text:
Setting Up and Using a Dual-Drum Sander
A related matter is whether having the lumber company leave 1/64"-1/32" on each face for me to clean up on the drum sander after they resaw and plane S2S will be enough, especially since I can't really control the condition of their planer and their blades, or how they're likely to hog it through the planer. Any suggestions?
Forum Responses
From contributor E:
I have to agree with the previous post. Unless you find a good deal on something used, you're not going to find the quality of machine you're describing in that price range. I bought what I thought was the best for the money, Powermatic's dual drum sander. But it is about $2400 and has two speeds, not variable. Great machine, though. I also started with the Performax 16/32 and would not recommend it for what you are doing. It needs too much fine tuning every time you set up a different grit. Plus it's only a single drum.
Best bet... outsource to a local shop.
From contributor P:
I have the General 24" drum sander, and I'm happy with it. I was using a Performax 24" before that, and it was half the machine for twice the money. The General is still less than $2000.
I use my General sander as a single drum machine, specifically because it's too hard to precisely dial in the second drum, if you're moving it up and down to go from roughing to finish. It's easier to change paper instead. When I got my machine from the dealer, the infeed drum was out by .013" over the width of the drum. I was able to put shims under the upper support brackets to even that out, and now I'm within .001" over 24", repeatedly. I sand everything from narrow strips for edgebanding to 24" wide planks. It would take too long to try to get the second drum within this kind of tolerance, although if you never moved it once it was dialed in, it should stay there.
As far as surfacing goes, taking off 1/64" from each face is easy to do (not all at once), as long as that will clean up the surface left by your supplier's planer. That's something that can only be determined by experience. Mahogany is soft and easy to sand - I do a lot of sanding using only 150 grit, which will surface it down nicely without leaving deeper scratches. If you have to remove significant tearout, 120 grit will go faster, but you're correct in that you have to go slowly (slow feed speed and light passes), use that sandpaper cleaner stick often, and definitely hook the system up to a decent dust collector. You're going to need optimally a 6" duct split into two 4" hoses just above the sander to pull all the dust out.
I have much fewer problems with burned paper now that I've gotten more experience with the machine, but I've learned that once you get a burned stripe, you have to deal with the problem immediately, as it will only get worse if ignored. Sometimes the burned particles can be picked out with an awl or scraper, sometimes it's just better to replace the paper itself. Keeping the paper clean, using a slow feed, and replacing the paper before it's used up seems to be the best approach. There was a review of this machine in American Luthier a couple of years back, in which it was referred to as the hidden gem of drum sanders. The reviewer replaced the conveyer belt with a sandpaper one, to increase accuracy, and he tells exactly how he did it. You might be able to get a reprint of this from the publisher, or a back issue of the magazine.
From the original questioner:
I appreciate your response. I've read mostly favorable reports about the General, and it seems that for the price, it's the best thing out there. The negatives I've read: no separate conveyor belt switch, height of pressure rollers not adjustable, and rubber belt requires a lot of roller pressure, which makes it more difficult to remove cupping, especially in the 1/4" thicknesses I use. Yet overall, the machine seems to outrank others in its price range. A couple of other things come to mind: Is the drum itself soft or hard, and do the edges of material come out rounded or fairly crisp?
At American Luthier they are trying to locate the article about converting the conveyor belt to sandpaper. I'm not sure if this would involve eliminating part or all of the existing rubber, or building right on top of it - I can see where keeping the drums parallel to the belt might be tricky. I'd appreciate any further input on the above.
From contributor P:
I'll try to answer your questions as best I can. First, there is a separate switch for the conveyer feed, as well as a dial for conveyer speed. The reviewer who said there wasn't was mistaken. Pressure roller height is not adjustable, but roller pressure is. As far as removing cupping is concerned, that's what a jointer is for. You can remove cupping with the sander if you use a sanding board under the stock with shims to build up the space under the cup, which will get one face flat, and then flip the stock, and sand the second side parallel to the first. Actually, at the 1/4" thickness you are using, I doubt if any conveyer system by itself would hold the stock so delicately that it would take the cup out instead of just flattening the stock out by roller pressure. The sanding drums are aluminum with no rubber or Velcro covering. Edges come out crisp.
The article in American Luthier is on pg. 36 of issue #70, summer 2002. Its title is "General 15-250MI: The Hidden Gem of Thickness Sanders". The author removed the rubber conveyer belt completely, and replaced it with a sandpaper-type as on the Performax sanders. He gives step-by-step directions, as well as a source for the belt.
I would try the machine with the stock conveyer belt first and see how it works before going to all that trouble. Perhaps using a sanding board under the stock would solve all the problems. He says as much in the article, but also says that he didn't want to continue using one. He had problems with dust pickup, but was splitting one 4" line into two 4" lines above the machine, which won't work. You can't get enough airflow through a 4" duct; like I said in my earlier post, you should go for a dust collector which can drive a 6", which usually means a 2 hp collector or larger. Going with inadequate airflow and blaming the sander is not addressing the real problem. Adjusting the first drum parallel to the bed is very easy. I ran two strips of stock through the machine at opposite sides of the belt at the same time, and used my digital caliper to check them when they came out. The difference in thickness equals the amount out of parallel. The bed assembly is supported above the lower body of the machine by 4 angle brackets, 2 on each side. Take some shim stock equal in thickness to the difference in stock thickness determined above, and insert it under the two brackets under the side of the bed which produced the thicker measurement. Double check your results with two pieces of stock again, and fine tune if necessary. Worked for me.
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When you woke up this morning I sincerely doubt you realized that Christmas is seven months away. Well it is! I'm not a huge Christmas person but I do like to make sure that I can give meaningful gifts for my close friends and family. I normally buy for eight to ten people in any given year and normally spend anywhere from $20 to $50 on each person. If I had kids I bet I'd be spending a lot more.
In order to get meaningful gifts I try to buy as many gifts ahead of time as possible. However, like a lot of people I know, I am a procrastinator and normally end up buying most of my gifts in December. This can mean spending a lot of money in a short time frame and I don't know about you but my budget could easily get blown on Christmas.
My monthly spending allowance is much less than what I plan to spend on gifts so how do I get the money to buy my gifts at the last minute in December? I'm going to save in advance! For simplicity's sake let's pretend that I said I was planning on spending $700 for Christmas this year. In order to save that much money I would need to save $100 each month until Christmas. Most people would agree that sounds much more reasonable than coming up with $700 out of nowhere in December. I get two paychecks a month so that makes it even more manageable at only $50 a paycheck.
Talking about saving for Christmas is easy, but actually doing it is another story. Here are a couple ways you can effortlessly save for Christmas without having to think about it after you have it set up.
The first requires you to set up a new savings account. If you have a bank like ING Direct this process is as simple as a few clicks and about 2 minutes. If you have a more traditional bank or credit union stop by a local branch and ask to have a new account set up. Next, set up a direct deposit for the desired amount to automatically get part of your paycheck deposited into your Christmas account. Most employers should allow this. I can set mine up myself where I work but you may have to talk to your Human Resources department and fill out a simple form.
Another method would be to set up an automatic transfer out of your checking account and into your savings account the day after your paychecks get deposited. At ING Direct this is called an Automatic Savings Plan. All you have to do is set it up and forget it until Christmas and then you'll have your desired amount ready for buying gifts!
If that all sounds like too much work for you I can understand that. This method is even easier, but a little less secure. After your paycheck is deposited withdraw your desired amount from your checking account and put the Christmas money in a secret place that you know you won't touch. You can hide it in a jar in your kitchen or in a safe hidden in a closet. The location is up to you but make sure it is somewhere you won't be tempted to spend it, somewhere you will remember, and somewhere not out in the open so it doesn't get "borrowed from" or stolen.
Now that you are fully aware Christmas is only seven months away hopefully you have a great idea how to pay for it this year. This should help reduce any Christmas stress and more importantly Christmas debt.
Have you ever saved in advance for Christmas? How did it work out? Any other tips you'd like to share?

subset: commoncrawl


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Kanye West Called Out for Antisemitism by the Anti-Defamation League
Kanye West may be in trouble again after making comments that didn't sit well with the Anti-Defamation League. During his Nov. 26 interview with Power 105's 'The Breakfast Club,' Yeezy tried to explain the politics of working with corporate America and it veered into why Obama is having problems establishing his policies at the White House.
"Man, let me tell you about George Bush and oil money and Obama and no money," said West. "People want to say Obama can't make these moves or he's not executing. That's because he ain't got those connections. Black people don't have the same level of connections as Jewish people. Black people don't have the same connection as oil people."
The MC's remarks provoked the ire of the ADL who issued a statement Monday (Dec. 2) denouncing his claims.
If the comments are true as reported, this is classic anti-Semitism," Abraham H. Foxman, the League's national director, said in a statement. "There it goes again, the age-old canard that Jews are all-powerful and control the levers of power in government. As a celebrity with a wide following, Kanye West should know better. We hope that he will take responsibility for his words, understand why they are so offensive, and apologize to those he has offended."
As of press time, West has yet to respond to the ADL's statement.
What do you think? Should Kanye West apologize for his remarks? Tell us in the comments below.
Listen to Kanye West's Interview with Power 105.1's 'The Breakfast Club'


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Public Warned Against Slash-And-Burn Agriculture
Mr Eric Amaning Okoree, Chief Executive Officer, National Biosafety Authority (NBA), has said that the practice of slash-and-burn was as unfavourable as climate change, and both are threats to the sustainability of agriculture.
He said when someone sets a parcel of land on fire, soil organisms and the insects that form the major parts of pollinators were burnt.
Mr Okoree said these soil organisms, which prepare the soil for it to be able to hold water for the plants to absorb, were thus eliminated.
He said, in this regard, the very basis of agriculture is being destroyed through such practices.
Mr Okoree said as a result of such practices the types of crops and food produced decades ago could not be produced these days; adding that 'agriculture and lives are threatened because of the situation in which we find ourselves'.
Mr Okoree made this known in his remarks at the "Ask About Genetically Modified Organisms (GMOs) Engagement" with the National Biosafety Authority (NBA), a forum organised by the Alliance for Science Ghana.
The forum sought to create a platform for farmers, scientists, journalists and students to have an encounter with officials of the NBA.
Mr Okoree said scientists have found that in certain cases, crop plants could not grow well because they do not have the necessary natural and capabilities to do so and modern biotechnology could impact that capability for the plant to grow.
'Who will expect that rice can grow in a saline soil; a soil which has salt but modern biotechnology has taken genes from a plant that grows in a saline environment and put it in rice, and now rice can be grown in a saline area along the shore', he said.
Mr Okoree said the Authority does not release GMOs in the country but serves as the regulator of modern biotechnology and as such grants permits for GMOs to be researched on.
Mr Richard Ampadu-Ameyaw, a Senior Scientist at the Council for Scientific and Industrial Research- Science and Technology Policy Research Institute (CSIR-STEPRI), said food security issues, low production, land degradation and climate change were factors that needed more attention so far as agriculture was concern.
He said there were technologies, however, the reality was that the technologies were not able to meet the challenges of today, therefore, there was a need for another technology; and one of them was biotechnology with its associated GMOs.
Mr Ampadu-Ameyaw, who doubles as the National Coordinator for Open Forum Agricultural Biotechnology (OFAB), urged Ghanaians to go for biotechnological products and GMOs; adding that 'the farmer has the right to cultivate what he or she wants but we should not also stop others from making their own choice'.
Ms Slyvia Tawiah Tetteh, a member of Alliance for Science Ghana, said Ghana was working towards introducing Genetically Modified Foods into the country's food chain.
She said work was ongoing on Bacillus thuringiensis (Bt) cowpea, and nitrogen use efficient, water use efficient and salt tolerant (NEWEST) rice for release onto the market sometime soon.
She said, however, the debate over whether Ghana should accept GMO foods or otherwise has continued unabated, and a lot of concerns had been raised about the technology.
Ms Tetteh re-affirmed their confidence in Alliance for Science Ghana with regards to the assurances that scientists all over the world had repeatedly stated that GMOs were safe, and that it is a crucial tool that could help revolutionalise Ghana's agricultural sector.
Alliance for Science Ghana is a network of farmers, scientists, communications persons, students and other well-meaning Ghanaians working to ensure improved food and environmental security in the country.
The Alliance works with agric sector stakeholders to enhance access to agricultural innovation as a means of ensuring food security, improving environmental sustainability, and raising the quality of life for farmers.


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Camille Lee
"I saw you in Ames, Iowa, USA
in 1986 while I was a student
at Iowa State. All I knew about
you before the concert was a
clip I saw on TV. WOW! I
remember how much I was
mesmerized 14 years ago.
Now I'm a mother of 2 and I
play your music for them. I
hope you will come to the
Midwest US again."


text:
this hotel is currently unavailable
Overview of Shades
SHADE Bungalows is ideal for singles, couples and families. Very tropical , decorated with Thai antiques from the owner's collection. Every room has its own character. Feel relaxed and feel the friendliness of samui people We are located on Bophut Beach. The most charming beach of Samui, a local fishing village on the beach only 200 meters away from our resort . Village is full of Thai and European fine dinning restaurants you can pick one everyday and enjoy the best dinning experience with great sunset view in fisherman village bay


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Spotted Dead Nettle, Spotted Deadnettle, Creeping Lamium, Spotted Henbit
Lamium maculatum
|Lamiaceae (lay-mee-AY-see-ee) (Info)
|Lamium (LAY-mee-um) (Info)
|maculatum (mak-yuh-LAH-tum) (Info)
|Lamium album var. maculatum
Water Requirements:
Drought-tolerant; suitable for xeriscaping
Average Water Needs; Water regularly; do not overwater
Sun Exposure:
Partial to Full Shade
Good Fall Color
Provides Winter Interest
Foliage Color:
6-12 in. (15-30 cm)
9-12 in. (22-30 cm)
USDA Zone 3a: to -39.9 �C (-40 �F)
USDA Zone 3b: to -37.2 �C (-35 �F)
USDA Zone 4a: to -34.4 �C (-30 �F)
USDA Zone 4b: to -31.6 �C (-25 �F)
USDA Zone 5a: to -28.8 �C (-20 �F)
USDA Zone 5b: to -26.1 �C (-15 �F)
USDA Zone 6a: to -23.3 �C (-10 �F)
USDA Zone 6b: to -20.5 �C (-5 �F)
USDA Zone 7a: to -17.7 �C (0 �F)
USDA Zone 7b: to -14.9 �C (5 �F)
USDA Zone 8a: to -12.2 �C (10 �F)
USDA Zone 8b: to -9.4 �C (15 �F)
USDA Zone 9a: to -6.6 �C (20 �F)
USDA Zone 9b: to -3.8 �C (25 �F)
USDA Zone 10a: to -1.1 �C (30 �F)
USDA Zone 10b: to 1.7 �C (35 �F)
Where to Grow:
Can be grown as an annual
Bloom Color:
White/Near White
Bloom Characteristics:
This plant is attractive to bees, butterflies and/or birds
Bloom Size:
Bloom Time:
Late Spring/Early Summer
Mid Summer
Other details:
May be a noxious weed or invasive
Soil pH requirements:
7.6 to 7.8 (mildly alkaline)
Patent Information:
Propagation Methods:
By dividing the rootball
Seed Collecting:
This plant has been said to grow in the following regions:
Brea, California
El Cajon, California
Merced, California
Monterey, California
Sunnyvale, California
Denver, Colorado
Parker, Colorado
Brookfield, Connecticut
East Canaan, Connecticut
Jonesboro, Georgia
Chicago, Illinois
Wichita, Kansas
Cumberland, Maryland
Detroit, Michigan
New Baltimore, Michigan
Saint Cloud, Minnesota
Saint Paul, Minnesota
Jackson, Mississippi
Mathiston, Mississippi
Sparks, Nevada
Munsonville, New Hampshire
Nashua, New Hampshire
Tamworth, New Hampshire
Brookside, New Jersey
Binghamton, New York
East Amherst, New York
Penn Yan, New York
Dayton, Ohio
Painesville, Ohio
Youngstown, Ohio
Hillsboro, Oregon
Sandy, Oregon
Lewisburg, Pennsylvania
Osceola Mills, Pennsylvania
Schwenksville, Pennsylvania
Rock Hill, South Carolina
Clarksville, Tennessee
Murfreesboro, Tennessee
Oak Ridge, Tennessee
Salt Lake City, Utah
Bellingham, Washington
Spokane, Washington (2 reports)
Appleton, Wisconsin
Muskego, Wisconsinshow all
Gardeners' Notes:
On Mar 21, 2016, coriaceous from ROSLINDALE, MA wrote:
A great perennial for shade in New England. This doesn't survive long south of Z8.
On Dec 7, 2013, VJD from Oak Ridge, TN wrote:
Planted this in September on the north side, and even though we've had freezing temps, it's still lovely and flowering! So far I love it.
On Sep 16, 2012, Bhamster from Bellingham, WA wrote:
This plant grows nicely in a sheltered, shady, nutrient-poor patch of my back yard that is bone-dry in summer and constantly damp in winter. It is very well-behaved under these conditions. It was here when we moved in 15 years ago, and has shown no inclination to expand its territory.
On Jun 18, 2012, Clary from Lewisburg, PA (Zone 6b) wrote:
After several years I have removed this plant from the garden. It spread much too rapidly by long creeping shoots that rooted along the way; it didn't make or stay in a clump. It also crept over and smothered out its neighbors. I like plants intermingling but this deadnettle began to behave like an invasive weed. To my dismay, when I tried to dig it out it had created a dense mat of roots that required a shovel to remove and took a good deal of soil with it. The mat of roots also seemed to be a good environment for pillbugs.
On May 25, 2009, sseebart from Sunnyvale, CA wrote:
I've had great luck with this plant in shady locations. It spreads rapidly and blooms easily. Less luck with these in pots. After even a mild winter, they were gangly and needed quite a bit of cleaning up. A quick transplant to a shady spot in the garden brought them right back, however.
On Apr 14, 2009, Rosiegardener from Hillsboro, OR wrote:
Impressive plant. Looks fantastic in containers. Excellent cold hardiness. I will post an image of one in a coco fiber hanging basket with icicles hanging all over it and a mound of snow on top. That plant didn't die! I tidied it up and it looks great this spring. I use this plant in hanging baskets only, so I have no experience with invasiveness. I believe it could be, though, after seeing how hardy it is.
On Aug 6, 2007, gardenchicago from Chicago, IL wrote:
I love the orchid frost purple lamium. I am in Chicago, planted a hillside, that is in morning sun, and it has flowered all summer.
On May 21, 2006, kmenzel from Saint Paul, MN wrote:
Very pretty, but can be invasive (seeds) when it's happy. I have tried many cultivars of lamium, and this one is now taking over my yard! It has even spread UPHILL into my neighbor's yard!
On May 3, 2005, PurplePansies from Deal, NJ (Zone 7a) wrote:
Lamium is not a extremely impressive specimen plant but it is nice as a shade groundcover.... it has flowers that are fine (and may not flower much in shade) but the attraction is the variegated leaves.....If you get one of the prettier cultivars you will be even more satisfied with your plant.... not a "knock your socks" off plant but then again those are (mostly) relegated to sun aren't they? I perfectly nice groundcover for shade ..... spreads nicely in moist shade and I recommend it as such. :)
On May 2, 2005, ladyannne from Merced, CA (Zone 9a) wrote:
Mine was labeled Orchid Frost, yet has white flowers? I love this ground cover, it is lush and full, readily recovers from dog steps and water hoses. It gets a few hours of filtered morning sun and does extremely well, borderlines on invasive.
On Mar 24, 2005, nevadagdn from Sparks, NV (Zone 7a) wrote:
I've successfully killed this plant more times than I can count. I killed it in Kansas (zone 5), and I've killed it in Nevada. Shade, no shade, doesn't matter.
On Nov 17, 2004, designart from Schwenksville, PA (Zone 6a) wrote:
Love Dead Nettles for foliage and extended blooming period. In PA many people have problems with the plants dying out during a wet summer like we had in 2004. Sometimes they will come back the following year. They did okay in deep shade but died out after about 5 years. In more sun they seem much happier.
On Sep 28, 2004, pokerboy from Canberra,
This is a great hardy plant that thrives in my garden in zone 8b/9a on neglect. I don't deadhead this plant after it flowers in early Spring, I don't water it much, maybe once a fortnight and the Lamium is in midday and morning sun and it thrives. It likes to support itself on the 1.2 metre retaining wall a family friend has got. Great plant for all situations. Most of which anyway. pokerboy.
On May 25, 2003, SunshineSue from Mississauga, ON (Zone 6a) wrote:
I've had very good results with all types of Lamium, sun or shade, in ground as well as in pots & hanging baskets. No special requirements other than watering.
On Feb 3, 2003, Crimson from Clarksville, TN (Zone 6b) wrote:
I loved this plant, I just started it in summer 2002 and it kept blooming until after most other plants were killed by the hard frost... I'm hoping it spreads, it's foliage is very lovely. (zone 4)
On Mar 10, 2001, Terry from Murfreesboro, TN (Zone 7a) wrote:
Semi-evergreen member of the mint family. Silvery foliage brightens shady spots. Flowers appear from late spring to early summer; colors range from white to shell pink to dark lavender, depending on variety. May send a second, smaller flush of flowers in cooler fall temperatures.


text:
This week I am launching into a homebrew project that was recommended to me by a good friend in Vermont, I am referring to VK3YE's "Beach 40" DSB Transceiver. Peter Parker/ VK3YE has put together a very nice little QRP Double Sideband Transceiver with a very modest number of discrete parts. The Beach 40 has been featured in SolderSmoke Podcast and website and is outline in several YouTube Videos done by Parker right from the beaches in Australia!
I haven't built anything from scratch yet and I am going to do this in Manhattan style building techniques, which is somewhat ironic in a cheeky way as I live in Manhattan. It looks like I am nearing the acquisition of most of my parts, including the copper boards and little square things from the internet.
In building a homebrew rig for the first time. I do have some soldering experience with several kits two CW transceivers from Hendrick's QRPkits.com and two Rockmite kits from Dave Benson/K1SWL at Small Wonder Labs. Dave has closed Small Wonder Labs, cheers to him for making nice affordable kits that let me cut my teeth so to speak.
I live in Manhattan as I said before and I don't really have a dedicated bench so we have to see how I do on the dining room table, also I don't have any sophisticated bench gear. All I have is a multimeter and a soldering iron.
I am very excited by this project because of it's simplicity and also because I haven't done a voice transceiver yet, the thing with CW rigs is that you need to know CW to actually enjoy using them. As amazing as Peter Parker has been at creating this rig I think that there are lot's of gaps that can be filled in terms of documentation and mentoring. This would be an excellent rig to build for the new General class ticket holder that may not have a lot of money to spend on gear and also would like to go portable or even pedestrian as Peter Parker terms his walking excursions.
So may the radio Gods be with us and hopefully we can breath life into our own gear and as Bill says put the "spirit into the machine".


text:
Fill the glass with ice, put in one shot of each Parrot Bay and a splash of Absolut Peach, fill with OJ, and top with cranberry juice and a splash of 7-up. Garnish with any colorful fruit, I use an orange and cherries.
This drink recipe was submitted by one of our beautiful readers, liqrep!
There aren't any comments for Sex With The Manager yet. Why not mix one up and be the first to leave your thoughts?
Select the two fuzzy animals from this list:
Serving size: this recipe
*: Percent daily values are based on a 2,000 calorie diet.
Your daily values may be higher or lower depending on your diet needs.
‡: Does not include Absolut Apeach, Captain Morgan's, Captain Morgan's Parrot Bay, Captain Morgan's Parrot Bay, Orange Juice
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text:
Snipers in Syria: A Free Syrian Army fighter holds the head of a mannequin up to a hole in a wall of a Syrian Army base to attract and locate a sniper during heavy fighting in the Arabeen neighbourhood of Damascus February 3.
Kenya shopping mall: An injured woman cries for help after gunmen stormed the Westgate shopping mall in Nairobi September 21.
California fire: Fire-fighters spray water near a burning house in the Twin Pines Road area where the Silver Fire raged, near Banning, California August 7, 2013. The fire blackened more than 5,000 acres.
Gun violence: Ronnie Chambers Jr. looks at his mother Tahitah Myles (obscured) as she collapses during the funeral for his father Ronnie Chambers, 33, a victim of gun violence, in Chicago February 4.
Killer: TJ Lane takes off his shirt to show 'Killer' spelled out on his shirt at his sentencing hearing in Cleveland, Ohio, March 19. Lane was sentenced to life without parole for killing three students in a shooting rampage.
Banksy: A dog urinates on a new work by British graffiti artist Banksy on West 24th street in New York City, October 3, 2013.
Plane crash: Asiana Airlines Boeing 777 plane after it crashed while landing at San Francisco International Airport in California July 6, killing two people.
Man's best friend: Dan McManus and his service dog Shadow hang glide together outside Salt Lake City, Utah, July 22. McManus suffers from anxiety and Shadow's presence and companionship help him to manage.
Syria: A member of the 'Ansar Dimachk' Brigade, part of the 'Asood Allah' Brigade which operates under the Free Syrian Army, uses an iPad to fire a homemade mortar at one of the battlefronts in Damascus Sept 15.
Cruise ship disaster: The Costa Concordia lies on its side next to Giglio Island August 26, has lain partly submerged in shallow waters off the Tuscan island of Giglio since the accident in January 2012.
9/11: A man walks through the 9/11 Empty Sky memorial at sunrise across from New York's Lower Manhattan and One World Trade Center in Liberty State Park in Jersey City, New Jersey, September 11.
Syrian weapons: Issa, 10, carries a mortar shell in a weapons factory of the Free Syrian Army in Aleppo, September 7.
No hands: Angelica Murillo, 37, at her home in Tegucigalpa August 21. Angelica's husband attacked her in 2008, accusing her of having an affair, and cut off her two hands with a machete while trying to kill her.
Israeli clashes: An Israeli policeman drags an ultra-Orthodox man during clashes in the town of Beit Shemesh, near Jerusalem August 12.
Running of the bulls: A 31-year-old runner gets gored by a bull on Estafeta Street during the sixth running of the bulls at the San Fermin festival in Pamplona July 12.
Snowden: An empty passenger seat believed to be reserved by former U.S. spy agency contractor Edward Snowden June 24.
Germany floods: A garden with a swimming pool is inundated by the waters of the Elbe river during floods near Magdeburg in the federal state of Saxony Anhalt, Germany, June 10.
In hard times: A man walks his dog past a vacant shop, with graphics pasted to the outside to make it look like a working butcher's shop, in the village of Belcoo, Northern Ireland June 3, 2013. Local councils in Northern Ireland have painted fake shop fronts and covered derelict buildings with huge billboards to hide the economic hardship being felt in towns and villages near the golf resort where G8 leaders have met in June.
Free Syrian army: Abboud (L), 12, and his brother Deeb, 14, stand with their weapons behind sandbags in Aleppo's Sheikh Saeed neighbourhood, September 28, 2013. Abboud and Deeb, both school-going children before the civil war, joined the Free Syrian Army after the deaths of two of their brothers and an uncle in the conflict.


text:
For the Veggie Biker and many an older California sprocket jockey, Bicycle Rodeos at our elementary schools are how we learned to use bicycles for everyday getting about.
The Veggie Biker's wife, who was educated in Missouri, still remembers Robert Louis Stevenson–Right, Left, Stop. However, our children, educated in Oklahoma, had no bicycle rodeos–or any other training. They learned bicycling astride a 16-inch bike pointed downhill with dear old dad puffing alongside. They have bikes, but do not use them for daily transportation.
Education through Bicycle Rodeos not only promotes bicycle safety, it promotes love of bicycles.
We can do something about this. With the new emphasis on active transportation–bikes, buses, trains and sneakers–we can add the bicycle rodeo to our school's curriculum and the city's active transportation program.
The U.S. government-sponsored program is already in place. We just need to promote it locally. We just need to volunteer.


text: Grants will 'not be feasible' Wed, 06/07/2017 - 9:44am Four Bowman City Council members - Carla Patten, Clay Rooker, Wade Bridges and Rachel Felice - signed a letter to ...PLEASE LOG IN FOR PREMIUM CONTENT Our website requires visitors to log in to view the best local news. Not yet a subscriber? Subscribe today! Read more about Grants will 'not be feasible'


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PlayStation Plus Free Games for June Announced, PS4 Getting Two Titles
From June onwards, Sony will streamline PlayStation Plus to have two titles for PS4, PS3, and Vita.
From June onwards, Sony will streamline PlayStation Plus to have two titles for PS4, PS3, and Vita.
Q-Games' James Mielke returns to answer your questions about living and working in Japan.
Fan vote to determine cream of 2011's downloadable PS3 and PSP crop; winners to go on sale for 30 to 50 percent off starting next week.
Q Games dubbed biggest earner for Sony-published titles, while Playdead's platformer sets high bar for third parties.
Ms. 'Splosion Man proves that she's an independent woman in her own game.
Ubisoft's sleeper hit of 2003 gets its chance at redemption this week on Xbox Live Arcade.
Sony's virtual marketplace adds colorful shooting game, add-on content for Visceral Games' horror title; ModNation Racers now available as full-game download; Vagrant Story hits PSOne Classics; demos for MLB 2K11 ...
Q Games president announces sequel to last year's shoot-'em-up adventure; promises new features to the series.
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Company Profile :-
Sun Microsystems, Inc. was a company that sold computers, computer components, computer software, and information technology services and that created the Java programming language and the Network File System (NFS). Sun significantly evolved several key computing technologies, among them Unix, RISC processors, thin client computing, and virtualized computing. Sun was founded on February 24, 1982. At its height, Sun headquarters were in Santa Clara, California (part of Silicon Valley), on the former west campus of the Agnews Developmental Center.
On January 27, 2010, Sun was acquired by Oracle Corporation for US $7.4 billion, based on an agreement signed on April 20, 2009. The following month, Sun Microsystems, Inc. was merged with Oracle USA, Inc. to become Oracle America, Inc.
Sun products included computer servers and workstations built on its own RISC-based SPARC processor architecture as well as on x86-based AMD's Opteron and Intel's Xeon processors; storage systems; and a suite of software products including the Solaris operating system, developer tools, Web infrastructure software, and identity management applications. Other technologies include the Java platform, MySQL, and NFS. Sun was a proponent of open systems in general and Unix in particular, and a major contributor to open source software. Sun's main manufacturing facilities were located in Hillsboro, Oregon, and Linlithgow, Scotland.
There are 3 rounds.
- Aptitude
- Technical & HR
- Communication
Placement Papers :-
- Sun Microsystem Placement Paper 1
- Sun Microsystem Placement Paper 2
- Sun Microsystem Placement Paper 3
Download Sun Microsystem Placement Paper PDFs, Sun Microsystem Technical Sample Paper PDFs, Sun Microsystem Aptitude Sample Paper PDFs, Interview Sample Papers with solutions
The typical HR questions are :
- About yourself?
- Why Sun Microsystems?
- What do you know about Sun Microsystems?
- Willingness to work?
All the best from JobSamosa team!!!!


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Cooking time: 8-10 minutes
1 cup all-purpose flour
1/2 teaspoon salt
1/3 cup lard
1/3 cup plus 1 tablespoon shortening
2 tablespoons cold water (2 to 3 tablespoons)
- Code: Select all
Mix flour and salt. Cut in lard until particles are size of small peas.
Sprinkle with water, 1 tablespoon at a time, tossing with fork until all
flour is moistened and pastry almost cleans side of bowl (1 to 2 teaspoons
water can be added if necessary).
Gather pastry into ball; shape into flattened round on lightly floured
cloth-covered surface.
Roll pastry 2 inches larger than inverted pie plate with floured
cloth-covered rolling pin. Fold pastry into fourths; unfold and ease into
plate, pressing firmly against bottom and side.
Trim overhanging edge of pastry 1 inch from rim of plate. Fold and roll
pastry under, even with plate. Flatten pastry evenly on rim of pie plate.
Press firmly around edge with tines of fork, dipping fork into flour
occasionally to prevent sticking. Or build up edge of pastry. Place index
finger on inside of pastry edge and knuckles (or thumb and index finger)
on outside. Pinch pastry into V shape; pinch again to sharpen. Fill and
bake as directed in recipe.
For Baked Pie Shell: Heat oven to 475º. Prick bottom and side thoroughly
with fork. Bake 8 to 10 minutes or until light brown; cool.


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The Octopus Eats its Own Leg
Feb 3 – May 6, 2018
Murakami's signature character, Mr. DOB was born in 1993, intentionally designed as a graphic icon to that has been swiftly developed into a multidimensional and multifunctional figure that Murakami has used in all facets of his production. In Klein's Pot, the artist mapped advanced scientific theories onto the face of Mr. DOB, and named the series of paintings after a mathematical theory where one is unable to distinguish between 'inside' and 'outside' surfaces. DOB became an endless compositional machine that continues to fuel the artist's painterly imagination to this day.
Takashi Murakami, Klein's Pot A, 1994–97 (detail), acrylic on canvas mounted on board in plexiglass box
Colección Pérez Simón, Mexico, © 1994–97 Takashi Murakami/Kaikai Kiki Co., Ltd. All Rights Reserved
The Octopus Eats Its Own Leg
Takashi Murakami: The Octopus Eats Its Own Leg is the first major retrospective of Takashi Murakami's paintings to be shown in Canada. Spanning three decades of the artist's career—from his monumental paintings of the 1980s to new, never-before-seen works—this critical survey reveals the consistent themes and profound engagement with history that have guided the artist's practice. More than fifty paintings and sculptures in the exhibition highlight a dedication to craftsmanship and uninhibited imagination mining a diverse field of conceptual and cultural references extending from folklore to art history and popular culture.
The exhibition takes its title "The Octopus Eats Its Own Leg" from an ancient Japanese parable, tako ga jibun no ashi wo kurau. It refers to a situation in which one survives for the time being by feeding on or sacrificing oneself. The octopus eats its own leg to survive, but does so knowing the tentacle will regenerate. The phrase symbolises the cyclical nature of Murakami's practice and the creative output of the Kaikai KiKi studio. Murakami is the octopus: he consumes history, culture and even his own oeuvre and fame to persevere as an artist.


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Holocaust Memorial Day Student Poems
Holocaust Memorial Day was created on 27 January 2000, when representatives from 46 governments around the world met in Stockholm to discuss Holocaust education, remembrance and research. At the end of this meeting, all attendees signed a declaration committing to preserving the memory of those who have been murdered in the Holocaust. This declaration became the statement of commitment which is still used as a basis for HMD activities today. The date was chosen as it is the anniversary of the liberation of Auschwitz.
Every year, some of our students study the Holocaust and the subsequent atrocities then produce powerful, poignant poems and creative writing to form the basis of our annual Holocaust Memorial Day atrium display. The work is also used in assemblies throughout the week. This year, some of our Year 7 girls decided to send the poem they were inspired to write, to the patron of the Holocaust Memorial Day Trust, His Royal Highness the Prince of Wales!
Student Poems
Auschwitz by Ellie Lowery, Drew Sayers, Malik Scott and Hermione Tulip Year 7
Auschwitz by Morgan Bolton, Hope Dunn and Jodi Johnson
Iby Knill by Naeve Renforth, Emma Selwood and Alysha Young Year 7
Incredible by Cameron Bromley, Joseph Elliot, Jessica Johnson and Jaidan Malpass Year 8
Khmer by Michael Newman, Bethany Ramm and Kyron Scott Year 10
Lice by Gabrielle Dixon, Josh Hornsby and Brandon McSkelly Year 9
Lifeless by Kyrin Curtis, Paige Maddison, Joana Temperley and Nikki Wright Year 8
Nicholas Winton was an ordinary man by Surapet Puphuak Year 7
Auschwitz by Kieran Allaker, Matthew Jordan and Jay Sutton Year 8


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Chicago Mayor Rahm Emanuel unveiled a proposed budget on Wednesday that aims to close a projected $636 million+ shortfall. The budget proposes a major reorganization of the city's advisory councils and the elimination of its LGBT liaison, reports Windy City Times.
Under the subcategory "Commission on Human Relations," Emanuel has proposed having directors/community liaisons for the Advisory Council on Gender and Sexuality and the Advisory Council on Equity; each individual would receive an annual salary of $86,796.
However, in establishing these positions, several other councils would no longer exist, including those pertaining to African affairs; Arab affairs; Asian affairs; Latino affairs; immigration and refugees; women; and gay and lesbian issues. ( The directors/liaisons of those departments were being paid the same amount in the new proposal: $86,796. ) The aforementioned councils would be housed under gender and sexuality as well as equity. The Advisory Council on Veteran Affairs remains intact.
One individual whom the budget proposal negatively affects is Director of Lesbian, Gay, Bisexual and Transgender Issues Bill Greaves, who will lose his job. Windy City Times obtained an email that Greaves sent to members of that particular advisory council. "At a meeting this morning Commissioner [ of the Department of Human Relations Mona ] Noriega informed me that as of January 1 I will no longer be employed by the City of Chicago," the email states. "There will be reorganizations of the Advisory Councils, which we can discuss at our meeting October 19.
"I have been told by the Commissioner that my only responsibilities between now and the end of my tenure as Director are to request letters from the Mayor for the Hall of Fame inductees and to assist with the transition."
The new has not been welcomed:
Beth Kelly, chair of the council, called both Greaves' proposed departure and the reorganization of councils an "affront to LGBT communities in Chicago" and a "symbolic erasure." "I think it's really important for people to think about the practical and symbolic consequences of this proposal should it come to fruition," Kelly said. "It sends a message that Chicago does not care about this community." [...]
Kelly also argued that there are practical concerns in reorganization. Kelly worries that the changes will undo progress the LGBT community has made with the Chicago Police Department and that by not naming "LGBT" specifically, the community will be less likely to report incidents of discrimination.
Chicago was one of the first large cities in the U.S. to name an official LGBT liaison. The city's first mayoral LGBT liaison was appointed by the late Mayor Harold Washington in February 1984. Greaves has served as mayoral LGBT liaison since 2000.
Emanuel's proposed elimination of the LGBT advisory position follows several similar local moves. Veteran Black lesbian activist Vernita Gray was laid off from the Cook County State's Attorney's office earlier this year. Gray was the prosecutor's LGBT liaison.
In an ironic twist of fate, the city's human relations commissioner and the person responsible for executing the re-organization is openly lesbian. Mona Noriega is the veteran Latina lesbian activist who was considered for a vacant city council post in 2010. Noriega would have become Chicago's first openly lesbian alderman but was not chosen. Noriega was appointed human relations commissioner in September.
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Chicago Latina Lesbian Considered for City Council
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- Research article
- Open Access
Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations
© Illes et al; licensee BioMed Central Ltd. 2009
- Received: 31 March 2009
- Accepted: 6 August 2009
- Published: 6 August 2009
The present work was performed to investigate the ability of two different embryonic stem (ES) cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs), progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far.
While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2), only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 2-4 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated vGlut2 expression within presynaptic vesicles. Also those NPCs that had migrated away from adherent neural aggregates maintained their ability to generate a synchronously oscillating neuronal network, even if they were separated from adherent aggregates, dissociated and re-plated.
These findings suggest that the complex environment within niches and aggregates of heterogeneous neural cell populations support the generation of fully mature neurons and functional neuronal networks from ES cell-derived neural cells. In contrast, homogeneous ES cell-derived NPCs within monolayer cultures exhibited an impaired functional neuronal maturation.
- Embryonic Stem
- Enhance Green Fluorescent Protein
- Neural Precursor
- Microelectrode Array
- Secondary Cluster
Functional biological neuronal networks in vitro can be derived from primary tissue-derived neurons and represent populations of synaptically interconnected cells capable of generating synchronous electrophysiological activity [1–6]. Electrophysiological recordings of entire neuronal networks can be achieved by microelectrode arrays (MEAs) which can detect electrophysiological activity of spatially distributed neuronal populations by multi-electrode recordings over several weeks or months [5, 7–10]. In vitro generated neuronal networks that represent the basic principle for brain activity can be used to analyze the electrophysiological development and quality of interconnected neuronal populations of different cell sources as well as their reactions to pharmacologically active compounds. The formation of functional neuronal networks requires fundamental properties of neural populations such as the ability to exhibit adequate neurite outgrowth, synaptogenesis, neurotransmitter production and release, action potentials as well as the generation of glial cells supporting neuronal functions. If only one of these fundamental properties is absent, the function of neuronal networks might be heavily impaired or even entirely blocked.
Recently, we and others demonstrated that also embryonic stem (ES) cell-derived neural precursor populations are able to generate functional neuronal networks [11, 12]. We illustrated that the development of an ES cell-derived neuronal network is characterized by distinct stages of specific electrophysiological activity that finally results in spatially distributed synchronously oscillating bursts [11]. The rationale for using ES cell-derived instead of primary tissue-derived neural populations is the increased homogeneity and controllability of in vitro generated functional neuronal networks whose reactions, for instance, to different pharmacological compounds strongly depend on the standardization of the cell culture. Furthermore, the possibility of measuring the electrophysiological function of in vitro generated neuronal networks permits to verify the quality of different precursor-derived neuronal populations prior to transplantation within the scope of regenerative medicine.
In recent years, the so called niche-independent development of neural precursor cells in monolayer cultures instead of precursor development within niches of aggregated cells of different developmental stages, also called neurospheres, was regarded as the most efficient way to synchronize and control neural precursor cell development in vitro. In order to improve standardization and homogeneity of ES cell-derived neuronal networks, we also applied the niche-independent protocols [13] for monolayer neural precursor cells (MNPs) that were generated initially from serum-free, floating cultures of embryoid body-like aggregates (SFEBs). We were able to produce highly homogeneous cultures of MNPs that adopted a radial glia-like cell stage and differentiated under controlled conditions into astrocytes and oligodendrocytes as well as mature NeuN+ neurons that also expressed GABA or vesicular-glutamate-transporter-2 (vGlut2). While dense MNP cultures, differentiated for at least 28 days, were able to generate spontaneously spiking neurons as expected, they were, however, not able to generate functional, synchronously oscillating neuronal networks as demonstrated by the MEA technology. Instead, we could show that the development of functional neuronal networks was reliably possible when heterogeneous neural cell populations derived from aggregated neural precursor cell-enriched SFEB (nSFEB) niches were used. As comparison of the neuronal morphology between MNP- and nSFEB-derived neuronal populations revealed shortcomings of MNP-derived neurons with respect to neurite morphology and maturity of glutamatergic neurons, we speculate that heterogeneous neural cell populations within neural aggregates provide factors which support the generation of functional neuronal networks.
Neural differentiation of ES cells
The ES cell lines SV-129 (ATCC, Millipore, Germany), R1 [14] and an ES cell line constitutively expressing enhanced green fluorescent protein (EGFP) derived from transgenic C57BL/6J mice ubiquitously expressing EGFP under the influence of the chicken β-actin promoter [15] were used in this study. Undifferentiated ES cells were grown under feeder-deprived conditions in the presence of 1,000 U/ml leukemia inhibitory factor (LIF, Chemicon, Temecula, CA, USA) and 20% fetal bovine serum (FBS, HyClone, Thermo Fisher Scientific, Germany) in ES cell medium described elsewhere [16]. Neural differentiation of immature ES cells was performed by modified protocols according to Watanabe (2005) and Nat (2007). Briefly, ES cell colonies were harvested without dissociation and re-plated on bacterial dishes (Greiner, Germany) in ES cell medium containing 10% FBS without LIF [17]. After one day, free-floating embryoid bodies had developed and were re-plated in neural induction medium (NIM) consisting of DMEM/F12 and neurobasal medium (1:1) (both from Gibco-BRL), 2 mM Glutamax (Gibco-BRL) and N2 supplement according to Johe and colleagues [18] as well as B27 supplement (Gibco-BRL). After 7 days, serum-free, floating cultures of embryoid body-like aggregates (SFEB) had developed consisting predominantly of neural precursor cells [19–21]. SFEBs were used to generate neural precursor cell-enriched SFEBs (nSFEBs) or adherently growing monolayer neural precursors (MNP).
For the generation of nSFEBs, free-floating SFEBs were cultivated for 7 to 14 days in the presence of neural proliferation medium (NPM) consisting of DMEM/F12, 2 mM Glutamax, fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF) (20 ng/ml each, PeproTech) and N2 supplement.
For the generation of secondary neural cell populations derived from nSFEBs, nSFEBs were plated on laminin coated dishes and kept under the influence of EGF and FGF-2 for 5 to 7 days. During that time a dense cell layer of mixed neural cells had spread out from the nSFEB aggregates. Entire populations including the compact nSFEB aggregates and the population that had spread out were detached by accutase (Chemicon, Germany) treatment for 20 minutes at 37 degrees, slightly triturated while leaving the nSFEB aggregates intact and then transferred to conical tubes and allowed to sediment for 10 minutes. Single cells within the supernatant, referred to as secondary neural cell populations derived from nSFEBs (SNPs), were collected and transferred on PDL/laminin-coated substrates for further characterization.
For the generation of MNPs, SFEBs were seeded on gelatin (0,1%, Sigma-Aldrich) coated plastic dishes (Greiner) in the presence of neural stem cell expansion medium (NS-A medium, Euroclone, Milan, Italy) supplemented with modified N2 and 20 ng/ml of both EGF and FGF-2. After 4-6 days in culture, cells migrating away from attached SFEBs were selectively enriched by multiple passages according to Conti and colleagues (2005).
Differentiation of nSFEBs, SNPs and MNPs
For propagation, free-floating nSFEBs were passaged once a week under the influence of EGF and FGF-2. MNPs were cultivated on gelatin coated dishes in NS-A medium under the influence of EGF and FGF-2.
For differentiation, nSFEBs (5-10 aggregates/100 μl), dissociated SNPs or MNPs (40.000 cells to 80.000 cells/100 μl) were seeded on poly-D-lysine (PDL, 15 μg/ml, Sigma) and laminin (15 μg/ml, Sigma) - coated glass coverslips or microelectrode arrays (Multi Channel Systems, Reutlingen, Germany). For successful long-term attachment of cells on MEAs, PDL coating was performed for 1 day and laminin coating for 5 days at 4°C. To improve cell survival and induce a latent neurogenic program [22], cells were kept for further 6-10 days after plating under the influence of FGF-2 only, before also this growth factor was gradually removed from the medium to induce terminal differentiation. For long-term culture, ES cell-derived neuronal networks were kept in DMEM/F12 supplemented with N2, B27 and Glutamax.
Immunocytochemistry and quantification
For immunocytochemical investigations, cultured cells were washed in phosphate-buffered saline (PBS), pH 7.3 and fixed for 15 min in 4% paraformaldehyde before incubation with 1% normal goat serum (NGS, Sigma) in PBS for one hour. Primary antibodies were monoclonal mouse antibodies to βtubulin III (1:750, R&D Systems), glial fibrillary acidic protein (GFAP, 1:1000, DAKO) nestin (1:750, Chemicon), myelin basic protein (MBP, 1:1000, Chemicon), NeuN (1:500, Chemicon), synaptophysin (1:100, Sigma), GABA (1:1000, Sigma) and vesicular glutamate transporter 2 (vGlut2, 1:10000, Chemicon). They were applied at 4°C over night. After washing in PBS, appropriate secondary antibodies coupled to Cy2, Cy3 or Cy5 (1:750, Dianova, Hamburg, Germany) were applied for one hour at room temperature. Cell cultures were counterstained for one minute with 4',6-Diaminodino-2-Phenylindol (DAPI, 2 μg/ml, Serva) to visualize cell nuclei. Images were collected with a confocal-laser scanning microscope (LSM 510 Zeiss, Germany).
For quantification of neurons, astrocytes or oligodendrocytes, percentages of NeuN+, GFAP+ or MBP+ cells from all DAPI+ cells were counted with "Nucleolus Counter Plugin" and "Cell Counter Plugin" provided by ImageJ software.
Microelectrode array recordings
To measure the electrophysiological activity of nSFEB-, SNP- and MNP-derived neurons, microelectrode arrays (MEAs) were used with a square grid of 60 planar Ti/TiN electrodes (30-μm diameter, 200-μm spacing) and an input impedance of <50 kÙ according to the specifications of the manufacturer (Multi Channel Systems, Reutlingen, Germany). Signals from all 60 electrodes were simultaneously sampled at 25 kHz, visualized and stored using the standard software MC_Rack provided by Multi Channel Systems. Spike and burst detection was performed off-line by custom-built software (Result, Düsseldorf, Germany). Individually for each channel, the threshold for spike detection was set to 6.2 standard deviations (SDs) of the average noise amplitude during a 10% "learning phase" at the beginning of each measurement. An absolute refractory period of 4 ms and a maximum spike width of 2 ms were imposed on the spike detection algorithm. All spike waveforms were stored separately and visually inspected for artifacts. Burst detection was performed individually for each electrode by comparing the actual temporal clustering of spikes with a Poisson process of independently occurring spikes at the same mean firing rate (null hypothesis). Given the mean firing rate (MFR) of each electrode, the probability P(N, Δt|MFR) of finding at least N spikes within the time span Δt was calculated using the Poisson distribution. An observed cluster of N>3 spikes occurring within Δt was considered as burst, if its Poisson probability P(N, Δt|MFR) was below 0.005. Note that although bursts frequently occurred as population events, i.e. synchronized across many electrodes, they were detected separately for each electrode allowing for different mean firing rates. Bursts were also verified for plausibility by visual inspection. For the quantification of firing synchrony across two electrodes spikes were collected in 10 ms wide bins. Bins were then dichotomized to contain either zero spikes or at least one spike. For two electrodes (1, 2) with s1 (s2) bins containing spikes on electrode 1 (2), c12 bins with coincident spikes on both electrodes, and a total of N bins in the recording, there is an expected proportion pexp = ((N - s1)·(N - s2) + s1·s2)/N2 of coincident bins (both either empty or with coincident spikes), if firing occurred independently on both electrodes. Cohen's kappa statistic κ then captures the excess part of the proportion of observed coincident bins pobs over the expected proportion pexp: pobs = pexp + κ·(1-pexp) [23]. Kappa values lie in the range -pexp/(1-pexp) to +1. The average kappa of all electrode pairs with a firing rate of at least 50 spikes per minute was calculated as measure of overall synchrony of a recording.
Statistical analysis
MEA recordings for the different groups (nSFEB, SNP, MNP) were repeated at least 10 times for each group with at least 3 independent cell cultures. To verify electrophysiological differences of aggregates, clusters and interjacent cells, at least 826 electrodes, respectively, were ascribed to the spatially different compartments of individual cultures and electrophysiologically analyzed. Data were analyzed with one way analysis of variance (Anova) followed by Bonferroni's multiple comparison test. A P value less than 0.05 (*) or 0.01 (**) was considered to be statistically significant. For quantification of cell type-specific marker expression, at least 1000 cells in three independent cultures, respectively, were counted in duplicates for each time point and were analyzed by unpaired t-test.
Data of spikes/minute and kappa values were analyzed by paired t-test. Data are presented as mean ± standard error of means, n refers to the number of recordings. All data analyses have been performed with GraphPadPrism, version 4.0.
nSFEB-derived migrating progeny consists of a mixed neural cell population
In accordance to previous publications [13, 21], one day after plating nSFEBs on laminin coated substrates, two different cell populations were observed that migrated radially, leaving the edges of the adherent nSFEB aggregates. The first population displayed an elongated-flat morphology and most of these cells were nestin+/GFAP-, while only very few of them were nestin-/GFAP+ (Figure 1b, c). The second population was bipolar and round shaped and predominately βtubulin+ (Figure 1b, c).
Seven days after plating, the elongated-flat cell population had migrated a considerable distance away from the edges of the nSFEB aggregates and generated a flat cell layer of nestin+/GFAP-, nestin+/GFAP+ and nestin-/GFAP+ cells (Figure 1b, c). Interestingly, in addition to the flat cell layer and the βtubulin+ population (Figure 1c, iv and 1vi), secondary cell clusters appeared on top of the flat cell layer (Figure 1c, v). These secondary clusters consisted of round nestin+/GFAP- cells that were smaller than the elongated-flat cells. In addition, the nestin immunoreactivity in round nestin+/GFAP- cells was considerably stronger in comparison to nestin+/GFAP- and nestin+/GFAP+ elongated-flat cells (Figure 1b, c).
After further cultivation in the presence of growth factors, secondary clusters of round nestin+/GFAP- cells gave rise to larger structures that we termed secondary neural aggregates (Figure 1d, v, vi). They comprised βtubulin+ neurons, nestin+/GFAP- cells and nestin-/GFAP+ astrocytes (Figure 1e, viii, ix). The flat cell layer of migrating elongated cells and the secondary neural clusters and aggregates were referred to as the secondary neural cell population (SNP), which consisted of a mixed neural phenotype. Most importantly, we and others [21] were not able to detect contractile tissue indicative for mesodermal differentiation or epithelia like cells indicative for endodermal differentiation within the SNP during the first 7 days after plating nSFEBs under the influence of FGF-2 or EGF/FGF2 (data not shown). Residual non-neural cells, however, were detected within nSFEBs and also within the SNP at later time points (>14 days after plating) during terminal differentiation (data not shown).
The secondary neural cell populations (SNPs) derived from plated nSFEBs can selectively be harvested
To isolate SNPs derived from primary nSFEB aggregates on a laminin substrate at early time points and thus to eliminate non-neural cells determined to become, for instance, contractile tissue or epithelial-like cells, we first detached the entire cell population including primary nSFEB aggregates and SNPs and separated those populations by gentle trituration and sedimentation of nSFEB aggregates. The SNPs were then again dissociated and plated on a laminin coated substrate and characterized by immunocytochemistry and MEA recordings.
One day after plating dissociated SNPs, a mixed neural cell population could be observed which consisted of elongated-flat nestin+/GFAP-, nestin+/GFAP+, nestin-/GFAP+ cells and processes-bearing βtubulin+ cells (Figure 1d, e). Already at this early time, we again observed secondary clusters similar to those found in nSFEB cultures. They consisted of small round nestin+/GFAP- cells which were located on top of the elongated-flat neural cells (Figure 1e, i, ii). Three days after plating dissociated SNPs, secondary clusters adopted a rosette-like structure composed of nestin+/GFAP- and βtubulin+ cells with radially extending neurites. Interestingly, secondary clusters did not exhibit GFAP+ cells yet (Figure 1e, v + vi). Seven days after plating dissociated SNPs, secondary clusters gave rise to larger secondary neural aggregates, similar to those observed within nSFEB cultures. They were composed of nestin+/GFAP-, nestin-/GFAP+ cells and, particularly at the edges, βtubulin+ neurons (Figure 1e, viii + ix). At the same time, the cell population in between secondary clusters or aggregates comprised nestin-/GFAP+ flat cells (Figure 1d, v; 1e, vii) and βtubulin+ neurons (Figure 1d, ii, iv; e, iii). Importantly, all independently predifferentiated SNP cultures, no formation of contractile tissue was observed.
MNPs consist of a pure radial glia-like cell population
In contrast to neural aggregate cultures (nSFEB or SNP), MNPs were cultivated on gelatin-coated substrates in neural stem cell medium (NS-A) supplemented with EGF and FGF-2 with 2-3 passages per week (Figure 1a). As revealed by immunocytochemical analyses, all MNPs were homogeneously nestin+ (Figure 1f) Pax6+/Vimentin+/RC2+ (data not shown). After cultivation of MNPs for 7 days on a laminin substrate under the influence of FGF-2, few cells expressed GFAP or βtubulin (Figure 1f, ii-iv). This indicated that the gelatin substrate in combination with EGF and FGF-2 was able to keep MNPs in an immature radial glial state and that a laminin substrate together with FGF-2 alone was not able to completely prevent differentiation.
nSFEB- and SNP-, but not MNP-derived cells are able to generate synchronously oscillating neuronal networks
We then verified the functional activity of MNP-derived neuronal populations. While electrodes abundantly detected spontaneous spike activity and trains of spikes commencing 14 days after initiating the differentiation, synchronous bursts were absent during an observation time of up to 3 months in 20 independent MEA cultures (Figure 3c). Importantly, also the application of bicuculline did not elicit synchronous network activity in MNP-derived neuronal populations (Figure 3b, iii). As also the seeding density of neuronal populations is known to be of importance with respect to functional network generation [7, 24, 25] we plated nSFEB-, SNP- and MNP-derived neural cell populations at varying seeding densities. Similar to primary tissue-derived neuronal networks [7, 24, 25] higher seeding densities of nSFEB or SNP cultures changed the development of synchronous neuronal networks. However, varying seeding densities did not improve the functional activity of MNP-derived cultures.
Comparison of mean activity levels (spikes per minute) and degrees of network synchrony (kappa values) of nSFEB-, SNP- and MNP-derived neuronal networks revealed major differences. Twenty-eight days after initiation of differentiation, the mean spike rates in nSFEB or SNP cultures were 42-fold or 52-fold higher, respectively, than in MNP cultures (Figure 3c, i). Furthermore, kappa values at this point in time were clearly indicative of synchronous network activity in nSFEB and SNP cultures. Kappa values in MNP cultures were expectedly low suggesting asynchronous activity. This is in line with the comparably low overall activity levels of MNP cultures that were hardly consistent with synchronous bursting (Figure 3c, ii).
To measure developmental changes of network activity between 14 and 28 days after initiating the differentiation, spike rate ratios (spike rates at 28 days/14 days) or kappa ratios (kappa values at 28 days/14 days) were calculated for nSFEB-, SNP- or MNP-derived network activity. While spike rate ratios revealed no major differences between groups, kappa ratios suggested an increase in synchrony of network activity merely of nSFEB or SNP cultures (Figure 3c, iii + iv).
Percentages of active electrodes in nSFEB- and SNP-derived populations are regionally different
An overview of the distribution of active electrodes in different ES cell-derived neural population is given in figure 2 with active electrodes depicted in white and inactive electrodes in black. In the present study, most electrodes that detected spike activity (active electrodes) recorded multiphasic spikes, suggesting a somatic origin of those signals [4].
As nSFEB and SNP cultures exhibited a heterogeneous spatial distribution of cells with primary nSFEB aggregates (only in nSFEB cultures), secondary neural aggregates (in nSFEB and SNP cultures) and the interjacent cell population consisting of a flat cell layer (Figure 2), we accordingly analyzed the spatial distribution of active electrodes which were participating in synchronous burst activity.
We observed that most active electrodes were localized at the edges of primary nSFEB or secondary neural aggregates (edges: 75% ± 14.4% vs. centers: 7.2% ± 7.4%, p < 0.001) and that fewer active electrodes were localized within the interjacent cell population (interjacent cell population: 25.8% ± 10.6%).
Neural aggregate and MNP cultures contain considerable numbers of neurons and glial cells
Both MNP and neural aggregate cultures comprised nestin-/GFAP+, nestin+/GFAP-, and nestin+/GFAP+ cells with diverse morphology, process-bearing βtubulin+ neurons and multi-branched MBP+ oligodendrocytes at 28 days after the initiation of differentiation (Figure 4a, b). After initiating the differentiation by gradual withdrawal of FGF-2, the homogeneous population of radial glia-like MNPs differentiated into equally distributed βtubulin+ neurons, GFAP+ astrocytes and MBP+ oligodendrocytes (Figure 4a). Laser scanning microscopy revealed that secondary neural aggregates comprised a core and edge region (Figure 4c). The core region was composed of mainly nestin+ cells (Figure 4c, i-vi; yellow box), whereas edges appeared to harbour higher numbers of GFAP+, βtubulin+ and few MBP+ cells (Figure 4c, i-vi; white box).
Seven days after initiating the differentiation, 27.52% ± 1.547% of all MNP-derived cells were NeuN+ neurons. The number of neurons remained stable until 28 days after initiating the differentiation (31.52% ± 2.340%, Figure 4d, i).
Numbers of NeuN+ neurons in neural aggregate cultures were separately quantified within secondary neural aggregates and interjacent cells. Similar to the distribution of βtubulin+ cells, we could barely find any NeuN+ cells within centers of secondary aggregates, in comparison to the edges where we found 47.9% ± 6.8% NeuN+ neurons at 7 days after initiating the differentiation (Figure 4c, viii). At 28 days after initiating the differentiation, percentages of NeuN+ cells tended to decrease at the edges towards 30.8% ± 4.4% (Figure 4d, i). The interjacent cells in between secondary aggregates contained only 2.7% ± 0.3% NeuN+ neurons at 7 days after initiating the differentiation. After 28 days, percentages of NeuN+ neurons tended to increase towards 12.5% ± 2.5% (Figure 4d, i). This quantitative constellation suggests either a dynamic migration of cells between aggregates and the interjacent area or ongoing neurogenesis.
Considerable numbers of nestin+ cells within the centers of secondary aggregates indicate that antibodies do have access to this dense cellular structure (Figure 4c ii, v, iii, vi).
Thus, both neural aggregate (nSFEB or SNP) and MNP cultures contained considerable numbers of neurons. While NeuN+ neurons in MNP cultures were evenly distributed, NeuN+ neurons in neural aggregate cultures were predominantly located at the edges of secondary neural aggregates and to a lower percentage within the interjacent population. However, also those regions within neural aggregate cultures that showed lower neuronal percentages (interjacent population) were found to be functionally integrated within synchronous network activity (see above), suggesting that not only the mere number of neurons within cultures is responsible for different degrees of electrophysiological activity.
As it is known that astrocytes are able to regulate synaptogenesis, neurite outgrowth and neuronal maturation [26, 27], and that myelinating oligodendrocytes impact on action potential propagation, we also analyzed the numbers of GFAP+ astrocytes and MBP+ oligodendrocytes within neural aggregate and MNP cultures. As described above, GFAP+ or MBP+ cells were found predominantly within the interjacent cell population and at the edges of secondary neural aggregates (Figure 4b, iii; c, i, vii). While a reliable quantification of GFAP+ or MBP+ cells within the densely packed edges of secondary aggregates was not possible, we found that the interjacent cells showed no significant differences in percentages of GFAP+ astrocytes or MBP+ oligodendrocytes in comparison to MNP cultures at 7 or 28 days after initiating the differentiation (Figure 4d, ii). In contrast to MNP cultures, the distribution of cells within neural aggregate cultures was not homogeneous limiting the validity of a quantitative comparison.
Neural aggregate and MNP cultures differentiate into neurons positive for GABA or vGlut2
As differentiation into inhibitory GABAergic and excitatory glutamatergic neurons represents a prerequisite for the generation of functional neuronal networks, we immunocytochemically verified the expression of GABA and vGlut2 in neural aggregate (nSFEB or SNP) and MNP cultures after 28 days of differentiation.
In neural aggregate cultures, we found the typical punctuated vGlut2 staining localized on βtubulin+ neurites, a distribution which is known to correspond to vesicular glutamate transporters on synapses. However, vGlut2 labeling in differentiated MNP cultures was localized cytoplasmically (Figure 5b). Due to the different cellular localization of vGlut2+ structures in both groups, a reliable quantification of mature glutamatergic neurons that obviously do not exist within MNP cultures was not possible.
Neural aggregate and MNP cultures reveal differences in neurite morphology
While βtubulin+ neurons within neural aggregates (Figure 6a, i) and within the interjacent cell population (Figure 6a, ii; b, i + iii) of neural aggregate cultures (nSFEB or SNP) developed a dense network of neurites as soon as 14 days after initiating the differentiation, MNP-derived neuronal populations were not able to generate a morphologically dense network of neurites (Figure 6b, ii + iv).
We detected functional discrepancies between different ES cell-derived neuronal populations by using the MEA technology: (i) a population of homogeneous, radial glia-like cells [13] termed "monolayer neural precursors" (MNP), and (ii) a heterogeneous neural cell population comprising neural stem/progenitor cells, committed neural cells as well as mature cells like neurons and glial cells termed "secondary neural cell population" (SNP). Previous studies have demonstrated that ES cell-derived heterogeneous neural cell populations cultivated as neurospheres or as adherently growing cultures are unstable and alter their differentiation potential with time by adopting a gliogenic fate after few passages [16, 28]. Whether this phenomenon is mediated by an intrinsic program or rather by the environment consisting of more committed neural cells which serve as a niche to induce enhanced glial maturation is still under debate. It was an important step forward when new protocols were introduced allowing to generate stable ES cell-derived homogeneous radial glia-like cells which can be enriched in nearly unlimited numbers as niche-independent, adherently growing neural stem cells without losing their neurogenic fate [13]. However, we demonstrate that neurons derived from homogeneous, radial glia-like MNPs were only able to show spontaneous spike activity without generating synchronously oscillating neuronal networks. In contrast, neurons derived from heterogeneous SNPs reliably generated functional network activity with synchrony even increasing with age. Interestingly, both neural cell populations were initially generated from serum-free, floating cultures of embryoid body-like aggregates (SFEBs) before cells were split in accordance with the two different protocols. This suggests that the subsequently applied cell culture procedures were responsible for the functional differences.
MNP- but not SNP-derived neurons remain within immature neuronal stages
As both neural aggregate- and MNP-cultures differentiated into sufficient numbers of neurons, astrocytes and oligodendrocytes, the mere quantity of those cells appeared not to be responsible for differences in functional network generation. We could demonstrate that electrophysiological network activity was not limited to the edges of neural aggregates that comprised ~31% mature NeuN+ neurons at 28 days after initiating the differentiation. Indeed, electrodes situated outside neural aggregates also participated in network activity although the interjacent cell population situated here contained only ~13% mature NeuN+ neurons. Thus, neuronal percentages amounting to approximately 32% within dense MNP cultures at 28 days after initiating the differentiation are indicative of other causes for the missing network activity such as impaired neuronal maturity. This assumption is substantiated by the following observations.
Our morphological analyses suggest that MNP-derived neurons in general seem to remain within an immature stage as they sent out only sparsely ramified and short neurites despite a differentiation period for up to 3 months. Such delayed maturation is in accordance with patch-clamp experiments demonstrating that in MNP-derived neurons single action potentials cannot be evoked until 30 days of differentiation [13] while aggregate-based ES cell-derived neurons exhibit evoked repetitive firing already after 12 days of differentiation [17, 29, 30]. Furthermore, in contrast to SNP-derived neurons which exhibited a punctuated pre-synaptic vGlut2 labeling on βtubulin+ neuronal processes, in line with a mature neuronal morphology, we demonstrated that MNP-derived neurons showed a cytoplasmic vGlut2 expression similar to immature embryonic neurons in vivo [31]. A proper quantification of glutamatergic neurons in different groups by means of this marker was not possible as vGlut2 antibodies labeled presynaptic structures in aggregate cultures and cytoplasmic structures in MNP cultures preventing an acceptable comparison. It has to be determined in future studies by direct labeling of glutamate, if a reliable quantification of glutamatergic neurons within aggregate or MNP cultures is feasible. However, this approach has to take into consideration that, particularly in neural precursor populations, glutamate might be of metabolic, neurotransmitter or even precursor origin [32, 33]. Our morphological analyses specifically of GABAergic neurons also suggested an immature phenotype in MNP-derived cultures while aggregate-derived cultures exhibited GABAergic neurons of mature morphology with ramified and long neurites. The mere quantities of GABAergic neurons, however, were similar in both cultures. Thus, as network activity depends on the presence of mature excitatory and inhibitory neurons, our morphological results are in line with impaired neuronal network function. To further examine neuronal populations with respect to the functional balance of inhibitory or excitatory neurotransmitter systems, we pharmacologically modulated network activity and observed that the inhibition of GABAergic transmission in neural aggregate cultures (nSFEB or SNP) transformed asynchronous stage 2 activity into synchronous stage 3 activity, whereas MNP-derived neuronal populations remained within asynchronous stages. This functional constellation also suggests that MNP cultures miss morphological prerequisites supporting synchronous network activity.
SNPs represent a heterogeneous neural cell population with the ability to generate functional neuronal networks in vitro
In accordance with previous studies, we demonstrated that pure neural cell populations migrating away from neural aggregate edges on a laminin substrate can be found only within the first 7 days after attachment of nSFEBs. This period of fairly pure neural cell migration is followed by non-neural cells or residual ES cells that also migrate away from nSFEBs after further adherent cultivation [13, 21]. This observation is in line with reports of residual Oct-4+ cells and non-neural cells within nSFEB aggregates [19–21]. To purify neural populations, we selectively harvested migrating cells within the first week after attachment of nSFEBs by applying a sedimentation technique and termed this purified population the secondary neural cell population (SNP). We demonstrated that dissociated SNPs gave rise to neural populations that (i) were devoid of residual ES cells indicated by negative Oct-4 immunocytochemistry (data not shown), contractile tissue and epithelial-like cells in more than 130 cultures from three independently pre-differentiated ES cell populations, and (ii) generated functional neuronal network activity.
Interestingly, we showed that round nestin+/GFAP- cells, which were found in the periphery of adherently growing nSFEBs a few days after attachment, reorganized to neural rosette-like structures on top of a cellular monolayer consisting of nestin+/GFAP-, nestin+/GFAP+ and nestin-/GFAP+ cells. Thereby, the cellular monolayer might contain mixed types of neural precursors and astrocytes which might serve as a neurogenic niche for neural rosette-forming nestin+/GFAP- cells similar to postnatal astrocytes [34, 35]. This suggests that SNP-derived neural rosettes comprised neuroepithelial cells displaying several in vivo properties and giving rise to neural aggregates [36–40]. Laser-scanning microscopy revealed that only the edges of neural aggregates comprised higher numbers of mature neural cells (NeuN+ neurons, GFAP+ astrocytes or MBP+ oligodendorcytes) while aggregate centres predominantly contained immature nestin+ neural cells. In agreement with this, neural aggregates revealed low electrophysiological network activity within their centres (~7% of all electrodes) and high network activity at their edges (~75% of all electrodes). The development from neuroepithelial cells organised in rosette-like structures towards neural aggregates might lead to a hypothesis that immature neural cells remain within the centre of neural aggregates, virtually constituting the niche of functional neuronal progeny.
In previous studies, we demonstrated that a prolonged cultivation of ES cell-derived neural cell populations under the influence of FGF-2 resulted in the formation of substrate-adherent ES cell-derived neural aggregates (SENAs) that appeared to be similar to those secondary neural aggregates that were described in the present study. Interestingly, SENAs did not form teratomas after transplantation into an animal model of Huntington's disease [15]. We hypothesize that both, SENAs and secondary neural aggregates, can be derived from those neural rosettes by different protocols [15]. If secondary neural aggregates are also devoid of teratoma forming cells remains to be verified.
Do MNPs exhibit intrinsically impaired neurogenesis or miss the right environment for proper neuronal maturation?
Interestingly, we also found clustering of MNP cells at higher seeding densities, but those were devoid of rosette formation, showed an even distribution of mature and immature cells and contained only short-ramified neurons after the initiation of differentiation. It remains to be determined, if these radial glia-like cells are able to organize as neural rosettes in co-culture with astrocytes [34, 35] or if they have lost the ability to recapitulate early neural stage development resulting in a restricted developmental potential. A possible reason for inhibited maturation in gelatine-cultivated monolayer precursors might also be shortcomings in the expression of extracellular matrix (ECM) molecules in comparison to the heterogeneous neural cell populations. Recent reports demonstrated that different ECM molecules such as fibronectin and laminin exert strong influences on ES cell-derived neural stem and progenitor cells resulting in the expression of different transcription factor profiles, as well as diverse morphologic and electrophysiological properties of their differentiated mature neuronal progeny [29]. Furthermore, as different subclasses of GFAP+ cells are known to considerably support the electrophysiological maturation of neurons [27] and enhance or inhibit neurite outgrowth [26], it might be speculated that the different types of GFAP+ cells which we found at early stages of neural aggregate development but not within homogeneous MNP cultures are important for network function. Thus, niche-independent neural development might provide a high degree of cellular homogeneity at the cost of the proper milieu to generate functionally mature neurons in vitro. This speculation of course will have to be substantiated by additional investigations. As the ability of precursor-derived neurons to form functional neuronal networks represents a prerequisite to properly restore functional deficits in succession to neuronal degeneration, our data are directly linked to the field of regenerative medicine.
The MEA technology can be used to unmask functional differences of neural precursors
The data presented here demonstrates that the MEA technology detects functional differences in neural precursor cell development that exceed the functional verification of single neurons. So far, functional neuronal analyses by intracellular recordings have shown that conventional ES cell-derived neural precursors differentiate into electrophysiologically active neurons [13, 29, 41]. We confirmed this by applying the MEA technology to ES cell-derived neuronal networks, suggesting that the description of synchronous network activity represents a functional feature that should be additionally taken into consideration in order to characterize properties of precursor-derived neuronal populations. Furthermore, it could be shown that multi-site extracellular recordings were able to spatially resolve functional differences within individual neural cultures that developed compact, three-dimensionally arranged cellular aggregates as well as the interjacent cellular monolayers that were evenly distributed in between those neural aggregates.
Our investigations provide evidence that ES cell-derived neural precursors derived from heterogeneous neural aggregates give rise to neurons that are able to generate functional neuronal networks between 2-4 weeks after initiating the differentiation in vitro, whereas neurons derived from a homogeneous monolayer of radial glia-like cells do not exceed the stage of uncorrelated spontaneous activity during a differentiation period of up to 12 weeks under the cell culture conditions used in the present study. If MNP-derived neuronal populations adopt a functional state under modified cell culture protocols in vitro or after transplantation in vivo remains to be determined. Nonetheless, our findings indicate that niche- and aggregate-based differentiation clearly improves the functionality of ES cell-derived neural populations in vitro.
We are grateful to Jessica Opatz for critically revising the manuscript, Adrian Hülsewede and Christine Holland for excellent technical support. This work was supported by the "Bundesministerium für Bildung und Forschung" (BMBF, 01GN0503).
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