Mg) and fixed in 2% paraformaldehyde/PBS on ice for 10 min. After fixation, the cells were washed three times with PBS(−). Nonspecific binding was blocked with 2% BSA/PBS for 1–3 hr. Samples were incubated at 4°C overnight with mouse monoclonal biotinylated anti-human GPA antibody (R&D systems) diluted to 1∶200 in blocking solution. At the same time, SYTO16 solution (Invitrogen), which is a cell membrane-permeable fluorochrome dye that stains nucleic acids, was added to the staining mixture at a final concentration of 0.5 µM. After extensive washing with PBS(−), the cells were incubated with Alexa 647-conjugated streptavidin for 30 min to 1 hr to detect the biotinylated anti-human GPA antibody bound to the cells. After washing, the cells were observed with an LSM780 laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
Benzidine staining was performed using the Peroxidase Stain DAB Kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer's instructions. After staining, coverslips were mounted using Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA) in order to identify nucleated and enucleated cells.
Establishment of Immortalized Erythroid Progenitor Cell Lines from Human iPS Cells
Initially, we attempted to establish immortalized human erythroid progenitor cell lines from human ES cells and iPS cells using essentially the same protocol as for the successful establishment of MEDEP cell lines from mouse ES cells [8]. Three human ES cell lines and two human iPS cell lines were used in these experiments but none yielded abundant erythroid cells although some hematopoietic cells were induced. Thus, the protocol for establishing MEDEP cell lines failed to establish immortalized erythroid progenitor cell lines from human pluripotent stem cell lines.
Next, we investigated whether enforced expression of the transcription factor TAL1 might enable establishment of immortalized erythroid progenitor cell lines. TAL1 plays essential roles in early hematopoiesis [13] and differentiation of erythroid cells and megakaryocytes [14], [15]. In an earlier study, we also showed that its expression improved the efficiency of inducing hematopoietic cells from common marmoset ES cells [12]. Therefore, we forced expression of TAL1 in human iPS cell lines (HiPS) [16] and established sub-lines, HiPS-TAL1. HiPS-TAL1 cells showed a significant improvement in the efficiency of induction of hematopoietic cells when grown on OP9 feeder cells in the presence of insulin-like growth factor-II (IGF-II) and vascular endothelial growth factor (VEGF) (Figure 2). Long-term cultures of HiPS-TAL1 cells were also initiated on OP9 cells in the presence of stem cell factor (SCF), erythropoietin (EPO) and thrombopoietin (TPO). The cells proliferated continuously and no longer required OP9 cells after they had been in culture for approximately three months. As a result, we were able to establish six immortalized cell lines that proliferated continuously for more than 1 year. However, these cell lines expressed few hematopoietic cell markers and did not differentiate into more mature cells (data not shown).
Figure 2. Effect of enforced expression of a transcription factor, TAL1, on induction of hematopoietic cells from human iPS cells. HiPS, human iPS cells (HiPS-RIKEN-3A).
HiPS-TAL1, HiPS cells expressing TAL1 (HiPS-RIKEN-3A-TAL1). The cells were analyzed after the induction of differentiation of hematopoietic cells for 15 days. (A) Fold increase of production of hematopoietic cells from HiPS-TAL1 cells compared to HiPS cells. (B) Flow cytometer analysis. CD71, transferrin receptor. Glycophorin A (GPA), an erythroid specific marker. Percentages of GPA-positive cells are indicated in the figure. (C) Morphology of the cells derived from HiPS-TAL1 cells. Scale bar indicates 50 µm. The comparison of HiPS-RIKEN-4A and HiPS-RIKEN-4A-TAL1 showed similar results.
Immortalized erythroid progenitor cell lines can be established by transformation of erythroid progenitor cells with the HPV16-derived proteins HPV16-E6/E7 [17]. These cell lines do not produce mature erythrocytes, such as enucleated RBCs, possibly because continuous expression of HPV16-E6/E7 inhibits terminal differentiation of the cells.
In order to test this possibility, we used a Tet-inducible expression system [18], [19] to control expression of HPV16-E6/E7 in iPS-derived hematopoietic progenitor cells and investigated whether such expression inhibited establishment of immortalized erythroid progenitor cell lines. Hematopoietic cells were induced from HiPS-TAL1 cells that had been in culture for 16 days (see Materials and methods); the cells were infected with the lentiviral vector containing the Tet-inducible expression system for HPV16-E6/E7. One day after viral infection, the cells were transferred to a new culture on OP9 cells in the presence of SCF, EPO and dexamethasone (DEX) for 3 days. The cells were then cultured in the presence of SCF, EPO, DEX and doxycycline (DOX) over a prolonged period with regular changes of medium. DOX was used as a substitute for Tet, and the presence of DOX induced expression of HPV16-E6/E7. The cells proliferated continuously for more than 1 year; thus, we succeeded in establishing immortalized cell lines. Two cell lines were established from two independent trials using two different HiPS-TAL1 cell lines. We designated these cells as human iPS cell-derived erythroid progenitor (HiDEP) cell lines. HiDEP-1 and HiDEP-2 were established from HiPS-RIKEN-4A and HiPS-RIKEN-3A, respectively.
Establishment of Immortalized Erythroid Progenitor Cell Lines from CD34-positive Hematopoietic Cells in Human Umbilical Cord Blood
As it would be more convenient if immortalized human erythroid progenitor cell lines could be established without recourse to ES or iPS cells, we investigated whether our protocol could be applied to the CD34-positive hematopoietic stem/progenitor cells in umbilical cord blood. CD34-positive cells were collected from umbilical cord blood and cultured in the presence of SCF, TPO and FLT3-ligand (FLT3-L) for 1 day; the cells were then infected with the lentiviral vector containing the Tet-inducible expression system for HPV16-E6/E7. One day after viral infection, the cells were cultured with SCF, EPO and DEX for 4 days. Non-adherent cells were then collected and cultured in the presence of SCF, EPO, DEX and DOX over a prolonged period with regular changes of medium. The cells proliferated continuously for more than 1 year; thus, we also succeeded in establishing immortalized hematopoietic cell lines from umbilical cord blood cells as well. In total, three cell lines were established from three independent trials using three different cord blood samples. We designated these cells as human umbilical cord blood-derived erythroid progenitor (HUDEP) cell lines, HUDEP-1, HUDEP-2 and HUDEP-3.
Dependency on Externally Supplied Culture Factors
The HiDEP and HUDEP cell lines varied in their dependency on externally supplied culture factors (Table S1); thus, for example, the survival and proliferation of HiDEP-1 cells were dependent on DOX (HPV16-E6/E7) and EPO, and partially dependent on DEX but not on SCF (Figure 3A) and the HUDEP-1 cells were dependent on DOX and SCF and partially dependent on EPO but not on DEX (Figure 3B). After confirmation of these dependencies, all cell lines were cultured in the presence of essential factors alone for each cell line.
Figure 3. Dependency of the established erythroid progenitor cell lines on externally supplied culture factors.
(A) The survival and proliferation of HiDEP-1 cells are dependent on DOX (HPV16-E6/E7) and EPO and partially dependent on DEX. (B) The survival and proliferation of HUDEP-1 cells are dependent on DOX (HPV16-E6/E7) and SCF and partially dependent on EPO. (A, B) DOX, doxycycline; expression of HPV16-E6/E7 is induced by DOX. SCF, stem cell factor. EPO, erythropoietin. DEX, dexamethasone. ALL, cells were cultured in the presence of DOX, SCF, EPO and DEX. –DOX, –SCF, –EPO, –DEX, cells were cultured after deprivation of DOX, SCF, EPO and DEX, respectively. Dependencies of other cell lines on externally supplied culture factors are summarized in Table S1.
To date, all of the cell lines have continued to proliferate vigorously with no indication of slowdown in cell division rates. We also confirmed that cultures could be re-established after freeze-thaw cycles for all cell lines. Below, we describe the characteristics of the cell lines after continuous culture for more than 6 months and after more than 80 cell division cycles.
Expression of Cell Surface Molecules
Flow cytometric analyses demonstrated that both HiDEP-1 and HiDEP-2 cells expressed the erythroid-specific cell surface marker glycophorin A (GPA) at a high level (Figure 4A), while other hematopoietic cell markers, such as CD11b, CD33, CD34, CD41a and CD45, were not detectable (Figure S3A). CD36 and c-KIT (CD117), markers of immature erythroid cells, were detected at very low levels (Figure 4A and Figure S3A).
Figure 4. Flow cytometer analyses of the established erythroid progenitor cell lines.
(A) Representative results of HiDEP cells. (B) Representative results of HUDEP cells and HUDEP-1 expressing TAL1, HUDEP-1-TAL1. (A, B) GPA, glycophorin A. CD71, transferrin receptor. c-KIT, the receptor of SCF. CD36, a marker of immature erythroid cells.
All three HUDEP cell lines expressed CD71, glycophorin A, CD36 and c-KIT (Figure 4B). In contrast to the HiDEP cell lines, HUDEP cell lines expressed CD33 and CD45 at various levels, although non-erythroid markers such as CD11b and CD41a were not detectable (Figure S3B). This difference between HiDEP and HUDEP cells might be due to the fact that HiDEP cell lines were established from cells expressing TAL1. To determine whether this was the case, we forced expression of TAL1 in HUDEP-1 cells at 7 days after induction of HPV16-E6/E7 expression and established the cell line HUDEP-1-TAL1. However, the phenotype and characteristics of HUDEP-1-TAL1 cells were quite similar to those of HUDEP-1 cells (Figure 4B and Figure S3B).
Induction of Differentiation
Next, we examined whether HiDEP and HUDEP cells could differentiate into more mature stages and produce enucleated RBCs. We found that differentiation could be induced by culturing the HiDEP and HUDEP cells in an erythroid differentiation medium in the presence of EPO alone (see Materials and methods).
Production of Hemoglobin
Upon centrifugation, the HiDEP-1 cells produced a red cell pellet even before the induction of differentiation (Figure 5A) suggesting a continuous and abundant production of hemoglobin. In contrast, upon centrifugation, the HUDEP-1 cells yielded a light orange cell pellet before the induction of differentiation and a red cell pellet after the induction of differentiation (Figure 5B) suggesting that hemoglobin synthesis was upregulated following differentiation.
Figure 5. Cell pellets of the established erythroid progenitor cell lines.
(A) HiDEP-1 cells before the induction of differentiation. (B) HUDEP-1 cells before and after the induction of differentiation. HUDEP-1 cells were cultured in erythroid differentiation medium on OP9 feeder cells to maintain cell viability during the differentiation process. All other cell lines also showed red cell pellets after the induction of differentiation.
Functional Analysis of Hemoglobin Produced in HiDEP and HUDEP Cells
We used a Hemox-Analyzer to measure the oxygen binding and dissociation abilities of the hemoglobin produced in mature cells derived from the HiDEP and HUDEP cells [20], [21]. Although the oxygen binding and dissociation curves obtained with HiDEP- and HUDEP-derived cells differed between cell lines, they showed similar curves to those obtained with adult peripheral blood or umbilical cord blood (Figure 6). The variation among the curves may be due to the types of hemoglobin expressed in each cell line, i.e., adult type hemoglobin, fetal type hemoglobin or mixture of both types (see below). Apart from this variation, the data indicate that HiDEP and HUDEP cells can produce hemoglobin with oxygen binding and dissociation abilities equivalent to red blood cells produced in vivo.
Figure 6. Oxygen-carrying abilities of hemoglobin produced in the established erythroid progenitor cell lines.
Oxygen equilibrium curves were determined using an automated apparatus. Following the induction of differentiation, the cells were subjected to the analyses. CB, umbilical cord blood. PB, peripheral blood of adult.
Analysis of Gene Expression
Gene expression profiles were analyzed by quantitative RT-PCR (qRT-PCR) before and after the induction of differentiation in the HiDEP and HUDEP cells lines. The erythroid-specific markers GATA1, EKLF, GFI1B, TAL1 and EPOR were detected in all cell lines and their expression profiles before and after differentiation were similar to those of cultured erythroid cells derived from umbilical cord blood (Figure 7). This was also the case for c-MYB and SOX6, which are markers of definitive erythroid cells [22], [23], [24]. In addition, we detected upregulation of the erythroid membrane genes, Band 3, Band 4.1, Ankyrin-1 and α-Spectrin in all cell lines after differentiation (Figure 7).
Figure 7. Gene expression profiles of the established erythroid progenitor cell lines estimated by quantitative RT-PCR analysis.
Analyzed genes are indicated. FL, results with cDNA derived from human fetal liver. Relative expression was evaluated compared to that of FL. CB, cord blood. CB 6, 10 and 16, results with cDNA derived from cultured erythroid cells, i.e., CD34-positive cells in CB were induced to differentiate into mature erythroid cells for 6, 10 and 16 days, respectively, using the previously reported method [4]. B and A, Before and 2 days after induction of differentiation of HiDEP and HUDEP cells.
After differentiation, we found upregulation of α-globin in all cell lines, upregulation of β-globin only in the HUDEP-2 cell line, and upregulation of γ-globin in all cell lines except HUDEP-2. Of note, BCL11A, a repressor of γ-globin expression [25] and critical mediator of globin switching [26], was abundantly detected only in the HUDEP-2 cell line in which β-globin was up-regulated after differentiation (Figure 7).
Cell Viability and Cell Size
After the induction of differentiation in HiDEP-1 cells, the proportion of viable cells decreased but more than half of the cells were still viable 14 days after differentiation (Figure 8A). The average cell size after differentiation decreased compared to undifferentiated cells and the proportion of cells with a diameter less than 10 µm increased from about 20% to approximately 40% (Figure 8A) indicating the increase of very mature erythroid cells.
Figure 8. Analyses of cell viability and cell size during the induction of differentiation of the established erythroid progenitor cell lines.
Cell viability and cell size were estimated by an automated cell counter. (A) Results of HiDEP-1 cells. (B) Results of HUDEP-1 cells. (A, B) <10 µm (%), the percentages of cells with a diameter less than 10 µm. Among all HiDEP and HUDEP cell lines, HiDEP-1 cells most efficiently differentiated into more mature cells.
After the induction of differentiation in HUDEP-1 cells, the frequency of viable cells was considerably reduced compared to HiDEP-1 cells (Figure 8B). However, among viable cells average cell sizes decreased rapidly for two days after differentiation compared to undifferentiated cells, and the proportion of cells with a diameter less than 10 µm increased from about 10% to about 50% (Figure 8B) indicating the increase of very mature erythroid cells. To improve viability, the cells were cultured on OP9 feeder cells and a morphological analysis was performed.
Morphological Changes during Differentiation
Before the induction of differentiation, both HiDEP-1 and HUDEP-1 cells had round, erythroblast-like morphologies (Figure 9A, B). After the induction of differentiation in HiDEP-1 cells, enucleating cells (arrowheads in Figure 9A) and enucleated cells (black arrows in Figure 9A) were observed from about 7 days after differentiation. Since the cultures contained various types of cell and cell components such as enucleated cells, extruded nuclei after the enucleation process, nucleated cells, dead cells, and cell debris, it was very difficult to accurately calculate the frequency of enucleated cells even by flow cytometrical and morphological analyses. However, at approximately 12 days after the induction of differentiation a considerable number of enucleated cells were present (Figure 9A). The second cell line, HiDEP-2, could also differentiate into more mature cells and produce enucleated RBCs after the induction of differentiation; however, the efficiency of differentiation was lower compared to HiDEP-1 (data not shown).
Figure 9. Morphological analyses during the induction of differentiation of the established erythroid progenitor cell lines.
(A) HiDEP-1 cells before (Day 0) and 6, 10 and 12 days after the induction of differentiation (Day 6, 10 and 12). Scale bar in Day 0 indicates 50 µm and other scale bars indicate 10 µm. (B) HUDEP-1 cells before (Day 0) and 2, 6 and 9 days after the induction of differentiation (Day 2, 6 and 9). HUDEP-1 cells were cultured in erythroid differentiation medium on OP9 feeder cells to maintain cell viability during the differentiation process. All scale bars indicate 50 µm. (A, B) Black arrows and arrowheads show enucleated and enucleating cells, respectively. Blue arrows indicate cell debris. Among all HiDEP and HUDEP cell lines, HiDEP-1 cells most efficiently produced enucleated cells.
After the induction of differentiation in HUDEP-1 cells, the morphology of the cells clearly changed and mature and enucleating cells (arrowheads in Figure 9B) and enucleated cells (black arrows in Figure 9B) were observed from about day 6 of differentiation. Although the numbers of enucleated cells were much lower than those induced from HiDEP-1 cells, we did observe reproducible production of enucleated cells. The other HUDEP cell lines, HUDEP-2 and HUDEP-3, could also differentiate into more mature cells and produce enucleated RBCs after the induction of differentiation; the efficiencies of differentiation were almost identical to HUDEP-1 (data not shown).
Confirmation of Enucleated Cells
In general, in vitro produced enucleated RBCs are spherical reticulocytes and not fully mature biconcave cells. To confirm that these enucleated cells were reticulocytes, we performed supravital staining, immunostaining and benzidine staining. The supravital staining demonstrated the presence of reticulocytes (arrows in Figure 10A). In addition, we observed GPA-positive enucleated cells (pink cells in Figure 10B) and benzidine-positive enucleated cells (brown cells in Figure 10C).
Figure 10. Confirmation of enucleated cells.
As a representative, the cells induced from HiDEP-1 cells were subjected to the analyses. (A) Supravital staining. Arrows indicate reticulocytes. (B) Immunohistostaining with glycophorin A antibody (pink) 12 days after the induction of differentiation. Nuclei are stained with SYTO16 (green). An arrow shows an enucleated cell. (C) Benzidine staining 10 days after the induction of differentiation (brown, left panel). After benzidine staining, cell nuclei were labeled with DAPI to distinguish nucleated and enucleated cells (blue stained nuclei in right panel). Arrows or arrowheads show enucleated and enucleating cells, respectively. (A–C) Scale bars indicate 10 µm.
We previously succeeded in establishing MEDEP cell lines from mouse ES cells and were able to induce enucleated RBC production in these cell lines. However, the protocol developed for mouse cell lines failed to establish immortalized erythroid progenitor cell lines from human ES cells and human iPS cells. This difficulty may be related to the fact that in general it is more difficult to establish immortalized human cell lines than immortalized mouse cell lines. The failure of the original protocol led us to alter our strategy for establishing human erythroid progenitor cell lines.
The principal change to our strategy was to use inducible expression of HPV16-E6/E7 in transfected hematopoietic stem/progenitor cells. Through this approach, we developed a method that enabled establishment of immortalized human erythroid progenitor cell lines that possess the ability to differentiate. In iPS cells, the enforced expression of TAL1 supported the process of obtaining cell lines with hematopoietic potential by increasing the number of induced erythroid cells. To date, we have successfully established immortalized cell lines in all trials with this method. All of the immortalized cell lines we described in this study could differentiate into more mature RBCs including enucleated RBCs. The hemoglobin produced in these cell lines possessed similar oxygen binding properties to the hemoglobin in normal RBCs produced in vivo. To our knowledge, this is the first report describing the establishment of immortalized human erythroid progenitor cell lines able to produce enucleated RBCs ex vivo. Although erythroid progenitor cell lines have previously been established using HPV16-E6/E7, these cell lines did not produce enucleated RBCs [17]. One possible explanation for this failure is the continuous expression of HPV16-E6/E7 in these cells. Since efficient differentiation of cells generally requires cell cycle arrest, the continuous expression of HPV16-E6/E7 might have inhibited terminal differentiation of the cells.
The cell lines obtained in this study displayed different efficiencies for producing enucleated RBCs in vitro as is illustrated by comparison of HiDEP and HUDEP cells. We previously found that the efficiency of enucleated RBC production by MEDEP cells improved in vivo after transplantation [8]. Therefore, it is highly likely that HiDEP and HUDEP cells would also be able to produce enucleated RBCs more efficiently in vivo than in vitro.
Although we suspect that the cell lines established here might be able to produce enucleated RBCs more abundantly in vivo, the main goal of our work is to develop a means of producing abundant enucleated RBCs ex vivo. There are two reasons for this emphasis. First, enucleated RBCs pose no risk of tumorigenicity and, therefore, can be transfused without hazard into the recipient. This is not the case for transplantation of nucleated cells derived from immortalized cells such as ES cells and iPS cells. Enucleated RBCs can be selected by size, e.g., by filtration, and contaminating nucleated cells can be eliminated by irradiation without affecting the structure and function of RBCs. Currently, such irradiation is routinely used in the clinic before transfusion of RBCs in order to eliminate any lymphocytes. Second, transplantation of progenitor cells requires compatibility for major histocompatibility antigens [8]. This is not the case for enucleated RBCs, which mainly require the compatibility of ABO and RhD blood phenotypes. One of the HUDEP cell lines developed here, HUDEP-3, possesses an O/RhD(+) phenotype (Figure S4, S5 and Table S2); thus, RBCs produced ex vivo from this cell line could potentially be transfused into the vast majority of the Japanese population, since approximately 99% of Japanese possess an RhD(+) blood phenotype. Needless to say, cell lines that produce O/RhD(−) RBCs will have more widespread utility as these RBCs can be transfused into the vast majority of people around the world.
Our next aim is to establish cell lines that have a high yield (>90%) of enucleated RBCs after induction of differentiation. The underlying basis for the different characteristics of the various cell lines, such as the efficiency of enucleated RBC production, remains uncertain. Possibly, they may have been immortalized at different stages of erythroid differentiation; the mechanisms of immortalization at different stages of differentiation remain to be studied. Until these mechanisms are elucidated, our experience with the generation of MEDEP, HiDEP and HUDEP cell lines suggests that establishment of useful cell lines will necessitate numerous trials. At present, we do not think that hematopoietic cells derived from iPS cells are a better source than cord blood cells for establishing cell lines. Indeed, HiDEP-1 cells demonstrated a high efficiency of production of enucleated RBCs, while HUDEP-2 cells could produce adult type hemoglobin consisting of α and β globin. We certainly believe it will be feasible to establish HiDEP and/or HUDEP cell lines able to produce adult type hemoglobin and also to produce enucleated RBCs with a high efficiency. With respect to the production of adult type hemoglobin, the results shown in Figure 7 suggested that enforced expression of BCL11A in the source cells may support the establishment of cell lines that produce adult type hemoglobin. In a similar manner to the exogenous expression of TAL1 and HPV16-E6/E7, the introduction of BCL11A will have no consequence for clinical applications if the ultimately established cell lines can produce abundant enucleated RBCs.
Of note, the enucleated cells produced in vitro from HiDEP and HUDEP cells were spherical (Figure 9) and were reticulocytes (Figure 10A). We previously reported that the enucleated RBCs produced from hematopoietic stem/progenitor cells present in cord blood in vitro were also spherical reticulocytes [4]. Generally, the majority of in vitro produced RBCs seem to be reticulocytes; however, it has recently been reported that reticulocytes produced in vitro can complete maturation in vivo, i.e., following transplantation, into biconcave cells [27]. Therefore, it is likely that the reticulocytes produced in vitro from HiDEP and HUDEP cells could also complete maturation in vivo after transfusion.
The method developed to establish the HiDEP cell line involved use of the mouse derived-OP9 cell line as feeder cells during the early stages of establishment and also use of fetal bovine serum (FBS). These xenogeneic factors need to be eliminated before the application of cells in the clinic. There are two reasons for believing this elimination is possible. First, the xenogeneic factors derived from OP9 cells will disappear after long term culture in the absence of OP9 cells. The HiDEP cells ceased to have a requirement for OP9 cells after two or three months in culture and they could be maintained in the presence of humoral factors alone. Notably, HUDEP cell lines could be established in feeder cell-free conditions, i.e., without use of OP9 cells. Second, as we have previously demonstrated, FBS can be replaced with human serum without compromising the production of abundant enucleated RBCs from hematopoietic stem/progenitor cells in cord blood [4]. In addition, all HiDEP and HUDEP cells could be maintained in serum-free conditions using a commercially available medium (see Materials and methods). Thus, with respect to HUDEP cell lines they can be established in a feeder cell-free and serum-free conditions throughout all procedures.
Changes to the characteristics of immortalized cell lines after long term culture can sometimes be an obstacle to their use. However, it is also a fact that many immortalized cell lines, such as human cancer cell lines, stably maintain their characteristic properties even after long term culture. Of note, MEDEP-BRC5, one of the MEDEP cell lines we previously reported [8], has maintained the ability for high rates of production of enucleated RBCs in vitro even after continuous culture for nearly 2 years; in this cell line, more than 50% of the cells are enucleated RBCs after induction of differentiation in vitro [28].
We are now confident that the method described in this study can reproducibly and robustly establish immortalized human erythroid progenitor cell lines, such as the HiDEP and HUDEP cell lines, able to produce enucleated RBCs ex vivo. Establishment of HiDEP cell lines from iPS cells derived from people possessing very rare blood phenotypes, such as Rh-null, may open a way to produce such rare types of RBCs ex vivo.
Map of the CSII-EF-RfA lentiviral vector plasmid.
https://doi.org/10.1371/journal.pone.0059890.s001
(TIF)
Map of the CSIV-TRE-RfA-UbC-KT lentiviral vector plasmid.
Summary of flow cytometry analyses. Expression of the indicated markers was analyzed. c-KIT, the receptor of SCF. GPA, glycophorin A. (A) Results from HiDEP cells. (B) Results from HUDEP cells.
Analysis of blood types in HUDEP and HiDEP cells. (A) The results with respect to the ABO gene. (B) The results with respect to the RhD gene. (A, B) A detailed description of the typing of the cells can be found in Lu et al., Blood 112; 4475–4484 (2008).
Characterization of phenotype with respect to RhD antigen. Expression of RhD antigen was clearly detected in HiDEP-1-derived cells. In contrast, HUDEP-3-derived cells did not show abundant expression of RhD antigens. These results indicated that RhD antigens might be induced depending on the stage of maturation of the cells since HiDEP-1 produced mature red blood cells at a higher rate (see manuscript).
Table S1.
Factor dependency of iPS and cord blood-derived erythroid progenitor cell lines.
(DOC)
Blood phenotypes of the established erythroid progenitor cell lines.
We thank Dr. Kiyono for providing the HPV16-E6/E7 DNA, Dr. Miyawaki (RIKEN BSI) for providing the phKO1-S1 plasmid, and all members in the Cell Engineering Division for help, discussion, or secretarial assistance.
Conceived and designed the experiments: RK KT YN. Performed the experiments: RK NS. Analyzed the data: RK KS KM TH YN. Contributed reagents/materials/analysis tools: HM. Wrote the paper: RK YN.
1. Anstee DJ (2010) Production of erythroid cells from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPS). Transfus Clin Biol 17: 104–109.
2. Douay L, Lapillonne H, Turhan AG (2009) Stem cells–a source of adult red blood cells for transfusion purposes: present and future. Crit Care Clin 25: 383–398, Table of Contents.
3. Migliaccio AR, Whitsett C, Migliaccio G (2009) Erythroid cells in vitro: from developmental biology to blood transfusion products. Curr Opin Hematol 16: 259–268.
4. Miharada K, Hiroyama T, Sudo K, Nagasawa T, Nakamura Y (2006) Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells. Nat Biotechnol 24: 1255–1256.
5. Neildez-Nguyen TM, Wajcman H, Marden MC, Bensidhoum M, Moncollin V, et al. (2002) Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat Biotechnol 20: 467–472.
6. Lu SJ, Feng Q, Park JS, Vida L, Lee BS, et al. (2008) Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood 112: 4475–4484.
7. Lapillonne H, Kobari L, Mazurier C, Tropel P, Giarratana MC, et al. (2010) Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Haematologica 95: 1651–1659.
8. Hiroyama T, Miharada K, Sudo K, Danjo I, Aoki N, et al. (2008) Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells. PLoS One 3: e1544.
9. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872.
10. Kurita R, Oikawa T, Okada M, Yokoo T, Kurihara Y, et al. (2008) Construction of a high-performance human fetal liver-derived lentiviral cDNA library. Mol Cell Biochem 319: 181–187.
11. Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A (2004) Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. The Biochemical journal 381: 307–312.
12. Kurita R, Sasaki E, Yokoo T, Hiroyama T, Takasugi K, et al. (2006) Tal1/Scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset (Callithrix jacchus) embryonic stem cells. Stem Cells 24: 2014–2022.
13. Robb L, Lyons I, Li R, Hartley L, Kontgen F, et al. (1995) Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc Natl Acad Sci U S A 92: 7075–7079.
14. Mikkola HK, Klintman J, Yang H, Hock H, Schlaeger TM, et al. (2003) Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene. Nature 421: 547–551.
15. Hall MA, Curtis DJ, Metcalf D, Elefanty AG, Sourris K, et al. (2003) The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S12. Proc Natl Acad Sci U S A 100: 992–997.
16. Fujioka T, Shimizu N, Yoshino K, Miyoshi H, Nakamura Y (2010) Establishment of induced pluripotent stem cells from human neonatal tissues. Hum Cell 23: 113–118.
17. Wong S, Keyvanfar K, Wan Z, Kajigaya S, Young NS, et al.. (2010) Establishment of an erythroid cell line from primary CD36+ erythroid progenitor cells. Exp Hematol 38: 994–1005 e1001–1002.
18. Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 89: 5547–5551.
19. Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, et al. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268: 1766–1769.
20. Shirasawa T, Izumizaki M, Suzuki Y, Ishihara A, Shimizu T, et al. (2003) Oxygen affinity of hemoglobin regulates O2 consumption, metabolism, and physical activity. J Biol Chem 278: 5035–5043.
21. Sakai H, Cabrales P, Tsai AG, Tsuchida E, Intaglietta M (2005) Oxygen release from low and normal P50 Hb vesicles in transiently occluded arterioles of the hamster window model. Am J Physiol Heart Circ Physiol 288: H2897–2903.
22. Yi Z, Cohen-Barak O, Hagiwara N, Kingsley PD, Fuchs DA, et al. (2006) Sox6 directly silences epsilon globin expression in definitive erythropoiesis. PLoS Genet 2: e14.
23. Dumitriu B, Patrick MR, Petschek JP, Cherukuri S, Klingmuller U, et al. (2006) Sox6 cell-autonomously stimulates erythroid cell survival, proliferation, and terminal maturation and is thereby an important enhancer of definitive erythropoiesis during mouse development. Blood 108: 1198–1207.
24. Tober J, McGrath KE, Palis J (2008) Primitive erythropoiesis and megakaryopoiesis in the yolk sac are independent of c-myb. Blood 111: 2636–2639.
25. Sankaran VG, Menne TF, Xu J, Akie TE, Lettre G, et al. (2008) Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 322: 1839–1842.
26. Sankaran VG, Xu J, Ragoczy T, Ippolito GC, Walkley CR, et al. (2009) Developmental and species-divergent globin switching are driven by BCL11A. Nature 460: 1093–1097.
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28. Hiroyama T, Miharada K, Kurita R, Nakamura Y (2011) Plasticity of cells and ex vivo production of red blood cells. Stem Cells Int 2011: 195780.
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text:
University of St Andrews Research
The Synthesis of [{n-Bu2Sn(S2N2)}2] and its use in the preparation of Organometallic Iridium Sulfur Nitrogen Complexes
FinalfullMS2N2_paper.doc (248.5Kb)
Aucott, SM
Slawin, Alexandra Martha Zoya
Woollins, John Derek
Liquid ammonia
Tin sulfur
Nitrogen reagent
Metathesis
Sulfur-nitrogen metallacycle
Iridium complexes
Coordination chemistry
QD Chemistry
The addition of [n-Bu2SnCl2] to a solution of [S4N3][Cl] in liquid ammonia gave after extraction of the dry reaction mixture the new tin disulfur dinitrido compound [{n-Bu2Sn(S2N2)}(2)] (1). Reaction of [{n-Bu2Sn(S2N2)}(2)] (1) with the pentamethylcyclopentadienyl (Cp*) iridium derivatives [{IrCl(mu-Cl)(eta(5)-C5Me5)}(2)] or [(eta(5)-C5Me5)IrCl2(PPh3)] gave different products, which were dependent on the reactant ratios. A 1:1 reaction between 1 and [{IrCl(mu-Cl)(eta(5)-C5Me5)}(2)] gave only [(eta(5)-C5Me5)Ir(S2N2)] (2) in moderate yield; the same product in higher yield was obtained from a 2:1 reaction between 1 and [(eta(5)-C5Me5)IrCl2(PPh3)]. Reaction of 1 and [(eta(5)-C5Me5)(2)IrCl2(PPh3)] (1:1 molar ratio) in the presence of NH4[PF6] gave the unusual bimetallic species [(eta(5)-C5Me5)IrCl(PPh3)(S2N2)Ir(eta(5)-C5Me5)][PF6] (3). The X-ray crystal structures of 1, 2, and 3 are reported.
Aucott , SM , Slawin , A M Z & Woollins , J D 2002 , ' The Synthesis of [{ n -Bu 2 Sn(S 2 N 2 )} 2 ] and its use in the preparation of Organometallic Iridium Sulfur Nitrogen Complexes ' Candian Journal of Chemistry , vol. 80 , pp. 1481-1487 . https://doi.org/10.1139/V02-085
Candian Journal of Chemistry
https://doi.org/10.1139/V02-085
© 2002. Canadian Science Publishing.
http://www.scopus.com/inward/record.url?scp=0036904344&partnerID=8YFLogxK


After playing Gentes, we thought all the goodness had come and gone for the year. But no, we are here to tell you that once again, we get blindsided by two games: Bastion and Sidereal Confluence. When will the madness end? How are we going to narrow down our selections for the Squirrely Awards? And on top of that, Marty goes to Pax Unplugged and I am sure he has more to add to the pile.
Bastion is a coop game from Z-Man that was out in 2015 and they have made it available for all to consume. We saw this at Gen Con and we were intrigued by this simple tower defense game. What seemed to be a straight forward coop game turned into a strategy game that caught us off guard. It went from go defeat this guy to now, wait, you need to do this, so I can do that and then he can do that because if you don't then this will happen and then this chain occurs and so forth and so on. Give this one a try if you like coop games.
Then the Excel spreadsheet game hit the table, Sidereal Confluence and man did it have us scratching our head and in the dark, but not in a bad way. It had player interaction, engine building, multiple strategies and depth, so much depth that we can't wait to get this back on the table again. Will have to say if it wasn't for the trading, this game would have floundered a bit, but with the trading, it took it up so many levels. In an engine builder, if your plan comes together, even if you don't win, there is still satisfaction and that is what happened here. For those that enjoy these types of games don't miss it.
But not everything was rosy on the table and we discuss that as well. And also, special guests come on the show as Marty heads to Pax Unplugged and has a slumber party with some people you may already know. Listen to these familiar voices talk about Necromunda.
Thanks for listening and all the support.
RDTN Episode 126: Gen Con 50 with special guest host, Rodney Smith from Watch It Played!
Our Gen Con report is here and it is a hefty one. It might have been shorter had a special guest not joined us, but he was forced to participate or we wouldn't let him go to bed. Rodney Smith joins us on this episode as we recount our events at Gen Con. The daily festivities are recounted and not much gaming because, well, Gen Con is about seeing the stuff that is coming and hearing about what is planned for the rest of the year. Also, it doesn't help that there are 60,000+ people at the Con and we are all trying to get to the hotness. Also, please don't forget to support Rodney on his crowd funding campaign.
Even with that many people, it never felt that crowded. Now, don't misunderstand, there was a crowd, but you could move and patience went a long way when you were on the floor. And when the vendor area closed, there were plenty of opportunities to attend other events, some you paid for, others were free.
We also want to say thank you to everyone who attended the RDTN meetup at the Omni Bar, it exceeded our expectations. The Strike tournament was a hit and the final battle was epic.
You can visit our Facebook page for pictures from our trip!
Surprise, surprise, surprise. That is right Renegade Studios is releasing Clank! in Space at Gen Con. Talk about keeping things under wraps and making a huge a splash, Renegade might have done it. If you like Clank and you like space themes, give this game a try. It builds on the mechanics of Clank!: The Deck-Building Adventure and adds some solid additions. One of those additions is the factions on cards where if cards from the same faction are played on a turn, their special abilities trigger. This is a solid component that it really can add strategy to the market as you look at what to buy during your turn. Was looking forward to seeing Thunderstone Quest at Gen Con, but this might still a little of that thunder in the deck building world.
The Climbers was released in 2008 and is being brought back to us gamers by Simply Complex which is part of Capstone Games in 2017. This is an easy to play game with a ton of strategy that even creates moments of take that. As you play the game, it is so cool to watch the tower grow and see it morph into a skyscraper. However, there are cases where players can almost be eliminated and thus not be fun for those left on the lower portions of the tower. This is one of those games that maybe for the older gamers, it will be a nice one to break out periodically, but this definitely has the staying power with younger gamers.
Gen Con is this week and we are very excited to be attending and for the first time, together. Hard to imagine, but Marty and I have never attended Gen Con at the same time. Last time that I was in attendance was in 2012, so I know I am in for a shock to the system. I joked during the show about being confused and lost, but how can you not be? This is a huge show and there will be so much to take in. If you can't make it to Gen Con, be sure to take part in Gencant which is now in its third year and headed up by our incredible friend @425Suzanne.
Thanks for listening and look forward to our next show where we data burst our thoughts on Gen Con.


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Aerodrums is working great with Superior drummer, but I can't seem to get the cymbal choke zones to send any midi.
If not, you can guess the next question ... any plans to?
You're correct, choking cymbals isn't supported yet in MIDI mode. This feature was labelled 'experimental' in our release notes because, of the various ideas we had to support choking, it wasn't clear which was the best. We wanted to see what our users thought about it before committing to it and adding support for MIDI, etc.
I'm guessing you like this feature and can use it reliably? Do you think you would prefer it to having a separate cymbal element that you can add to your kit, position where you want, and hit to trigger choked cymbal samples?
Thanks for the reply. I'm a beginner drummer but I definitely like the feature and used it when I was playing with Aerodrums' samples; it's essential for some songs.
Yes, I think a separate cymbal element could work better in terms of positioning flexibility. What I'm not sure about is actually having to hit it rather than quickly moving the stick into it. I found I could hit the left crash and choke in almost one movement. Hit then 'swipe' left.
How about starting a poll so some more advanced drummers can weigh in?
Thanks for the great work on Aerodrums btw, I'm really enjoying learning with it.
- There are various sized cymbal choke kit elements. "10-inch cymbal choke zone, ... ,20-inch cymbal choke zone etc"
Thanks a lot for the feedback. The main motivation behind the current choke feature was to allow you to use natural one-handed and two-handed cymbal choking gestures. It works but at the cost of using up some of the playable space for the cymbal choking zones. Some of the alternative ideas were to have a choke pedal, a choke zone element (what you described) and new elements that would just trigger choked cymbal samples. The main difference between the last two options is timing; the first ones lets you decide when to choke whereas in the second the timing is "baked" into the samples. Can you comment on how important timing is to you when choking crash cymbals...do you just hit and grab or is the grab timed?
I think the 'grab' needs to be timed ideally. Two songs I'm doing a poor job of learning at the moment start with choked cymbals: Jesus of Suburbia by Green Day and Always by Blink 182.
On the Green Day track you do four sustained cymbal hits then choke on the next beat, on the Blink 182, grab as fast as you can.
My Aerodrums kit has 5 cymbals and I've found 5 or 6 to be my individual limit where I can hit each one reliably without looking at the screen.
If each cymbal had a "choked" counterpart, I'd need to scale back to 3 normal cymbals and 2 choked. I still couldn't play the Green Day track with that though.
I don't even have my kit yet but I have been thinking about several elements of drumming that may not work with this concept. I am very excited to work with this product.
I am not an engineer but I am a long time and pretty capable drummer. What if choking the cymbal was simply holding the stick still in the "zone" of the cymbal strike would be? Again I am not familiar with the product yet. I do play many genres and I can't recall ever needing to choke my ride.
Does the program currently offer a way to choke cymbals?
There is an experimental cymbal choking feature in the current version of Aerodrums. Basically there are two adjustable zones on either side of you and whenever a drum stick enters one of these regions, all cymbals will be choked. To a certain extent this allows some natural choking gestures such as hitting a crash on the left of your kit with your right hand and choking it with your left hand (the stick will point towards the left and enter the choke zone). We're still undecided though if this is the best mechanic...we have other ideas that we'd like to experiment with.


In partnership with Apollos Partners, LLC, NABA Atlanta, is launching the NABA CPA Small Group Study Initiative to provide members direct support, resources, and accountability partners in a community committed to empowering their success.
This 9-Week program will connect NABA members who've registered to sit for the exam by November 2018 with a unique opportunity to build a stronger community while advancing their careers.
We are looking for 50 active NABA members to take part in this program designed to provide members with dedicated study space, support, encouragement, a plan, and accountability partners to help our members achieve their goals and pass the CPA Exam.
Applications Are Due by Friday, August 24th.
Participants must meet all program requirements.
Applicants will be notified of acceptance to the program no later than, Monday August 27th.


text:
package org.apache.hc.client5.http.impl.async;

import java.io.IOException;

import org.apache.hc.client5.http.HttpRequestRetryStrategy;
import org.apache.hc.client5.http.HttpRoute;
import org.apache.hc.client5.http.async.AsyncExecCallback;
import org.apache.hc.client5.http.async.AsyncExecChain;
import org.apache.hc.client5.http.async.AsyncExecChainHandler;
import org.apache.hc.client5.http.protocol.HttpClientContext;
import org.apache.hc.core5.annotation.Contract;
import org.apache.hc.core5.annotation.Internal;
import org.apache.hc.core5.annotation.ThreadingBehavior;
import org.apache.hc.core5.http.EntityDetails;
import org.apache.hc.core5.http.HttpException;
import org.apache.hc.core5.http.HttpRequest;
import org.apache.hc.core5.http.HttpResponse;
import org.apache.hc.core5.http.nio.AsyncDataConsumer;
import org.apache.hc.core5.http.nio.AsyncEntityProducer;
import org.apache.hc.core5.http.nio.entity.DiscardingEntityConsumer;
import org.apache.hc.core5.http.support.BasicRequestBuilder;
import org.apache.hc.core5.util.Args;
import org.apache.hc.core5.util.TimeValue;
import org.slf4j.Logger;
import org.slf4j.LoggerFactory;

/**
 * Request executor in the asynchronous request execution chain that is
 * responsible for making a decision whether a request that failed due to
 * an I/O exception or received a specific response from the target server should
 * be re-executed. Note that this exec chain handler will not respect
 * {@link HttpRequestRetryStrategy#getRetryInterval(HttpResponse, int, org.apache.hc.core5.http.protocol.HttpContext)}.
 *

 * Further responsibilities such as communication with the opposite
 * endpoint is delegated to the next executor in the request execution
 * chain.
 *

 *
 * @since 5.0
 */
@Contract(threading = ThreadingBehavior.STATELESS)
@Internal
public final class AsyncHttpRequestRetryExec implements AsyncExecChainHandler {

    private static final Logger LOG = LoggerFactory.getLogger(AsyncHttpRequestRetryExec.class);

    private final HttpRequestRetryStrategy retryStrategy;

    public AsyncHttpRequestRetryExec(final HttpRequestRetryStrategy retryStrategy) {
        Args.notNull(retryStrategy, "retryStrategy");
        this.retryStrategy = retryStrategy;
    }

    private static class State {

        volatile boolean retrying;
        volatile TimeValue delay;

    }

    private void internalExecute(
            final State state,
            final HttpRequest request,
            final AsyncEntityProducer entityProducer,
            final AsyncExecChain.Scope scope,
            final AsyncExecChain chain,
            final AsyncExecCallback asyncExecCallback) throws HttpException, IOException {

        final String exchangeId = scope.exchangeId;

        chain.proceed(BasicRequestBuilder.copy(request).build(), entityProducer, scope, new AsyncExecCallback() {

            @Override
            public AsyncDataConsumer handleResponse(
                    final HttpResponse response,
                    final EntityDetails entityDetails) throws HttpException, IOException {
                final HttpClientContext clientContext = scope.clientContext;
                if (entityProducer != null && !entityProducer.isRepeatable()) {
                    if (LOG.isDebugEnabled()) {
                        LOG.debug("{} cannot retry non-repeatable request", exchangeId);
                    }
                    return asyncExecCallback.handleResponse(response, entityDetails);
                }
                state.retrying = retryStrategy.retryRequest(response, scope.execCount.get(), clientContext);
                if (state.retrying) {
                    state.delay = retryStrategy.getRetryInterval(response, scope.execCount.get(), clientContext);
                    if (LOG.isDebugEnabled()) {
                        LOG.debug("{} retrying request in {}", exchangeId, state.delay);
                    }
                    return new DiscardingEntityConsumer<>();
                } else {
                    return asyncExecCallback.handleResponse(response, entityDetails);
                }
            }

            @Override
            public void handleInformationResponse(final HttpResponse response) throws HttpException, IOException {
                asyncExecCallback.handleInformationResponse(response);
            }

            @Override
            public void completed() {
                if (state.retrying) {
                    scope.execCount.incrementAndGet();
                    if (entityProducer != null) {
                       entityProducer.releaseResources();
                    }
                    scope.scheduler.scheduleExecution(request, entityProducer, scope, asyncExecCallback, state.delay);
                } else {
                    asyncExecCallback.completed();
                }
            }

            @Override
            public void failed(final Exception cause) {
                if (cause instanceof IOException) {
                    final HttpRoute route = scope.route;
                    final HttpClientContext clientContext = scope.clientContext;
                    if (entityProducer != null && !entityProducer.isRepeatable()) {
                        if (LOG.isDebugEnabled()) {
                            LOG.debug("{} cannot retry non-repeatable request", exchangeId);
                        }
                    } else if (retryStrategy.retryRequest(request, (IOException) cause, scope.execCount.get(), clientContext)) {
                        if (LOG.isDebugEnabled()) {
                            LOG.debug("{} {}", exchangeId, cause.getMessage(), cause);
                        }
                        if (LOG.isInfoEnabled()) {
                            LOG.info("Recoverable I/O exception ({}) caught when processing request to {}",
                                    cause.getClass().getName(), route);
                        }
                        scope.execRuntime.discardEndpoint();
                        if (entityProducer != null) {
                            entityProducer.releaseResources();
                        }
                        state.retrying = true;
                        final int execCount = scope.execCount.incrementAndGet();
                        state.delay = retryStrategy.getRetryInterval(request, (IOException) cause, execCount - 1, clientContext);
                        scope.scheduler.scheduleExecution(request, entityProducer, scope, asyncExecCallback, state.delay);
                        return;
                    }
                }
                asyncExecCallback.failed(cause);
            }

        });

    }

    @Override
    public void execute(
            final HttpRequest request,
            final AsyncEntityProducer entityProducer,
            final AsyncExecChain.Scope scope,
            final AsyncExecChain chain,
            final AsyncExecCallback asyncExecCallback) throws HttpException, IOException {
        final State state = new State();
        state.retrying = false;
        internalExecute(state, request, entityProducer, scope, chain, asyncExecCallback);
    }

}

redpajama_set_name: RedPajamaGithub