text:
"The only way to show real savings in health expenditures in the future is through efforts to reduce the prevalence of obesity and related health conditions," Finkelstein said.
Hoping to turn the tide of the obesity epidemic, the CDC is taking several steps it hopes will alert people to the problem and get Americans to make the changes need to reduce obesity.
Among the strategies the CDC is promoting are making healthy food more available, encouraging more choices of healthy foods, encouraging breast-feeding, encouraging physical activity and creating sites in communities that support physical activity, Dr. William H. Dietz, director of CDC's Division of Nutrition, Physical Activity, and Obesity, said during the press conference.
"These recommendations, I believe, set the foundation for the community interventions necessary to reverse this problem in the United States," Dietz said.
One of the biggest problems facing Americans is soaring consumption of soda and other sugar-sweetened drinks, which add almost 150 calories to the daily diet, Frieden said.
Frieden believes that taxing sodas and other sugar-sweetened drinks will help cut down on consumption and raise revenues that can be used to fight the obesity epidemic.
The upshot of Monday's meeting is that stemming the obesity epidemic is going to take a societal effort.
"Reversing obesity is not going to be done successfully with individual effort," Frieden said. "We did not get to this situation over the past three decades because of any change in our genetics or any change in our food preferences. We got to this stage of the epidemic because of a change in our environment and only a change in our environment again will allow us to get back to a healthier place," he said.
HHS head Sebelius agreed. "These are investments that can be made at the neighborhood, city, and stat
|Copyright©2009 ScoutNews,LLC.
All rights reserved


text:
Thanks for explaining.
The reason I asked is that I can never get the colors right in my black and white toned prints, even on my own display, probably because I don't have the proper setup and training to do it.
I'll follow with interest how this develops and have subscribed to the thread.
- Thomas

subset: commoncrawl


text:
Card City Nights
By Ludosity AB
Open iTunes to buy and download apps.
9/10 - GOLD AWARD "Fun, funny, and fundamentally unputdownable, Card City Nights is a fantastic mix of casual strategy and collectible card coolness"
- Pocket Gamer
"Effortlessly charming .. a simple yet rock-solid addition to the genre that's well worth a play."
- Jay Is Games
"The real hook is the CCG that serves as the game's engine. Fortunately, it's fantastic."
- Indie Game Reviewer
Card City Nights is an adventure through a city filled with card-nuts and lunatics. Battle for the 8 legendary cards to enter the biggest competition this city has seen yet.
Card City Nights is not a traditional card-trading game. It's an adventure game with zany characters to meet and weird locations to explore. In the center is our new card battle system that breaks away from the fantasy TCG mold with connecting cards to form combos.
* 8+ hours of gameplay in a singleplayer adventure
* Connect cards to form combos
* 180 unique cards with characters from the Ludosity universe
* No IAP's in this game! You collect booster packs by simply playing the adventure from start to finish
* An amazing soundtrack in a blend of Jazz and Hiphop
What's New in Version 1.06
+ A new optional character to challenge outside the House on Wheels! Look for the chicken suit.
+ Three new cards.
* Balance changes to existing cards.
- Speling fickses
Customer Reviews
Awesome game but random AI card deck?! Yea right.
Pretty awesome game, good music and extremely entertaining. I've never been into card bases games but man this one has drawn me in. Love it!
May have spoilers below.
One issue I kinda have with the game is the AI. Particularly the fiery women and that lemon girl. These two were crazy over powered with their decks. I doubt this the AI is choosing random cars. Every time I disable one of their key legendary or rare cards they pull a revive card?! EVERY SINGLE TIME :/ really?
An that lemon girl. I know she is cheating from the beginning she says so. But does she have an infinite amount of target opponent cards and health cards? They just keep on coming. Non stop. Haha. It's a good challenge and all but come on. It starts to get a little comical when every card is the exact type of card they need.
Ex. With both fiery woman and lemon girl. I disabled their legendary card 4 separate times. Can you guess what that AI pulled out in their next draw?? Yup an heart card that restores the card I just disabled.
Otherwise great game developer! Nice job. Will defintitely check out your other apps.
Good. Different
This has a different aspect than other card games have. You have to be strategic in the position of the cards and link up the abilities. This is different and interesting. I recommend this.
Unlike the other reviewer, i found the learning curve extremely easy. The mechanics and gameplay is very easy to understand...
I spent 15 hours playing through the storyline, and that's the most I've ever spent on ANY mobile game. Card City Nights is a true hidden gem and deserves more love. It is extremely addictive and fun, and there is plenty of humor tossed in to keep you entertained.
NOTICE: People are only giving 1 star because they're too freaking stupid to beat it.
- $1.99
- Category: Games
- Updated: Apr 16, 2014
- Version: 1.06
- Size: 156 MB
- Language: English
- Seller: Ludosity AB
- © 2014 Ludosity AB
Compatibility: Requires iOS 6.0 or later. Compatible with iPhone, iPad, and iPod touch.


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Updated 06/11/2013
It's easy to strike up a conversation with local foodies at this casual BYOB Asian street-food spot. And there's a lot to discuss: should you go for a bowl of udon noodles swimming in a chili-lime broth or the pho-spiced duck dumplings and "phat rice" (fried rice with diced pork belly and short rib)? Either way, it's hard to go wrong with anything chef Bill Kim creates in his tiny kitchen. But come early: despite the restaurant's out-of-the-way location in the residential Avondale neighborhood, seating at the four long communal tables is first-come, first-served, and spots fill up quickly.
3053 N. California Ave., Chicago, Illinois, 60618, United States
Website: www.urbanbellychicago.com
Restaurant Details:
Updated 06/11/2013
No Thanks


text:
Schrade® SCH302 Tanto Folding Framelock Flipper Knife
Item # WX2-297631
- Google+
- Details & Specs
Schrade® SCH302 Tanto Folding Framelock Flipper Knife.
Super durable Folding Knife that's solid, sturdy and ideally sized! 9Cr18Mov high carbon stainless steel tanto blade with hollow grinds and a plain cutting edge. Stainless steel framelock handle has a checkered pattern on the front, smooth back and is outfitted with a tip-down carry pocket clip. Both blade and handle are titanium coated stainless steel with a tactical gray finish. One-hand opening is provided by either thumb stud or the integrated flipper guard. Knife has a comfortable grip thanks to its ergonomically cut handle and smooth jimping where the thumb naturally rests.
- Overall length: 8.75"
- Blade length: 3.7"
- Cutting edge: 3.25"
- Blade width: 1.875"
- Closed length: 5"
- Handle width: 1"
- Handle thickness: 0.5"
- Weight: 10.4 ozs.
Don't miss this deal... order ONLINE today!
- Reviews
Schrade SCH302 Tanto Folding Framelock Flipper Knife is rated out of 5 by 1.Rated 5 out of 5 by dirtydog from Wow , nice blade ! The SCH 302 is a nice blade ! It's solid and tight, it was so tight to start with I had trouble unlocking it . But as with all new knives, I flipped it bunches and now it'd just right. I ran it on my steel a few times and it's razor sharp. The quality is great, better wear a belt if you carry it in your pocket because it has some weight, (which I appreciate ), or wear it in a sheath. I LIKE IT .Date published: 2014-12-13
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- Articles & Tips
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Browning Desert Fighter Fixed Tactical KnifeNon-Member $49.99 Buyer's Club $44.99


text: Mme. Broich | IBDB: The official source for Broadway Information IBDB: The official source for Broadway Information Official Broadway League sites: - sites - broadway.org broadwayleague.com broadwayfanclub.com kidsnightonbroadway.com stagespecs.org tonyawards.com Quick Search For: In This Category: All Show People/Org. Theatre Season Mme. Broich Designer Female Productions ProductionsDate of ProductionsThe Better 'Ole,[Special, Comedy, Extravaganza, Original] Costume Design by Mme. Broich Oct 19, 1918 - Oct 04, 1919 Find out where Mme. Broich and are credited together © 2001-2015, The Broadway League, All Rights Reserved. (Copyright and Disclaimer)


text:
Thunder forward Nick Collison has now drawn six offensive fouls, which is tied for second in the league behind Milwaukee's Mike Dunleavy with seven.
Collison nearly had a perfect night against the Sixers, shooting 5 for 6 from the field and 4 for 4 from the line. He finished with 14 points and five rebounds in 21:17.
Thunder shooting guard DeAndre Liggins recorded a triple-double as the Developmental League Tulsa 66ers opened their season with an 83-76 victory over the Texas Legends on Friday night at the SpiritBank Event Center in Bixby.
Liggins had 15 points, 11 rebounds and 10 assists. Thunder center Daniel Orton, a former Bishop McGuinness standout, had 10 points and 11 rebounds.
Texas is coached by former Oklahoma standout Eduardo Najera.
Philadelphia entered the contest first in the NBA in points allowed at 90.9 per game and the Thunder scored 85 through the first three quarters. … Dapper-looking Hall of Famer Julius Erving (Dr. J) was in attendance and received a thunderous ovation. … Thunder guards Reggie Jackson and Jeremy Lamb were shown on "kiss cam." … The Sixers had their three-game home winning streak snapped. … Philadelphia center Andrew Bynum, acquired in a three-way trade with the Los Angeles Lakers and Orlando Magic, still has not played this year due to a right bone bruise. There is no timetable for his return and he potentially could miss the entire season.
Thunder guard Kevin Martin, who shot a combined 3 for 16 from the field on the two-game road trip: "I think I left my offense back in Oklahoma City. So I'll go back tomorrow morning and get it out of my locker."


text:
Diversified Telecommunication Services
Company Overview of Alaska Communications Systems Holdings, Inc.
BOARD MEMBERS AFFILIATED WITH Liane J. Pelletier *
Chief Executive Officer, President, and Chairwoman
*Data is at least as current as the most recent Definitive Proxy.
MOST CONNECTED PEOPLE TO Liane J. Pelletier
|John Walter Meisenbach
|55 Relationships
|Richard B. McCune
|51 Relationships
|William Dexter Paine
|45 Relationships
|Michael T.C. Prior
|41 Relationships
|Michael T. Flynn
|40 Relationships
Sponsored Financial Commentaries
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To contact Alaska Communications Systems Holdings, Inc., please visit . Company data is provided by Capital IQ. Please use this form to report any data issues.
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Our data partners will research the update request and update the information on this page if necessary. Research and follow-up could take several weeks. If you have questions, you can contact them at email@example.com.


text:
32 Reviews
Very very nice, I halved the recipe but not the liquid that you add and that turned out good, used only 1 tablespoon of rice per vegetable. It was enough for the size of the tomatoes and the peppers I used. Could not get the cheese you mentioned in the recipe and used grated Halloumi cheese. The raw rice was perfectly cooked by the time we had the dish. The only thing I added was some dried oregano.
FINALLY.. !! My grandmother used to make these and I have been searching the world for the recipe that would capture this uniquely "greek/turkish" taste.
Well folks.. this is it !
Evelyn shows deep understanding of greek cuisine in her quantity of olive oil (don't be alarmed..) and , especially her step 10. ( Leave an hour or two in the oven..) This is very important as it allows a kind of caramelization which is.. everything ! without this last step mine used to taste like a warm pepper salad mixed with rice !
It has now become one of my family's favourites !
Just a tiny detail.. since the rice is not pre-boiled, it will expand.. so don't stuff them all the way to the top or they will overspill..
I also dribbled 1/2 tsp of olive oil on the mixture in each pepper after stuffing ( but then..I'm an olive-oil addict )
( Evelyn you are a national treasure !! )
Veggie Dornette #1 was home for the weekend and I thought I would surprise her with something besides lasagne (which she asks for each time she comes home). Peppers are plentiful in the market now, although it's not really tomato season. I did try this with 2 tomatoes, but I have to say that the recipe was 90% peppers. To make up for the missing tomato flesh I used some canned tomatoes, chopped up. Left out the mint, because I knew the kids would never touch it with mint (not very adventurous, my kids). I also left out the raisins, again, because the kids won't eat anything with "bugs" in it. Doubled up on the pine nuts instead, you can never have too many pine nuts (budget permitting). The only other change -- I didn't have (or even look for) mizithra or kefalograviera cheese, so I used a nice Cypriot haloumi instead. Ev, major yummy dish here!
Outstanding! Made exactly as written, used shredded parmesan, and it turned out PERFECT!!! Evelyn, you are a gem for sharing this, everyone LOVED IT! I will make this again and again.
Updated 08/27/12: I can not say enough great things about this recipe. You can not get results like this from a cookbook. Evelyn's instructions and measurements are like learning from your Greek yaya. I turn to this recipe again and again, and have made these probably 8 times - always perfect, always to rave reviews. Make them, you won't be disappointed.
Oh My! These were the best stuffed peppers and tomatoes I have ever had in my life. This recipe is amazing. If you are thinking of trying this, think no longer, this is a winner. I have never been so happy with a recipe in my life. I love Greek food and this is Greek. Absolutely amazing! There should be a special category for recipes like this. 10 stars and more. Thank you so much for this dish. My family and I are still fighting for the leftovers.
Great recipe. It's realy worth the time it takes to prepare. It's not the one I had in Santorini, but it's close.
These were very good the first day, although my husband wasn't crazy about them. I took a lefover pepper and a tomato as my lunch for work today, and BOY - this is the kind of dish that really improves after sitting a while! It was incredible! This would be a nice thing to make for a buffet dinner party.
absolutely "lick the plate"!!!!!!
Very tasty. I had to improvise a tiny bit with ingredients due to what I had on hand (and because I cut the peppers the way I typically do to remove the stem, oops!), but nothing serious. The only thing I'd change is to to possibly reduce the olive oil a little. I'll have to "sacrifice" myself for a few more experiments of this recipe to be sure. :)


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"That's too much bacon," said no one ever. Achin' for more bacon—that's the only bad thing about it. Once you snarf up hot n' crispy, fried pork belly, you just want more. (Keep calm, there's more bacon). Come to the swine side. With Traeger, every day can be a bacon day. Get bacon-wrapped recipes at: http://www.traegergrills.com/recipes
...Read More
Get ready, Tigard! Kenny the Pit Master is heading your way on the #TraegerRoadTrip. Stop by to say hi, sample phenomenal food and snap a pic of the Traeger Trailer. If you post it, hashtag it #TraegerRoadTrip on Facebook, Twitter, and/or Instagram, it will put you in the running for a free bag of 100% hardwood. We'll announce the winner on 7/16/15 on this post.
July 10th-12th, The Tigard Home Depot: 14800 SW Sequoia Parkway, Tigard, OR
Sampling food from 11am-3pm Get a pic of the truck and trailer from 9am-6pm Full Terms and Conditions: po.st/traegerroadtrip
Where should Kenny head next?
Share with your pals near Tigard.
Go whole hog with your 4th of July menu this weekend. From snout to curly tail, Boston butt to shank, and belly to fat back, whatever piece of pig you choose, it's all delicious and Traegers up to hog heaven. We like pork butts and we cannot lie. Get pork recipes for your fireworks feast here
Through the 4th of July weekend, when you buy a new Traeger, you get a FREE cover to protect your grill. Paired with free shipping on orders over $100 through 7/7, you've got even more reasons to celebrate with fireworks.
Kenny is heading up north to grill up a storm in Oregon City, the next stop on the #TraegerRoadTrip. Come to sample killer grub and take a quick pic of the Traeger Trailer, post it and hashtag it #TraegerRoadTrip on Facebook, Twitter, and/or Instagram. We will select one lucky hashtagger to win a free bag of Traeger pellets. We'll announce the winner on 7/2/15 on this post—so keep an eye out.
June 26th-28th, The Oregon City Home Depot: 2002 Washington St, Oregon City, OR
Sampling food from 11am-3pmGet a pic of the truck and trailer from 9am-6pmFull Terms and Conditions: po.st/traegerroadtrip
Where should the road trip head next?Like and share with your Oregon friends.
See what Kenny's got cooking in Carmel and Santa Cruz CA, the next stops on the #TraegerRoadTrip. Come to sample some food, and while you're there, take a quick pic of the Traeger Trailer, post it and hashtag it #TraegerRoadTrip on Facebook, Twitter, and/or Instagram—it will put you in the running for a free bag of 100% hardwood. We will announce one winner on June 18th on the original post.
Scarborough's Ace Hardware –290 Crossroads Boulevard Carmel, CA 93923 Scarborough Home Center – 9470 Highway 9 Ben Lomond, CA 95005
Sampling food from 11am-4pm Get a pic of the truck and trailer from 8am-5pm Full Terms and Conditions: po.st/traegerroadtrip
Congratulations to Brook Hargrave, winner of our Make Your Father's Day Giveaway! Her touching story won her a Lil' Tex Elite and Junior Elite duo for her and her wonderful dad. Thank you to everyone who participated by sharing inspiring stories of the admirable individuals that raised you! Head on over to Facebook to congratulate Brooke and see their story: https://www.facebook.com/TraegerPelletGrills Our Smokin' Rebates are available in-store and online through June 21st, so there's still time to make your father's day and get up to $100 back. See the rebates: po.st/rebates
If passionate doesn't even begin to describe your feelings for all things barbecue and a Traeger is your rig of choice—we've got a way for you to channel that energy into some extra scratch. If you can't stop talking about your latest and greatest Traeger creation and you're the first to hop on a thread to impart your hard-earned knowledge to the masses—it's time to turn your wood-fired obsession into grill gear. We are looking for Traeger Advocates to step up and represent on traegergrills.com. Get the details and apply here: http://bit.ly/1cQPp4z
Visit us in California this week and sample some killer grub. While you're there, if you snap a picture of the Traeger Trailer, post it and hashtag it #TraegerRoadTrip on Facebook, Twitter, and/or Instagram, it will put you in the running for a free bag of pellets. We will announce one winner on June 17th on the original post.
See what Pit Master Kenny's got cooking at these 6 locations in CA this weekend:
June 11th: Crown Ace – 2750 Dewey Rd, San Diego, CA 92106 June 12th: Crown Ace – 9045 Adams Ave. Huntington Beach, CA 92646 June 13th: BBQ Grill Outlet – 24396 Swartz Dr. Lake Forest, CA 92704 June 14th: Corning Ace Hardware – 2020 Solano St. Corning, CA 96021 June 15th: Richert Lumber Company – 5505 Sunol Blvd. Pleasanton, CA 94566 June 16th: Schrimper's Ace Hardware – 150 N Wilma Ave Ripon, CA 95366
Sampling food from 11am-4pm Get a pic of the truck and trailer from 8am-6pm Full Terms and Conditions: po.st/traegerroadtrip
It's race day in Baja and our friends at BBQ Island in AZ, the largest BBQ store west of the Rockies, are off-roading with the best of them. Mike West and JT Thomas of #bbqislandracing are tackling the Baja 500 course in their new, custom class 10 car, but they're no strangers to the course. BBQ Island Racing has been riding the 500 in Baja since 2006 in multiple different classes from UTV to the Current 4400/Ultra 4 Truck.
The Baja 500 course changes every year, but always begins and ends in Ensenada, just two hours south of San Diego. They'll be battling rough and unforgiving desert terrain in hopes of off-roading straight to the podium. Cheer them on and wish them luck on Instagram @bbqislandaz. Tell them @traegergrills sent you!


text:
- Filed Under
? Piketon pitcher Morgan Merritt 5 IP, 0 R, 2 H.
? Summary The Redstreaks were benefited by a solid performance by Merritt, and the offense jumped on the Warriors in a Scioto Valley Conference game. Adena never was able to generate any offense.
? Next game Piketon hosts Adena today.


text:
Agriculture positions in Togo
Looking for a job in agriculture in Togo? Find the agriculture job offers in Togo. Please post your job offer or create your CV here.
No job ads are available for the moment. Post your job ad here.
Job categories
Free advice and quotation service to choose an expat health insurance in Togo.>> Click here


text: What would the concequence of 2 black holes moved to a certain distance appart with one being say double or triple the mass of the other. Would the force (or could the force) be strong enough to warp space back so that it 'destroys' the event horision therefore returning the black hole to regular matter or whatnot. Can you reverse a black hole?


text:
- Details
*1:4 scale premium format figure (15") *Hand-cast polystone body *Hand painted *Fully costumed in expertly tailored real fabric clothing *Individually hand finished and hand numbered with the edition size *Great for display *Limited edition of 500 pieces *Brand new Sideshow Collectibles is proud to present Elvis the biggest-selling solo recording star of all time in this 1:4 scale Premium Format figure. This astonishingly lifelike mixed-media figure depicts The King of Rock n' Roll as he appears in his '68 Comeback Special one of his most remembered performances. The Elvis Presley Premium Format Figure features a hand-cast polystone body hand painted to exacting standards and fully costumed in expertly tailored real fabric clothing. Each piece is individually hand-finished and hand-numbered with the edition size. The Elvis Premium Format figure is sure to be the centerpiece of any Elvis collection and stand among the highest quality reproduction of the King of Rock n' Roll to date
- Additional Info
SKU SST-7188-C Age 12-18 years, 18+ Age Group Adult, Teen Manufacturer Sideshow Collectibles
- Reviews


text:
I've used the Hasselblad V system for a long time and recently got into the Mamiya TLR system. Both are great systems with high quality mechanicals, good optics, and extensive accessories. If you're carrying around a camera body and a bunch of lenses, the Mamiya system will be smaller and lighter. For the camera and 1-2 lenses, the differences in weight and bulk are marginal, with the Hasselblad lenses taking more space.
I've been delighted with the Mamiya TLRs and currently have both the C33 and C330 cameras. The C33 is heavier, but beautifully made camera with no plastic parts--I think it's my favorite of the line because of the design, solid build, and auto shutter cocking (absent on the lighter C2xx cameras). However, they're all nice cameras--and the lenses are available at a fraction of the price of the Hasselblad glass. The Hasselblad Zeiss lenses are better performers in absolute terms--especially wide-open and to the far corners of the frame. In practice, the Mamiya lenses are excellent--especially if you stop down a bit. You also gain 1-2 stops by the ability to shoot the TLR more easily at lower shutter speeds.
Try a Mamiya TLR first and see if it meets your needs. If you're happy, you'll have saved a lot of money.


text:
US Partner News Online
WPC 2015 for US partners
US Channel Chief
Cloud profitability and adoption
Training schedule
Stay informed as a US partner
Get support
Home | US Partner News Online | WPC 2015
Azure Partner Community | Office 365 Partner Community
The Microsoft Cloud OS provides a consistent platform for infrastructure, apps, and data, and allows IT to rapidly build and deploy applications, flexibly manage IT services, and support real-time analytics across all forms and data.
On October 7, Microsoft released the fall wave of enterprise products and services that help companies seize the opportunities of cloud computing and overcome today's top IT challenges. Read the post about this announcement by Satya Nadella, Executive Vice President, Cloud and Enterprise at Microsoft, on The Official Microsoft Blog. Read the Server and Cloud Platform blogs.
About this Guide
This guide provides Microsoft partners with links to the information and resources you need to understand the Microsoft Cloud OS vision, opportunity, and products, and engage with Microsoft. As new materials and content are available, we'll update this guide. Come back to it any time at http://aka.ms/cloud-os-partner-guide, and check out the companion page on the MPN portal at http://aka.ms/about-cloud-os.
This guide was updated on November 21.
The Microsoft Cloud OS Story
December 3 live webcast: Ahead of the Game with Microsoft You and your customers are invited to join us on December 3 for a free, one-hour, online event about Microsoft Virtualization and cloud services. You'll hear from Microsoft executives and experts who will talk about the features and benefits of Windows Server, Hyper-V, Windows Intune, and Windows Azure. Guest speakers from customers and partners that are deploying these products will talk about the value that solutions based on these products bring to their businesses.
Topics include:
Ends December 31 – Demonstrate your sales knowledge and be entered* to win up to $1,000
Sales and presales technical professionals who pass qualifying assessments through December 31 can earn entries to our sweepstakes. Official rules.
*No purchase necessary. Open only to authorized representatives of companies actively enrolled in the Microsoft Partner Network who are US residents (including Washington, DC), age 18 years and older. Game ends December 31.
Successful Cloud Partners: Higher, Faster, Stronger
An IDC study sponsored by Microsoft indicates that partners with more than 50 percent of their revenue related to the cloud benefit from higher gross profit, more new customers, increased revenue per employee, and faster overall business growth.
Cloud OS Product Lineup, Evaluation, and Internal Use
Download evaluation versions and virtual labs for Windows Server, System Center, SQL Server, Windows Azure, and Windows Intune from the TechNet Evaluation Center.
If you are a Microsoft Partner Network member with a competency or a Microsoft Action Pack subscription, you have internal-use software benefits. Access and use your benefits to run your business and gain hands-on experience with Microsoft products and technologies.
Demonstrate Expertise with an MPN Competency
The competencies below have requirements that allow you to demonstrate your expertise to build, deliver, and support solutions that include products in the Cloud OS family. To meet exam and assessment requirements for each competency, learning paths specify technical, sales, and licensing training courses and link to the exam or assessment.
Server Platform
Application Development
Application Integration
Application Lifecycle Management
Business Intelligence
Data Platform
Devices and Deployment
Identity and Access
Management and Virtualization
Training and Accreditations for Sales Professionals and Presales Technical Professionals
There are live in-person, live online, and on demand training courses from Microsoft that can help you increase your product knowledge and prepare for the customer conversation. You can earn an MPN Sales Accreditation to demonstrate your expertise, and your accreditation may benefit your company as well by helping to meet MPN competency requirements.
Training and Certification for Technical Professionals
There are in-person, live online, and on demand training courses from Microsoft that can help you increase your product knowledge and prepare for development and deployment of solutions that meet customer needs. You can earn Microsoft Certifications to demonstrate your expertise, and your certification may benefit your company as well by helping to meet MPN competency requirements.
Create Demand with Ready-to-Go Marketing
Campaigns from Ready-to-Go Marketing include customer-ready sales and marketing materials. You can also develop and manage events, and maintain your website with content syndication services.
Increase Sales Velocity
Immersion Conversations on the Microsoft Server and Cloud Partner Resources website (accessible by MPN members with a gold or silver competency):
Partner Incentives: Download the Windows Azure incentive guide.
Deployment Tools and Programs
Software Assurance Planning Services engagements are conducted by qualified partners who share best practices, analyze the customer's organizational environment, and help create comprehensive deployment and implementation plans.
Use Your MPN Membership Benefits and Support


text:
Obesity is associated with a higher risk of developing cancer and co-morbidities that are part of the metabolic syndrome. Adipose tissue is recognized as an endocrine organ, as it affects a number of physiological functions, and contains adipose tissue-derived stem cells (ASCs). ASCs can differentiate into cells of multiple lineages, and as such are applicable to tissue engineering and regenerative medicine. Yet the question of whether ASC functionality is affected by the donor's body mass index (BMI) still exists.
ASCs were isolated from patients having different BMIs (BMI-ASCs), within the ranges of 18.5-32.8. It was hypothesized that overweight BMI-ASCs would be more compromised in early adipogenic and osteogenic potential, and ability to form colonies in vitro. BMI was inversely correlated with ASC proliferation and colony forming potential as assessed by CyQUANT proliferation assay (fluorescence- based measurement of cellular DNA content), and colony forming assays. BMI was positively correlated with early time point (day 7) but not later time point (day 15) intracytoplasmic lipid accumulation as assessed by Oil-Red-O staining. Alizarin red staining and RT-PCR for alkaline phosphatase demonstrated that elevated BMI resulted in compromised ASC mineralization of extracellular matrix and decreased alkaline phosphatase mRNA expression.
These data demonstrate that elevated BMI resulted in reduced ASC proliferation, and potentially compromised osteogenic capacity in vitro; thus BMI is an important criterion to consider in selecting ASC donors for clinical applications.
Keywords:Adipose stromal/stem cells (ASCs); Body mass index (BMI); Osteogenesis; Proliferation; Colony formation; Cell size
The increasing epidemic of obesity within the United States has been associated with a higher risk of developing co-morbidities that are categorized as part of the metabolic syndrome that include dyslipoproteinemia (raised triglyceride and/or reduced high density lipoprotein cholesterol levels), diabetes mellitus, and cardiovascular and coronary artery diseases. Obesity has also been identified as a risk factor for an increased incidence of several forms of cancer, including colon and breast cancers [1-5]. A high incidence of these morbidities exists in obese individuals, both adolescents and adults [3,4,6]. Recent studies have also investigated the effects of obesity on the musculoskeletal system [7], and have identified obesity as an independent risk factor for increased bone fracture risk [8] and clinical implant failure following total joint replacement [9-14]. Obese individuals with higher body mass indices (BMIs; the ratio of body weight (kg) to height (m2)) exhibit lower relative bone area and bone mass compared to non-obese individuals [15]. While various explanations have been widely suggested, our knowledge relating the pathogenesis of obesity at the cellular level and its potential impact in tissue engineering and regenerative medicine applications is very limited.
Adipose tissue is a complex, highly active endocrine organ that secretes bioactive peptides, or adipocytokines, that are known to affect a number of physiological functions in the reproductive system, neuroendocrine system, rennin-angiotensin system, and in bone metabolism. [16-18]. Adipose tissue contains mature adipocytes, endothelial cells, cells of the immune system, and a small percentage of the adipocyte precursors termed adipose tissue-derived stem cells (ASCs). The ability of ASCs to differentiate into cells of the endodermal, mesodermal, and ectodermal lineages makes ASCs optimal candidates for applications in cellular therapies [19], including tissue engineering and regenerative medicine [20-23]. These applications potentially involve the repair of the musculoskeletal and other biological systems [24,25]. However, the growing interest in ASCs for cell therapeutics has led to questions about donor physiological conditions on ASC functionality (i.e. effects on viability, differentiation, and growth properties).
Recent reports have positively correlated BMI with ASC yields from adipose tissue and inversely correlated BMI with adipocyte size [26]. BMI has also been inversely correlated with bone-marrow derived mesenchymal stem cell (BMSC) cyclic tensile strain capacity, or mechano-response, and alkaline phosphatase activity in vitro, which suggested a possible compromise in the osteogenic differentiation potential of BMSCs from individuals with higher BMIs [5]. Similar studies on ASCs have not been conducted.
The current study investigated the proliferation ability, in vitro differentiation potential, relative cell volume and complexity, and colony forming potential of ASCs isolated from patients having different BMIs, within the ranges of 18.5-24.9 kg/m2, (designated lean BMI-ASCs), and 25–32.8 kg/m2, (designated overweight BMI-ASCs). It was hypothesized that overweight BMI-ASCs would be more compromised in the ability to proliferate, differentiate, and form colonies in vitro, thereby contributing to the problematic obesity-associated pathologies, and therefore BMI should be considered when using ASCs for regenerative medicine applications.
Percent serum and BMI inversely correlated with ASC growth
Cryopreserved ASCs isolated from lipoaspirates of women with different body mass indices (BMIs; Table 1), were cultured for up to 72 hours in ASC culture medium supplemented with 0 to 10% FBS. Cell growth was measured by MTT and CyQUANT cell proliferation assays. Growth data reflected an inverse relationship between BMI and ASC growth in vitro (Figure 1a,b). The largest effect was observed in 2% serum for 48 hrs (Figure 1a); however, growth was also compromised when ASCs were cultured 10% serum (Figure 1b). MTT data also revealed a time-dependent biphasic response in cell growth in which full recovery and maximum growth occurred at 72 hrs following a decline in higher BMI-ASC growth at 24 hrs and 48 hrs (data not shown). Non linear regression analyses using least fit ordinary squares supported the strong inverse relationship between both BMI (determination coefficient, R2 = 0.90; p < 0.05) and serum (R2 = 0.86; p < 0.05) on ASC growth (Figure 1c-f), where culture in the lowest percent serum (0%) reflected the strongest determination coefficient. Quadratic equations were used for nonlinear regression analyses, curve-fitting and subsequent R2 values (reported in Figure 1c-f).
Table 1. BMI-ASCs used in the study
Figure 1. Higher BMI-ASCs exhibited compromised growth when exposed to low serum in vitro. a, b. CyQUANT was performed following ASC exposure to low serum (0-10% FBS supplementation) culture conditions for 48 hrs. Percentage serum and BMI both significantly affect ASC growth. c-f. Regression analysis of BMI-ASC growth when exposed to low serum in vitro. Nonlinear regression analysis performed using least squares ordinary fit. R2 values indicate a strong correlation between BMI, percent serum exposure, and ASC growth. Values are reported as N ± SE; * p > 0.05, ** p > 0.001, *** p > 0.0001.
BMI negatively correlated with colony-forming unit potential
To further examine differences in BMI-ASC growth, colony-forming unit (CFU) assays were performed on the BMI-ASC donors (Table 1). These donors were grouped as follows: lean (BMI <25; mean BMI 22.2 ± 1.79, N = 5), and overweight (BMI >30; mean BMI 30.3 ± 2.17, N = 5). When grouped, the lean BMI-ASCs formed a significantly higher percentage of colonies (34.94 ± 1.46) compared to the overweight BMI-ASCs (28.26 ± 1.78); p < 0.05 (Figure 2b). Representative photomicrographs of CFUs are shown in Figure 2c. Annexin-V/PI staining and fluorescence-activated cell sorting (FACS) was used to determine whether the compromised growth in higher BMI-ASCs was accompanied by elevated apoptosis. There was no significant difference in apoptosis between lean and overweight BMI-ASCs at 24 and 48 hrs of culture with 2% or 10% FBS (data not shown). The percent early apoptotic cells did not exceed 5.49 ± 1.86%, and the percent late apoptotic cells did not exceed 4.12 ± 0.23% (mean ± SD).
Figure 2. BMI negatively correlated with ASC colony-forming potential in vitro. ASCs were plated at a concentration of 100 cells/mL in 6 well plates and cultured for 14 days. The number of colonies per plate divided by the cells plated x 100 was determined as the "% CFU". a. Ungrouped BMI-ASC colony-forming potential, and b. grouped BMI-ASC CFU potential. c. Visualization of colonies formed in 100 cm2 dishes. Values reported as N ± SE; * p > 0.05.
BMI did not affect late time point adipogenic differentiation in vitro
To investigate the effect of BMI on ASC differentiation, adipogenesis was induced by culturing ASCs in differentiation induction medium for three days followed by culture in maintenance medium until day 15. Differentiation was induced in both 3% and 10% serum. Lipid formation was assessed by percent intracytoplasmic incorporation of Oil Red-O (ORO) into monolayers at days 7 and 15 of adipogenesis. Oil-red-o staining at day 7 revealed a positive correlation between BMI and adipogenesis at early time points (as BMI increased, lipid accumulation increased; Figure 3a). Grouping of BMI-ASCs revealed that overweight BMI-ASCs had significantly higher Oil-Red-O staining (61.40 ± 5.139) compared to the lean BMI-ASCs (46.20 ± 2.70); p = 0.017 at day 7 (data not shown). Staining at day 15 revealed that BMI has no significant effect on ASC adipogenesis during late time points (Figure 3b). To further investigate the correlation between BMI and adipogenesis, nonlinear regression analyses were applied to adipogenesis data from days 7 and 15. R2 values reflected a correlation between BMI and adipogenesis at day 7 (R2 = 0.78; Figure 3c), and no correlation at day 15 (R2 = 0.57; Figure 3d). Representative photomicrographs of ORO staining in BMI-ASCs at day 15 are shown in Figure 3e.
Figure 3. BMI did not significantly affect adipogenesis potential in vitro. ASC adipogenesis was induced using differentiation induction medium for three days. ASC cultures were then switched to maintenance medium until day 15. Lipid formation was assessed by percent incorporation of oil red-o (ORO) into monolayers cultured in adipocyte differentiation medium for a. 7 days, and b. 15 days. c. Representative micrographs of ORO staining in BMI-ASCs at day 15. d, e. Regression analyses of differentiation data that was analyzed at days 7 and 15 using least squares ordinary fit. R2 value reflected no significant correlation at late adipogenesis timepoints (e; Day 15); however, early adipogenesis (d; Day 7) R2 value reflected a correlation between BMI and adipogenic potential. Values are reported as N ± SE; * p > 0.05, ** p > 0.001.
To determine whether cryopreservation was a factor in the observed absence of BMI effects on adipogenic differentiation, we induced adipogenesis in freshly isolated ASCs and cryopreserved ASCs at passage 1 from two of the same donors (mean age 49.5 ± 4.95; BMI 25.6 ± 1.56; Additional file 1: Figure S1a, b). ORO staining (Additional file 1: Figure S1e) demonstrated no significant difference in intracytoplasmic lipid accumulation prior to or following the freeze-thaw process.
Additional file 1: Figure S1. Cryopreservation does not significantly affect BMI-ASC osteogenic and adipogenic potential in vitro. BMI-ASCs that were freshly isolated or frozen and thawed, were induced into a. b. adipogenesis for 15 days or c. d. osteogenesis for 16 days, as described in the materials and methods section. Oil-red-o staining was used to evaluate adipogenic potential, and Alizarin red stain was used to determine ECM deposition. Quantification of e. ORO and f. ARS reflected no significant difference in adipogenic or osteogenic potential, respectively, in freshly isolated cells compared to frozen cells. Values reported as N ± SE.
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BMI negatively correlated with extracellular matrix mineralization
The effects of BMI and percent FBS on ASC osteogenesis at early and late time points was investigated by inducing osteogenesis using an osteogenic cocktail medium for 16 days, as described in the materials and methods. Differentiation was induced in both 3% and 10% serum. Calcium deposition was assessed by Alizarin Red staining (ARS) at days 8 and 16. ARS revealed an inverse correlation (as BMI increased, matrix mineralization decreased) between BMI and ASC osteogenic potential at both time points; this was most pronounced at day 16 (Figures 4a, b). Non linear regression analyses of the ARS data revealed that% FBS had no significant effect on BMI-ASC osteogenic potential, similar to that observed with adipogenesis (data not shown). To evaluate the correlation between BMI and osteogenesis, nonlinear regression analyses were performed on osteogenesis data from days 8 and 16 using the least squares ordinary fit. R2 values reflected a strong inverse correlation at both time points (day 8, R2 = 0.86; day 16, R2 = 0.96; Figures 4c and d, respectively). Representative photomicrographs of ARS in BMI-ASCs at day 16 are shown in Figure 4e. To determine whether cryopreservation was a factor in the observed effects on osteogenic differentiation, we induced osteogenesis in freshly isolated ASCs and cryopreserved ASCs from two of the same donors at passage 1 (mean age 49.5 ± 4.95; BMI 25.6 ± 1.56; Additional file 1: Figure S1c, d). ARS (Additional file 1: Figure S1f) demonstrated no significant difference prior to or following the freeze-thaw process.
Figure 4. BMI significantly affected ASC early and late osteogenesis potential in vitro. ASC osteogenesis was induced using a cocktail medium for 16 days. Calcium deposition was assessed by Alizarin red staining (ARS) at a. day 8, and b. day 16. c. Representative micrographs of Alizarin Red Staining (ARS) in BMI-ASCs at day 15. d, e. Regression analyses of differentiation data that was analyzed at days 8 and 16 using least squares ordinary fit. R2 value reflected a strong correlation at both early and late osteogenesis time points. Values reported as N ± SE; * p > 0.05, ** p > 0.001; *** p > 0.0001.
BMI negatively correlated with alkaline phosphatase mRNA expression
To confirm the results of Figure 4 demonstrating a significant effect of BMI on ASC osteogenesis, alkaline phosphatase mRNA expression was measured in additional ASC cultures after induction of osteogenesis. Alkaline phosphatase mRNA results confirmed that when grouped, overweight BMI-ASCs were significantly more compromised in osteogenic differentiation potential (N ± SE: 0.71 ± 0.18) compared to the lean BMI-ASCs (N ± SE: 4.4 ± 0.21; p = 0.017,s Figure 5a,b). Nonlinear regression analyses were performed on osteogenesis RT-PCR data using least squares ordinary fit. The R2 value again reflected a strong inverse correlation (R2 = 0.89) between BMI and osteogenic potential (Figure 5c).
Figure 5. Expression of alkaline phosphatase mRNA indicated that higher BMI was associated with reduced ASC osteogenesis capacity in vitro. ASC osteogenesis was induced using a cocktail medium for 9 days. RT-PCR was performed using primers and a probe to alkaline phosphatase for a measure of mRNA expression. ΔCT values were normalized to internal GAPDH. a. ungrouped averages of triplicates b. grouped averages of 'lean' (BMI <25 n = 5) and 'overweight' (BMI >25; n = 5) BMI-ASCs. c. Linear regression analyses of differentiation data that was analyzed at day 9 using least squares ordinary fit. R2 value reflected a strong correlation between BMI and osteogenesis. Values reported as N ± SE; * p = 0.017.
BMI did not affect ASC relative cell size and complexity in vitro
Recent studies indicated that mature adipocytes from overweight and obese individuals were smaller in size compared to adipocytes from lean individuals [27]. To determine whether ASCs might exhibit similar relationships between BMI and cell size and complexity, forward-scatter flow cytometry (to approximate cell size), and side- scatter flow cytometry (to approximate cell granularity, e.g. organelles) was performed on BMI-ASCs following culture in 2% or 10% FBS for 24 and 48 hrs. Grouping of forward and side scatter analyses revealed no significant difference in cell size or complexity between lean and overweight BMI-ASCs following 24 hrs of culture in both 2% and 10% serum (Additional file 2: Figure S2).
Additional file 2: Figure S2. BMI does not significantly affect cell size and complexity in vitro. Forward-side scatters were performed on BMI-ASCs post low serum exposure (2% FBS supplementation), and in ASC culture medium (10% FBS) for 24 hrs. a. Cell size (Forward scatter) 24 hrs post exposure. b. cell complexity (Side scatter) c. Representative forward-scatter and side-scatter plots of cellular events from normal BMI-ASCs (BMIs <25; n = 5) and overweight BMI-ASCs (BMIs >25; n = 5) in 10% serum. Values reported as N ± SE.
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The potential use of ASCs in tissue engineering and regenerative medicine has been well demonstrated in multiple pre-clinical animal models [24,28]. Although ASCs have been characterized based on immunophenotype, cell yield and differentiation properties [26,29-31], the effects of BMI on ASC functionality have not been fully elucidated. The present study demonstrated that cryopreserved human ASCs from patients with higher BMIs were compromised in colony-forming potential and growth under exposure to low serum concentrations. Overweight BMI-ASCs were also compromised in osteogenic differentiation capacity when compared to lean BMI-ASCs. Cryopreservation was not a factor in the observed differences, as both freshly isolated BMI-ASCs and cryopreserved BMI-ASCs were comparable in both osteogenic and adipogenic differentiation capacities. Moreover, in vitro adipogenesis data suggested that early time point intracytoplasmic lipid uptake was positively correlated to BMI. Exposure of ASCs to low serum uncovered differences among BMI-ASCs that were less apparent at higher serum concentrations. Although one individual donor exhibited variation in the trend of BMI and proliferation (e.g. BMI-ASC 29.4), the determination coefficients were sufficient to form conclusions about the correlation between proliferation and BMI.
Overweight BMI-ASC osteogenesis was reduced independent of serum levels. These results were similar to a published report that the BMSC osteogenic alkaline phosphatase response to mechanical strain was inversely correlated to donor BMI [5]. These findings paralleled clinical data demonstrating that obese patients possessed lower relative bone area and bone mass compared to lean individuals, when adjusted for body weight, [15], and this may contribute to an increased risk for fractures [8]. Other clinical studies have demonstrated an inverse correlation between body weight and bone density and peak bone mass. In addition, there is evidence of increased risk of implant failure following total joint knee replacement in obese patients [9-11]. An understanding of the impact of fat metabolism on bone precursors and osteogenic capacity will be necessary to advance ASC use in bone repair applications.
The present data suggests that overweight BMI dependent increase in early adipogenesis may be associated with a decrease in osteogenic capacity. Multiple studies suggest that when ASC fate is committed to the adipogenic pathway, osteogenesis is concomitantly down regulated, consistent with observations in BMSCs [25,32]. This balance is suggested to be partly modulated by the presence and amount of glucocorticoid, the glucocorticoid receptor pathway activity, crosstalk with the Jak/STAT3 pathway, and the subsequent activation of the AP-1 pathway [33,34]. Other studies have suggested that an inverse relationship exists between donor age and ASC osteogenesis [35], and that ASCs from male donors exhibit increased osteogenic potential compared to ASCs from female donors in vitro[36]. However, conflicting outcomes were reported in studies comparing donor age and BMSC osteogenic potential in vitro with one study demonstrating an inverse correlation [37] whereas other studies reported a positive correlation [38,39].
ASCs from overweight BMI patients (BMI >25) showed an increase in the intracytoplasmic Oil-Red-O staining at early adipogenesis time points, but no significant difference at later time points. Although an increasing trend was observed in early adipogenic potential that correlates to BMI, an individual donor effect may have contributed to the low adipogenesis in donor L070525 (BMI 30.65 kg/m2). Genes that are induced during both early and late stage adipogenesis have been extensively described [20,21,33,40-42]. Yu et. al., 2010 [42] performed time-dependent RT-PCR analyses of genes involved in adipogenesis in 4 donors, BMI 23.1 ± 1.4, age 39 ± 6 years. The adipogenic transcription factor C/EBPα, the adipokines adiponectin and leptin, and lipoprotein lipase (LPL) all displayed a time-dependent increase during adipogenesis, with the greatest percent increase occurring within the initial 3-day period; however BMI had no significant effect on the mRNA expression of the representative genes that were upregulated on day 3 of adipogenesis (PPARγ, Adiponectin, LPL, and aP2). This suggests that BMI has no overall effect on adipogenesis in vitro, and supports our data demonstrating no correlation between BMI and late adipogenesis. However, Schipper et. al. [43] reported age-dependent differences in ASC adipogenic potential and proliferative capacity in vitro. Further investigation is needed relating the impact of age and BMI on early and late ASC functionality.
It is widely accepted that hyperplasia follows adipocyte hypertrophy in patients during fat mass expansion which requires a gradual replenishment of the pool of adipocytes in obese individuals [44,45]. One study proposes that BMSCs are recruited and give rise to bone, muscle, and both white and brown adipose tissues in response to the appropriate developmental cues in vivo[46]. However, the ability of ASCs to differentiate into terminally differentiated cells of both white and brown adipose tissues adds further complexity to the interesting dynamic. The formation of newer, small mature adipocytes is thought to occur via differentiation of preadipocytes; however whether the preadipocyte originates from the fat mass, or from the recruitment of the circulating bone-marrow progenitor cells [47] remains to be determined. In this regard, it should be noted that Koh et. al. [28] demonstrated that BMSCs in bone marrow transplant mice became resident as phagocytic macrophages in adipose tissues and resembled trans-differentiated adipocytes, but did not express adipocyte markers.
The present study used cryopreserved ASCs that were passaged in vitro, not freshly isolated ASCs. Several studies have investigated the cryopreservation characteristics of ASCs and examined different methods of cryopreservation on ASC biology in vitro[41,48-50]. Thirumala et al., 2010 [22,51], showed that the post-thaw viability of ASCs differed significantly based on the method of freezing and rate of thaw, and determined that using an ethanol enclosed jacketed container, with 10% DMSO and 80% FBS in the cryopreservation medium, yielded the highest post-thaw viability. Other studies have examined the effects of various cryoprotective agents on cell viability, proliferation, and osteo- and adipogenic differentiation [47-49]. These studies indicated that proliferation and osteo- and adipogenic differentiation of ASCs could be maintained in vitro post-thaw. Our studies also demonstrated that cryopreservation did not significantly affect ASC adipogenic or osteogenic potential (Additional file 1: Figure S1, panels a-f).
Reports on the proliferation index of freshly plated ASCs versus cryopreserved ASCs are conflicting. James et. al., 2011 [39] compared ASCs freshly harvested from lipoaspirate to cryopreserved ASCs, and measured growth using Trypan blue dye exclusion assay. Their results demonstrated cryopreservation significantly affected in vitro proliferative capacity. However, studies by Deng et. al., 2008 [52], and Gonda et. al., 2008 [53] suggest that ASC proliferative capacity following cryopreservation is maintained. Calculations of ASC population doubling times in our studies coincide with reports by Deng et. al., 2008, and Gonda et. al., 2008 [52,53], as there was no significant difference observed between the doubling times of freshly isolated ASCs and cryopreserved ASCs (Additional file 1: Figure S1, panel g).
James et. al., 2011 [39] also compared ASCs freshly harvested from lipoaspirate to cryopreserved ASCs, and measured osteogenic differentiation (alkaline phosphatase staining and qRT-PCR), and adipogenic differentiation (alizarin red staining and qRT-PCR). The results demonstrated cryopreservation significantly affected osteogenic differentiation, both in vitro and in vivo. The use of recombinant proteins such as IGF and BMP, however, were used to mitigate the deleterious effects of the freeze–thaw process on osteogenic potential. It should be noted that BMI was not a tested factor, and no cytokines or signaling factors were added to their differentiation medium. A handful of cytokines and other factors are known to stimulate osteogenic differentiation in ASCs, including vitamin D, which was added in the differentiation medium used in the present study. In addition, the strong determination coefficients derived from performing the nonlinear regression analyses of the proliferation (r = 0.86-0.9) and osteogenic differentiation studies (day 8, r = 0.86; day 16, r = 0.96) suggest donor metabolic status may also have an effect on ASC functionality.
In vivo, near complete wound healing was observed in mice with calvarial defects that were engrafted with fresh human ASCs as compared to groups engrafted with cryopreserved ASCs which exhibited reduced healing [39]. Although freshly isolated ASCs may exhibit beneficial properties for wound healing and other applications, the majority of applications for ASCs will likely use cryopreserved ASCs, therefore it is important to understand the characteristics and limitations of cryopreserved ASCs. Future studies comparing the functionality of freshly isolated ASCs to cryopreserved ASCs within different BMI groupings are needed to determine whether the observed differences exist in freshly isolated cells.
Other possible contributing factors to the outcomes of this study are patient clinical characteristics that were unavailable from patient records, such as donor hormonal status, smoking and other undisclosed patient parameters. Regression analyses were conducted to determine whether a correlation exists between age, passage and the measured endpoints within the study. All R2 values were less than 0.2 and as such reflect no correlation between the known factors and the measured endpoints.
While ASCs are proving to be promising candidates for many applications in regenerative medicine and tissue engineering, understanding the phenotypic characteristics of these cells is essential to ensure product consistency and suitability towards specific applications. The present study demonstrated that in comparison to ASCs from lean individuals, ASCs that originate from individuals with higher BMIs had comparable adipocyte differentiation but reduced proliferation and osteogenic potential. Thus, ASCs derived from donors with overweight BMI may be less than optimal for applications that involve usage in biocompatible scaffolds for bone grafting.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Norcross, GA) unless otherwise specified.
Donor demographics
All tissue was obtained from the subcutaneous abdominal adipose tissue region of female, Caucasian patients. The tissue was acquired from elective procedures in local plastic surgery offices, with the patient's informed consent as approved by the Pennington Biomedical Research Center Institution Review Board. The primary cultures were prepared as described in Dubois et al., 2008 [54]. The tissues used were from 11 female donors of ages between 28 and 61 with a mean ± SD of 41.5 ± 8.61 years. The patients displayed a mean body mass index (kg/m2) (±SD) of 26.3 ± 4.62 (summarized in Table 1).
Isolation, collection, and culture of human ASCs
Both fresh and cryopreserved human ASCs were obtained from the Pennington Biomedical Research Center using the protocol described by Gimble et. al [21,40]. Briefly, liposuction tissues were transported to Pennington laboratory in saline solution within 2 h post-surgery. The tissue was washed at least three times with two volumes of Phosphate Buffered Saline (PBS) to remove blood. The tissue was then digested with one volume of PBS supplemented with 0.1% collagenase type I (Worthington Biochemicals, Brunswich NJ), 1% bovine serum albumin, and 2 mM CaCl2 for 60 min at 37°C with intermittent shaking. The floating adipocytes were separated from the stromal-vascular fraction (SVF) by centrifugation (300 × g) for 5 mins at room temperature. The supernatant, containing mature adipocytes was aspirated and discarded and the remaining pellet was identified as the SVF. The SVF cells were suspended and plated immediately in T225 flasks in ASC culture medium (DMEM/F-12 Ham's, 10% FBS [Hyclone, Logan, UT, http://www.hyclone.com webcite], 100 U penicillin/ 100 g streptomycin/0.25 g fungizone) at a density of 0.156 ml of tissue digest/sq cm of surface area for expansion and culture. This initial passage of the primary cell culture was referred to as passage 0 (P0). For cultivation of fresh ASCs for experiments, P0 ASCs were subjected to trypsinization with 5 mL 0.25% trypsin (Life Technologies, Grand Isle, NY) for 5 minutes. Trypsin digestion was stopped by the addition of an equal amount of ASC culture medium. P0 ASCs were then counted using trypan blue dye exclusion, and re-plated at the specified cell density required for each experiment, as described.
For cryopreservation, the ASCs were resuspended in cryopreservation medium (10% dimethylsulfoxide, 10% Dulbecco's modified Eagle's medium [DMEM]/F-12 Ham's, 80% fetal bovine serum [FBS]), frozen at 80°C in an ethanol-jacketed closed container, and subsequently stored in liquid nitrogen prior to thawing for individual assays. The cells were then replated and expanded in cell factories in the Adult Stem Cell Core at Tulane University. Patient donor information (donor number, body mass index, gender, and age) are shown in Table 1 and were used to categorize ASCs as cells isolated from individuals having either lean or overweight body mass indices (BMI-ASCs). Following expansion, freezing and thawing, cryopreserved P0 ASCs were counted using trypan blue dye exclusion, directly plated and used at passage 1 for all experiments, including the comparison between fresh and frozen cells.
Cell proliferation assays
Cell growth was measured by the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and CyQUANT proliferation assays. For the CyQUANT assay, cells from each BMI were seeded in triplicate in ASC culture medium into 24 multi-well plates (Falcon, BD Biosciences, San Jose, CA) at a density of 2.5×103 cells/cm2. Following 24 and 48 hrs of culturing in ASC culture medium supplemented with fetal bovine serum ranges of 0% to 10%, medium was removed from the plates, and the monolayers were rinsed with cold PBS. Plates were then frozen at −80°C overnight. The cells were thawed at room temperature and 200 μl of CyQUANT GR dye/cell lysis buffer (included in the CyQUANT kit, Invitrogen, Eugene, OR, USA) was added to each well. The fluorescence was measured using a Fluostar Optima microplate reader (Fluostar Optima, BMG Labtech; Durham, NC). The excitation maximum was 485 nm, and the emission maximum was 530 nm.
For the MTT assay, cells from each BMI were seeded in triplicate in ASC culture medium into 24 multi-well plates (Falcon, BD Biosciences, San Jose, CA) at a density of 2.5×103 cells/cm2. ASC culture medium supplemented with fetal bovine serum ranges of 0% to 10% was replaced with fresh ASC culture medium, and 10 μl of 12 mM MTT solution (Invitrogen, Eugene, OR, USA) was added to each well of the 24 multi-well triplicates. The plates were incubated for 4 h at 37°C. MTT formazan crystals were then solubilized by adding 150 μ1 100% dimethylsulfoxide (DMSO) to each well. Plates were then agitated on a plate shaker for 5 min., following which spectrophotometric absorbance at 540 nm was immediately determined using a scanning multi-well spectrophotometer (Fluostar Optima, BMG Labtech; Durham, NC). At least three independent sets of experiments were performed for each treatment.
Cell viability assessment
To quantify low serum exposure-induced apoptosis, a well-established annexin-V/ propidium iodide (PI) apoptosis staining was performed, and was evaluated by flow cytometry. The control consisted of ASCs treated in fresh ASC culture medium as previously defined (cells were seeded in triplicate into 24 multi-well plates at a density of 2.5×103 cells/cm2). Briefly, after 24 hr and 48 hr culture with 2% and 10% serum, both floating and attached cells were pooled, harvested by trypsinization (0.25% trypsin), washed in 10 mL of culture medium and resuspended in 100 μL of 1× annexin-binding buffer (included in annexin V-FITC/PI kit). Cells suspended in a volume of 100 μL were mixed with 8 μL of annexin-V-FITC and 8 μL of 100 μg/ mL propidium iodide (PI) and incubated in the dark at room temperature for 15 min. Apoptotic analyses for ASCs were performed on a fluorescence-activated cell sorter (FACS) flow cytometer (BD Biosciences, San Jose, CA) utilizing 488-nm laser excitation and fluorescence emission at 530 nm and >575 nm.
Apoptosis was characterized by phosphatidylserine (PS) translocation from the inner leaflet to the outer leaflet of the lipid bilayer, while the cell membrane remains intact. Annexin V-positive cells correspond to cells that have experienced PS translocation. PI staining of the cells indicates that the integrity of the cell membrane has been compromised and is used to distinguish living and early apoptotic cells from necrotic cells. Quadrant analysis was performed on the fluorescence dot plot to quantify the percentage of live, necrotic, and apoptotic cell populations, and reported in bar graph form.
Colony-formation units assay
100 ASCs in 1 ml ASC culture media were plated in triplicates in 6 well plates (10.4 cells/cm2) and cultured in a 37°C incubator with humidified 5% CO2. Following 14 days of culture, the media was removed, and washed 3 times with 1 mL PBS. 3.0% crystal violet (Invitrogen) in 100% methanol was added and the plates were incubated for 10 minutes at room temperature. The plates were then gently flushed with dH2O for 15 min. or until the background was clear. The plates with the stained colonies were examined under an inverted microscope and the number of colonies that were 2 mm diameter or larger were counted using a VersDoc Imaging system (Bio-Rad Laboratories, Hercules, CA). The number of colonies per plate divided by the cells plated × 100 was determined as the "% CFU".
Adipogenic differentiation
Adipogenic differentiation of ASCs was performed over a 12 day period as previously described [20]. Briefly, ASCs grown in ASC culture media until the culture reached 90-95% confluency. ASCs were then trypsinized and plated in 24-well plates in ASC culture media at 3×104 cells/cm2 for 24 hrs to allow attachment. On day 1 (24 hours after plating), the medium was removed and cells were incubated for three days in adipogenic differentiation medium (Dulbecco's modified Eagle's-Ham's F-12 medium supplemented with 3% or 10% fetal bovine serum, 15 mM HEPES (pH 7.4), biotin (33 μM), pantothenate (17 μM, Sigma), human recombinant insulin (100 nM, Boehringer Mannheim), dexamethasone (1 μM), 1-methyl-3-isobutylxanthine (IBMX; 0.25 mM), and rosiglitazone (1 μM). For the remaining 9 days of the adipocyte differentiation maintenance period, the medium was removed every 3 days and replaced with the same medium that did not contain IBMX and rosiglitazone (maintenance medium).
Intracytoplasmic lipid quantification
Lipid formation was assessed by incorporation of Oil-Red-O (ORO) (Sigma-Aldrich) into monolayers of ASCs cultured in adipocyte differentiation medium for 12 days. Quantitation of ORO incorporation was performed as previously described [20]. Briefly, 0.5% (w/v) oil red-o (ORO) (Sigma-Aldrich) was prepared in ethanol. 3 parts ORO and 2 parts PBS were then mixed to make a working solution. Monolayers of ASCs cultured in 12-well plates were rinsed 3 times with PBS and subsequently fixed in 10% (v/v) formalin (Sigma-Aldrich) for 15 minutes. The monolayers were then rinsed 3 times with PBS and then incubated in ORO working solution for 45 minutes at room temperature. Following aspiration of unincorporated ORO, monolayers were rinsed 4 times with PBS. Stained monolayers were visualized with phase contrast microscopy (Eclipse 800, Nikon; Tokyo, Japan). Incorporated ORO was extracted by incubating stained monolayers in 100% isopropanol for 10 minutes. The absorbance at 510 nm of each aliquot was then measured using a 96 well plate reader (Fluostar Optima, BMG Labtech).
Osteogenic differentiation
Osteogenic differentiation of ASCs was performed over a 16 day period as previously described [20]. Briefly, ASCs cultured in ASC culture media until cells reached 90-95% confluency. ASCs were then trypsinized and plated in 24-well plates in ASC culture media at 3×104 cells/cm2 for 24 hrs to allow attachment. On day 1 (24 hrs after replating), the medium was changed to BGJb medium (Fitton-Jackson Modification) supplemented with 10% fetal bovine serum, 100 μg penicillin streptomycin/mL, 10 nM dexamethasone, 10 mM b-glycerolphosphate and 50 μg/mL ascorbate-2-phosphate, and 10 nM 1,25-vitamin D3 (osteogenic medium). The cells were induced towards osteogenesis in this medium for approximately 14 days and the osteogenic medium was replaced every 2–3 days.
Mineralization quantification
40 mM Alizarin Red stain (ARS) (Sigma-Aldrich) was prepared in dH2O pH 4.1. ASC monolayers cultured in the 24-well plates were rinsed 3 times with Phosphate Buffered Solution (PBS) and fixed in 10% (v/v) buffered neutral formalin (Sigma-Aldrich) for 15 minutes. The monolayers were then rinsed 3 times with dH2O and incubated at room temperature in ARS for 20 minutes with gentle shaking. Following aspiration of unincorporated ARS, monolayers were rinsed with dH20 4 times. Stained monolayers were visualized with phase microscopy (Eclipse 800, Nikon; Tokyo, Japan). Quantitation of ARS incorporation was performed with cetylpyridinium chloride monohydrate (CPC) (Sigma-Aldrich) extraction. Briefly, 10% (w/v) CPC buffer was prepared in Na2P04 (pH 7.0). Stained monolayers were incubated in 1 ml of CPC buffer for 45 minutes. 200 μl aliquots of the extracted dye were then transferred to 96 well plates. The absorbance at 550 nm wavelength of each aliquot was then measured using a 96 well plate reader (Fluostar Optima, BMG Labtech; Durham, NC).
RNA isolation and reverse transcriptase polymerase chain reaction for alkaline phosphatase
Human ASCs were cultured under control conditions or were induced to undergo osteogenic differentiation for 9 days. Total RNA was isolated from the cells using Trizol (Molecular Research Center, Cincinnati, OH). One-step reverse transcriptase-polymerase chain reactions were performed with 200 ng of total RNA using iScript One-Step RT-PCR Kit for Probes (Bio-Rad Laboratories, Hercules, CA). 25 μl 2x RT-PCR reaction mix containing 0.5 mM of each dNTP (dATP, dCTP, dGTP, dTTP), magnesium ions, and iTaq DNA polymerase was added in a Master Mix with 500 nM forward and reverse primers and 250 nM probe for the amino terminal region of tissue non-specific alkaline phosphatase found in liver/bone/kidney (forward start site, 381: 5′-TCGCCTACCAGCTCATGCATAACA-3′; reverse start site, 509: 5′-TGAAGCTCTTCCAGGTGTCAACGA-3′; probe start site, 450: 5′-/56-FAM/TCAGGGACATTGACGTGATCATGGG/3BHQ_1/3′) or GAPDH (forward start site, 117: 5′-TCGACAGTCAGCCGCATCTTCTTT-3′; reverse start site, 210: 5-ACCAAATCCGTTGACTCCGACCTT-S3′; probe start site, 155: 5′-/56-FAM/AGCCACATCGCTCAGACACCATGGG /3BHQ_1/3′). Complete reaction mix was incubated in a real-time thermal detection system (Bio-Rad Laboratories, Hercules, CA). cDNA synthesis was performed using a 10 min 50°C cycle. iScript reverse transcriptase inactivation was performed using a 5 min 95°C cycle, and PCR cycling and detection were performed using 15 sec 95°C, and 30 sec at 60°C. The number of cycles performed was 40. mRNA expression was normalized to GAPDH control and reported as ΔΔCt values for each donor.
Statistical analysis
Statistical analysis of the data was performed using Graphpad Prism v5.0 software; the level of P value was set at 0.05. The Statistical analyses performed were as follows: 2-way analysis of variances (2-way ANOVAs), followed by Bonferroni's post tests were used for CyQUANT and MTT proliferation experiments, and differentiation experiments. The Student's one-sample t-tests were used for colony formation and viability experiments. Non linear regression analyses using least fit ordinary squares were also performed on proliferation, and differentiation, and the determination coefficients (R2 values) were given to evaluate the relationship between BMI and proliferation, or differentiation, respectively. The coefficient of determination is such that 0 ≤ R2 ≤ 1, and denoted the strength of the association between BMI and proliferation, or BMI and differentiation, where 1 represents the strongest correlation. Results were reported as a positive or inverse correlation. A positive correlation reflects an increase in growth, differentiation potential, or size as BMI increases. A negative correlation reflects a decrease in growth, differentiation potential, or size as BMI increases.
BMI: Body mass index; ASC: Adipose tissue-derived stem cell; ORO: Oil-red-O; ARS: Alizarin red staining; FBS: Fetal bovine serum; BMSC: Bone marrow-derived stem cell; CFU: Colony-forming unit; FACs: Fluorescence-activated cell sorting.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JMG provided BMI-ASCs and was instrumental in planning experiments. JWD expanded BMI-ASCs in the Tulane University Adult Stem Cell Core. HAT performed flow cytometry for forward and side scatter experiments. ESC provided monetary support. TPF performed the experiments and drafted the manuscript. BGR finalized the manuscript. All authors read and approved the final manuscript.
Authors' information
TPF is a doctoral student at Tulane University, in the Structural and Cellular Biology department under the Biomedical Sciences Program in New Orleans, LA. JMG is a Professor of Stem Cell Biology at Pennington Biomedical Research Center in Baton Rouge, LA, and an adjunct Professor to Tulane University in New Orleans, LA. JWD is the manager of the Adult Stem Cell Core at Tulane University in New Orleans, LA. HAT is the manager of the Flow Cytometry Laboratory in the Center for Stem Cell Research and Regerative Medicine at Tulane University in New Orleans, LA. ESC is an Associate Professor of Surgery and Director of Plastic Surgery Research at Tulane University in New Orleans, LA. BGR is an Associate Professor and Co-Vice Chair of the department of Structural and Cellular Biology at Tulane University, New Orleans, LA.
The authors thank the Tulane Center for Stem Cell Research and Regenerative Medicine, Pennington Biomedical Research Foundation for funding, Gang Yu and Xiying Wu for technical assistance, Dr. James Wade, Dr. Ann Reilley, and Dr. Michael Teague, their patients, and staff for the generous donation of adipose tissue lipoaspirates.
Bao W, Srinivasan SR, Berenson GS: Persistent elevation of plasma insulin levels is associated with increased cardiovascular risk in children and young adults. The Bogalusa Heart Study.
Circulation 1996, 93:54-59. PubMed Abstract | Publisher Full Text
Brown KA, Simpson ER: Obesity and breast cancer: progress to understanding the relationship.
Cancer Res 2010, 70:4-7. PubMed Abstract | Publisher Full Text
Csabi G, Tenyi T, Molnar D: Depressive symptoms among obese children 4.
Eat Weight Disord 2000, 5:43-45. PubMed Abstract
Csabi G, Torok K, Jeges S, Molnar D: Presence of metabolic cardiovascular syndrome in obese children 5.
Eur J Pediatr 2000, 159:91-94. PubMed Abstract | Publisher Full Text
Friedl G, Windhager R, Schmidt H, Aigner R: The osteogenic response of undifferentiated human mesenchymal stem cells (hMSCs) to mechanical strain is inversely related to body mass index of the donor 1.
Acta Orthop 2009, 80:491-498. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gutin B, Islam S, Treiber F, Smith C, Manos T: Fasting insulin concentration is related to cardiovascular reactivity to exercise in children 1.
Pediatrics 1995, 96:1123-1125. PubMed Abstract
Anandacoomarasamy A, Fransen M, March L: Obesity and the musculoskeletal system 1.
Curr Opin Rheumatol 2009, 21:71-77. PubMed Abstract | Publisher Full Text
Strotmeyer ES, Cauley JA, Schwartz AV, Nevitt MC, Resnick HE, Bauer DC, et al.: Nontraumatic fracture risk with diabetes mellitus and impaired fasting glucose in older white and black adults: the health, aging, and body composition study 1.
Arch Intern Med 2005, 165:1612-1617. PubMed Abstract | Publisher Full Text
Amin AK, Patton JT, Cook RE, Brenkel IJ: Does obesity influence the clinical outcome at five years following total knee replacement for osteoarthritis?
J Bone Joint Surg Br 2006, 88:335-340. PubMed Abstract | Publisher Full Text
Berend KR, Lombardi AV Jr, Mallory TH, Adams JB, Groseth KL: Early failure of minimally invasive unicompartmental knee arthroplasty is associated with obesity 4.
Clin Orthop Relat Res 2005, 440:60-66. PubMed Abstract | Publisher Full Text
Foran JR, Mont MA, Etienne G, Jones LC, Hungerford DS: The outcome of total knee arthroplasty in obese patients 2.
J Bone Joint Surg Am 2004, 86-A:1609-1615. PubMed Abstract | Publisher Full Text
Gillespie GN, Porteous AJ: Obesity and knee arthroplasty.
Knee 2007, 14:81-86. PubMed Abstract | Publisher Full Text
Smith BE, Askew MJ, Gradisar IA Jr, Gradisar JS, Lew MM: The effect of patient weight on the functional outcome of total knee arthroplasty.
Clin Orthop Relat Res 1992, 276:237-244. PubMed Abstract
Stern SH, Insall JN: Total knee arthroplasty in obese patients 3.
J Bone Joint Surg Am 1990, 72:1400-1404. PubMed Abstract | Publisher Full Text
Goulding A, Taylor RW, Jones IE, McAuley KA, Manning PJ, Williams SM: Overweight and obese children have low bone mass and area for their weight 3.
Int J Obes Relat Metab Disord 2000, 24:627-632. PubMed Abstract | Publisher Full Text
Dardeno TA, Chou SH, Moon HS, Chamberland JP, Fiorenza CG, Mantzoros CS: Leptin in human physiology and therapeutics 1.
Front Neuroendocrinol 2010, 31:377-393. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kotsis V, Stabouli S, Papakatsika S, Rizos Z, Parati G: Mechanisms of obesity-induced hypertension 3.
Hypertens Res 2010, 33:386-393. PubMed Abstract | Publisher Full Text
Kotsis V, Stabouli S, Toumanidis S, Tsivgoulis G, Rizos Z, Trakateli C, et al.: Obesity and daytime pulse pressure are predictors of left ventricular hypertrophy in true normotensive individuals 4.
J Hypertens 2010, 28:1065-1073. PubMed Abstract | Publisher Full Text
Gir P, Oni G, Brown SA, Mojallal A, Rohrich RA: Human adipose stem cells: current clinical applications.
Bunnell BA, Estes BT, Guilak F, Gimble JM: Differentiation of adipose stem cells 3.
Methods Mol Biol 2008, 456:155-171. PubMed Abstract | Publisher Full Text
Gimble JM, Guilak F, Nuttall ME, Sathishkumar S, Vidal M, Bunnell BA: In vitro differentiation potential of mesenchymal stem cells.
Transfus Med Hemother 2008, 35:228-238. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Jang S, Cho H-H, Cho Y-B, Park J-S, Jeong H-S: Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin.
BMC Cell Biology 2010, 11:25. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Lue J, Lin G, Ning H, Xiong A, Lin CS, Glenn JS: Transdifferentiation of adipose-derived stem cells into hepatocytes: a new approach 5.
Liver Int 2010, 30:913-922. PubMed Abstract | Publisher Full Text
Cowan CM, Shi YY, Aalami OO, Chou YF, Mari C, Thomas R, et al.: Adipose-derived adult stromal cells heal critical-size mouse calvarial defects 6.
Nat Biotechnol 2004, 22:560-567. PubMed Abstract | Publisher Full Text
Halvorsen YC, Wilkison WO, Gimble JM: Adipose-derived stromal cells–their utility and potential in bone formation 1.
Int J Obes Relat Metab Disord 2000, 24(Suppl 4):S41-S44. PubMed Abstract
Aust L, Devlin B, Foster SJ, Halvorsen YD, Hicok K, Du Laney T, et al.: Yield of human adipose-derived adult stem cells from liposuction aspirates 1.
Cytotherapy 2004, 6:7-14. PubMed Abstract | Publisher Full Text
Maumus M, Sengenes C, Decaunes P, Zakaroff-Girard A, Bourlier V, Lafontan M, et al.: Evidence of in situ proliferation of adult adipose tissue-derived progenitor cells: influence of fat mass microenvironment and growth 1.
J Clin Endocrinol Metab 2008, 93:4098-4106. PubMed Abstract | Publisher Full Text
Koh YJ, Kang S, Lee HJ, Choi TS, Lee HS, Cho CH, et al.: Bone marrow-derived circulating progenitorcellsfail to transdifferentiate into adipocytes in adult adiposetissues in mice.
Gronthos S, Franklin DM, Leddy HA, Robey PG, Storms RW, Gimble JM: Surface protein characterization of human adipose tissue-derived stromal cells 2.
J Cell Physiol 2001, 189:54-63. PubMed Abstract | Publisher Full Text
McIntosh K, Zvonic S, Garrett S, Mitchell JB, Floyd ZE, Hammill L, et al.: The immunogenicity of human adipose-derived cells: temporal changes in vitro.
Stem Cells 2006, 24:1246-1253. PubMed Abstract | Publisher Full Text
Patrick RW: Morbid obesity: considerations for the EMS provider.
Emerg Med Serv 2004, 33:34. PubMed Abstract
Gimble JM, Floyd ZE, Bunnell BA: The 4th dimension and adult stem cells: can timing be everything?
J Cell Biochem 2009, 107:569-578. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Carcamo-Orive I, Gaztelumendi A, Delgado J, Tejados N, Dorronsoro A, Fernandez-Rueda J, et al.: Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk 1.
J Bone Miner Res 2010, 25:2115-2125. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Orive IC, Gaztelumendi A, Delgado JTN, Dorronsoro A, Fernandez-Rueda J, Pennington DJ, et al.: Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and ap-1 cross-talk.
De Girolamo L, Lopa S, Arrigoni E, Sartori MF, Baruffaldi Preis FW, Brini AT: Human adipose-derived stem cells isolated from young and elderly women: their differentiation potential and scaffold interaction during in vitro osteoblastic differentiation 3.
Cytotherapy 2009, 11:793-803. PubMed Abstract | Publisher Full Text
Aksu AE, Rubin JP, Dudas JR, Marra KG: Role of gender and anatomical region on induction of osteogenic differentiation of human adipose-derived stem cells.
Ann Plast Surg 2008, 60:306-322. PubMed Abstract | Publisher Full Text
Majors AK, Boehm CA, Nitto H, Midura RJ, Muschler GF: Characterization of human bone marrow stromal cells with respect to osteoblastic differentiation 1.
J Orthop Res 1997, 15:546-557. PubMed Abstract | Publisher Full Text
Glowacki J: Influence of age on human marrow 3.
Calcif Tissue Int 1995, 56(Suppl 1):S50-S51. PubMed Abstract
Justesen J, Stenderup K, Ebbesen EN, Mosekilde L, Steiniche T, Kassem M: Adipocyte tissue volume in bone marrow is increased with aging and in patients with osteoporosis 2.
Biogerontology 2001, 2:165-171. PubMed Abstract | Publisher Full Text
Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C: Adipose-derived stem cells: isolation, expansion and differentiation 1.
Methods 2008, 45:115-120. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Goh BC, Thirumala S, Kilroy G, Devireddy RV, Gimble JM: Cryopreservation characteristics of adipose-derived stem cells: maintenance of differentiation potential and viability 1.
J Tissue Eng Regen Med 2007, 1:322-324. PubMed Abstract | Publisher Full Text
Yu G, Wu X, Dietrich MA, Polk P, Scott LK, Ptitsyn AA, et al.: Yield and characterization of subcutaneous human adipose-derived stem cells by flow cytometric and adipogenic mRNA analyses 101.
Schipper BM, Marra KG, Zhang W, Donnenberg AD, Rubin JP: Regional anatomic and age effects on cell function of human adipose-derived stem cells 2.
Ann Plast Surg 2008, 60:538-544. PubMed Abstract | Publisher Full Text
Bjorntorp P: Size, number and function of adipose tissue cells in human obesity.
Horm Metab Res 1974, Suppl 4:77-83. PubMed Abstract
Hirsch J, Batchelor B: Adipose tissue cellularity in human obesity 9.
Clin Endocrinol Metab 1976, 5:299-311. PubMed Abstract | Publisher Full Text
Crossno JT Jr, Majka SM, Grazia T, Gill RG, Klemm DJ: Rosiglitazone promotes development of a novel adipocyte population from bone marrow-derived circulating progenitor cells 2.
J Clin Invest 2006, 116:3220-3228. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gesta S, Tseng YH, Kahn CR: Developmental origin of fat: tracking obesity to its source 2.
Cell 2007, 131:242-256. PubMed Abstract | Publisher Full Text
Devireddy RV, Thirumala S, Gimble JM: Cellular response of adipose derived passage-4 adult stem cells to freezing stress.
Thirumala S, Gimble JM, Devireddy RV: Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium 1.
J Tissue Eng Regen Med 2010, 4:224-232. PubMed Abstract | Publisher Full Text
Thirumala S, Gimble JM, Devireddy RV: Evaluation of methylcellulose and dimethyl sulfoxide as the cryoprotectants in a serum-free freezing media for cryopreservation of adipose-derived adult stem cells 3.
Stem Cells Dev 2010, 19:513-522. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
James AW, Levi B, Nelson ER, Peng M, Commons GW, Lee M, et al.: Deleterious effects of freezing on osteogenic differentiation of human adipose-derived stromal cells in vitro and in vivo 9.
Stem Cells Dev 2011, 20:427-439. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Deng CH, Sun XZ, Gao Y, Luo DS, Liu GH, Huang YP: An experimental study of the culture, isolation, and biological characteristics of rat adipose tissue-derived stromal cells in vitro.
Gonda K, Shigeura T, Sato T, Matsumoto D, Suga H, Inoue K, et al.: Preserved proliferative capacity and multipotency of human adipose-derived stem cells after long-term cryopreservation.
Dubois SG, Floyd EZ, Zvonic S, Kilroy G, Wu X, Carling S, et al.: Isolation of human adipose-derived stem cellsfrom biopsies and liposuction specimens.
Methods Mol Biol 2008, 449:69-79. PubMed Abstract | Publisher Full Text


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Northwestern University's idea of, er, stimulation
Annual undergrad tuition at Northwestern University in Illinois tops $50,000 a year.
Some parents expecting a stimulating education for their children got a rude awakening this week. At least one professor took "stimulation" to new depths.
More than 100 Northwestern University students watched as a naked 25-year-old woman was penetrated by a sex toy wielded by her fiancee during an after-class session of the school's popular "Human Sexuality" class.
The woman said she showed up at the Feb. 21 lecture in the Ryan Family Auditorium in Evanston expecting just to answer questions, but was game to demonstrate. The course's professor on Wednesday acknowledged some initial hesitation, but said student feedback was "uniformly positive."
The p.r. flack for Northwestern defended the "lecture" and "demonstration" as an example of teaching that puts the high-priced school "at the leading edge."
At least one student who witnessed the XXX-rated demo had a more accurate description:
"I didn't expect to see a live sex show," said Justin Smith, 21, a senior economics and political science major who was in the after-class session. "We were told we were going to have some people talk to us about the fetish world and kink."
…After the demonstration, several students tried a different sex toy that gave a "titillating" but not painful shock, testing it out on their arms, said Ken Melvoin-Berg, who narrated the after-class lecture. Melvoin-Berg said the school paid him between $300 to $500 for his appearance.
Faith Kroll, the woman who stripped, was laying down on a towel when she was penetrated. When she arrived, she thought she just would be answering students' questions and showing off sex toys they brought, including whips, paddles and a clown wig.
…Melvoin-Berg said he met Prof. Bailey through a swinging couple who previously spoke to the class. Melvoin-Berg runs the "Weird Chicago Red Light District Sex Tour," which has participants playing games like "spot the ho" as they travel the city looking for prostitutes. He also teaches "Networking for Kinky People," a 3-hour version of the one hour lecture he gave at Northwestern.
Melvoin-Berg said the sex toy used was BDSM, but was "not like a pain thing…we wanted to make it poignant."
"I did mention this was going to be the best money their parents had spent on their education," he said.
What's next? A guest lecture with Charlie Sheen and his goddesses?
I bet a few of the parents of these students wouldn't mind taking a paddle to the clowns running this university-turned-S&M theater…
Super-competent former DHS head Janet Napolitano now protecting UC students from talk of American exceptionalism
June 11, 2015 09:14 PM by Doug Powers
Teach for America's professional, taxpayer-subsidized agitators
June 10, 2015 09:52 AM by Michelle Malkin
The College Board's sabotage of American history
June 3, 2015 08:37 AM by Michelle Malkin
Hillary Clinton helping build the Foundation for an affordable college education
June 1, 2015 11:31 AM by Doug Powers
Education Secretary Arne Duncan: Chicago street gang members hindered by lack of jobs
May 14, 2015 06:55 AM by Doug Powers
Categories: Barack Obama, Democrats, Education


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Battlestar Galactica 4x14: "Blood on the Scales"
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February 7 2009, 04:06 AM
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Re: Battlestar Galactica 4x14: "Blood on the Scales"
Seriously my only complaint is it went by so fast. It seems that in the end Zarek is truly a villan while Gaeta is just very misguided and miserable. I hope they keep killing off characters at this rate to keep things interesting!
It's been great to see these previous background characters get the focus in Season 4.5 so far. Dualla, Gaeta and Zarek have all come to the forefront and had their stories wrapped up or just ended. I like that.
Seeing Kelly, Gage, the quorum, Narcho, Connor, Laird, Seelix, Racetrack, Jaffee, Skulls, Vereem, etc really helped to put this episode over the top too. Having these secondary "background" characters helps propel BSG way ahead of shows like Voyager since it gives things a sense of realism seeing the same people over instead of random pilots and marines.
On another note the crack in the ship is clearly foreshadowing something to come. Awesome to see the ship fall apart. I only hope it goes down fighting like Pegasus!
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Monday, May 17, 2010
© Copyright 2015 Clayton News Daily
There is something thrilling and exciting about making a sale. Before I ever started working for Clayton News Daily, I sold suits for the now defunct S&K Menswear.
One of the things I enjoyed about selling suits was that you really got a chance to learn about the nature of people. After a while, you learn which people are apprehensive, which are open to new things, just by observation and paying attention to their body language.
Working as a journalist, you also learn a lot about the nature of people. By writing about current events, you learn a lot about people's fears, needs and prejudices.
However, sometimes, I miss the idea of having to win somebody over. Selling somebody an idea is one thing, but convincing somebody to purchase or accept an item you are selling, requires much more of a buy-in on the part of the seller and the recipient.
For the seller, there is a small sense of victory when you have worked your way into somebody's heart and convinced them that your product is worth their while.
I had a chance to dip back into my salesman roots this past weekend at the Geranium Festival in downtown McDonough.
Over the weekend, my newspaper company had a booth advertising our sister newspaper, the Henry Daily Herald. As a volunteer at the booth, one of my jobs was passing out promotional materials.
While the products I was "selling" were free, it is amazing how apprehensive people can be toward free stuff. Sometimes, you can make eye contact with someone from far away and tell that they are doing the best they can to avoid you.
One of the things that always helped me as a suit salesman was to be engaging. When someone enters your place of business, you only have a few seconds to greet them before they start making judgments.
If somebody stepped into my store, I usually shook their hand, got to know a little bit about them, and, if it was appropriate, tried to lighten the mood a little bit. It's amazing what can happen when somebody is comfortable and feels like you are paying attention to them.
Those sales skills helped me a lot over the weekend, as I was tasked with handing out packets of marigold seeds with the company logo on them.
Like a town crier, I reeled people in, describing in detail the benefits of marigold seeds. It sounds silly, but with handfuls of seed packets, I went to men, women, and children and did my best to win them over.
Women like flowers, so I told one man that he would be able to impress his gal with his sensitivity by planting a garden of marigolds.
I told some folks that you couldn't eat marigolds after you plant them, but they eventually smell really nice.
I told another person that while they might not be geranium seeds, the average person won't be able to tell the difference.
Mostly, I was being silly, but I followed the rules I had learned about making the sale. In the course of an hour, I must have won over at least 150 people who would have never thought twice about planting seeds.
While I enjoy the idea of helping people make decisions through writing, it was a lot of fun to make people consider a decision that they would normally pass up, if not for creative persuasion. If journalism doesn't work out, I know that I can always get a job selling marigolds.
Joel Hall covers government and politics for the Clayton News Daily. He can be reached via e-mail at email@example.com.
More like this story
- Geranium sale begins at Henry Extension Office ( April 7, 2010 )
- Extension Service prepares for Geranium Festival ( April 4, 2009 )
- Anticipation builds for annual Geranium Festival ( May 14, 2012 )
- We are somebody in The Lord Jesus! - Jim Bell ( July 23, 2011 )
- Geranium Festival today in McDonough ( May 17, 2008 )