2006 ASCB Summer Meeting Program - Boston, MA

Transcription

The American Society for Cell Biology 2006 Summer Meeting on Stem Cell Niches
July 15–18, 2006
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Boston University
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ascbinfo@ascb.org
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www.ascb.org
Table of Contents
Welcome ......................................................................................................... 4
Important Phone Numbers, Ground Transportation, Rental Cars .................. 6
General Information ...................................................................................... 7
Campus Map ................................................................................................... 9
Travel Awardees ............................................................................................. 9
Program Schedule .........................................................................................10
Speaker Abstracts ........................................................................................ 13
Things to Do in the Boston Area .................................................................. 18
Poster Abstracts ........................................................................................... 19
Disclaimer ......................................................................................................26
ASCB Annual Meeting Program .................................................................. 27
Author Index .................................................................................................28
The ASCB Gratefully Acknowledges the Support of
the Following Summer Meeting Sponsors:
AbCam
Biospherix, Ltd.
Fisher Scientific International, Inc.
Miltenyi Biotec
National Heart, Lung, and Blood Institute/NIH
National Institute of Neurological Disorders and Stroke/NIH
National Institute of Child Health and Human Development/NIH
R&D Systems
StemCell Technologies, Inc.
WiCell Research Institute
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July 15–18, 2006
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Boston University
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ascbinfo@ascb.org
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www.ascb.org
OFFICERS
MARY C. BECKERLE
President
THE AMERICAN SOCIETY FOR CELL BIOLOGY
8120 Woodmont Avenue, Suite 750, Bethesda, MD 20814-2762
TEL: (301) 347-9300
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FAX: (301) 347-9310
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ascbinfo@ascb.org
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www.ascb.org
BRUCE M. ALBERTS
President-Elect
ZENA WERB
Past-President
June 2006
JEAN E. SCHWARZBAUER
Secretary
Dear Colleagues:
GARY WARD
Treasurer
While there are many meetings related to stem cell biology, to my knowledge
there have not yet been any meetings focused specifically on stem cell niches.
Given the rapid progress in this area, I believe there is a need for this focused
meeting with prominent leaders in this area. We have deliberately kept this
meeting short so that speakers can attend the entire meeting. My hope is that
by including leading researchers who have made notable contributions to our
understanding of the environmental regulation of stem cell function in diverse
systems—including nematodes, flies, and mammals—that the meeting will
provide opportunities for cross-fertilization and discussion that are absent or
diluted at larger meetings.
Invited speakers were chosen to represent key areas of interest, and their
results and vision should stimulate vigorous discussion. The innovation
of young investigators is key to the future of this field, so I have selected a
number of talks from submitted abstracts. I expect the poster session, with
its first-rate science and relaxed atmosphere, will also stimulate even more
discussion and collaborations.
The success of a meeting hinges on active participation. All participants,
especially students and postdoctoral fellows, are encouraged to ask questions
and contribute to platform talks, poster sessions, and informal discussions.
I sincerely thank AbCam, Biospherix, Ltd., Fisher Scientific International,
Inc., Miltenyi Biotec, R&D Systems, StemCell Technologies, Inc., and WiCell
Research Institute for their corporate support. We would also like to thank
the National Heart, Lung, and Blood Institute, the National Institute of
Neurological Disorders and Stroke, and the National Institute of Child
Health and Human Development, all of the National Institutes of Health,
for their generous grant support for this meeting. I am indebted to the ASCB
staff, particularly Alison Harris, Trina Armstrong, Howie Berman, and Joan
Goldberg, for their efficient management and administration. Finally, I thank
Boston University for providing its conference facility and dormitories. We
hope you enjoy the exciting science, Boston, and the company of friends and
colleagues.
Sincerely yours,
•
JOAN R. GOLDBERG
Executive Director
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COUNCIL
KERRY S. BLOOM
JUAN S. BONIFACINO
DAVID R. BURGESS
JOHN S. CONDEELIS
PETER DEVREOTES
LINDA HICKE
CAROLINE M. KANE
SANDRA K. MASUR
BARBARA J. MEYER
ERIN KEANE O’SHEA
DAPHNE PREUSS
ANNE RIDLEY
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COMMITTEE CHAIRS
DON W. CLEVELAND
Constitution & By-Laws
TIM STEARNS
Education
GARY WARD
Finance
ZENA WERB
Inetrnational Affairs
ARSHAD DESAI
Local Arrangements
JEAN E. SCHWARZBAUER
Membership
LYDIA VILLA-KOMAROFF
Minorities Affairs
ELIZABETH H. BLACKBURN
Nominating
REX L. CHISHOLM
Public Information
LAWRENCE S.B. GOLDSTEIN
Public Policy
ANTHONY BRETSCHER
Annual Scientific Meeting
URSULA GOODENOUGH
Women in Cell Biology
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IMAGE AND VIDEO LIBRARY
KATHRYN E. HOWELL
Chair, Scientific Advisory Board
Sean J. Morrison
Organizer
University of Michigan
Harvey F. Lodish
Chair, External Advisory Board
CBE—LIFE SCIENCES EDUCATION
WILLIAM B. WOOD
Editor-in-Chief
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MOLECULAR BIOLOGY OF THE CELL
SANDRA L. SCHMID
Editor-in-Chief
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July 15–18, 2006
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Boston University
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ascbinfo@ascb.org
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www.ascb.org
Important Phone Numbers
Airport
Logan International Airport
(617) 561-1800
One Harborside Drive, Suite 200S
East Boston, MA 02128-2909
http://www.massport.com/logan for a list of airlines that service the airport.
Ground Transportation
Boston Cab
City Cab (617) 536-5100
ITOA
Metro Cab
Town Taxi
Star Shuttle, Inc.
(617) 536-5010
(617) 825-4000
(617) 782-5500
(617) 536-5000
(877) 970- STAR (7827)
Boston University
Campus Security
Office of Conference Services
10 Buick Street Residence Hall
(617) 353-2121
(617) 353-3520
(617) 358-0657
Rental Cars
Company
Toll Free
Number
Website
Alamo
(800) 462-5266
www.alamo.com
Avis
(800) 831-2847
www.avis.com
Budget
(800) 527-0799
www.budgetrentacar.com
Dollar
(800) 800-4000
www.dollar.com
Enterprise
(800) 261-7331
www.enterprise.com
Hertz
(800) 654-3131
www.hertz.com
National
(800) 227-7368
www.nationalcar.com
Thrifty
(800) 367-2277
www.thrifty.com
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ASCB Discount
Codes
T755300
1301817
July 15–18, 2006
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Boston University
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ascbinfo@ascb.org
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www.ascb.org
General Information
Accessibility for Persons with Disabilities
Boston University is committed to the importance of
the Americans with Disabilities Act requirements,
and the meeting facilities to be used for this conference
are all handicapped-accessible. Persons with special
needs should have been contacted by the ASCB
National Office. The ASCB will try to accommodate
any onsite requests.
Airport/Transportation
Logan International Airport is 6.5 miles from Boston
University and about a 15-20 minute drive. Air Lingus,
AeroMexico, Air Canada, Air France, Air Jamaica,
AirTran, Alaska Airlines, Alitalia, American West,
American, American Eagle, ATA Airlines, British
Airways, Cape Air, Continental, Delta Air Lines,
Finnair, Icelanair, Independence Air, JetBlue Airways,
KLM, Lufthansa, Midwest, Northwest, SATA, Song,
Swiss, TACA, United, US Airways and Virgin
Atlantic provide service to Boston, MA.
There are also three AMTRAK stations located in
Boston:
• Boston-South Station (BOS) located at Summer
Street & Atlantic Avenue
• Boston-North Station (BON) located at 126
Causeway Street
• Boston-Back Bay (BBY) located at 145 Dartmouth Street
Visit www.amtrak.com for more information or for
schedules.
Greyhound has bus terminals in the Boston area as
well; for more information, visit www.greyhound.com.
Other transportation options from the airport
to the BU campus can be found at www.massport.
com/logan/getti.html.
The ASCB will provide bus service to the airport
on Tuesday, July 18. Buses will depart from the front
of 10 Buick Street Residence following the conclusion
of the morning session.
ASCB Staff/Office, Onsite
The ASCB office at BU is located in the George
Sherman Union, East Balcony. Onsite Staff: Alison
Harris, Meetings Coordinator, and Howie Berman,
Technical Program Manager.
Badges
Your meeting badge must be worn at all times on
campus as it allows you access to the session room
and the poster room. Please remove your badge when
frequenting venues off campus.
Business Center
There is no onsite business center, but attendees can
use FedEx Kinko’s, located at 115 Cummington Street,
for copying and printing needs. For information and
pricing, call (617) 358-2679.
Cameras
Other than the official ASCB photographer’s, all types
of cameras and other recording devices are prohibited
at all posters and oral presentation sessions.
Check-in/Check-out
Guests who arranged for BU housing may check into
10 Buick Street beginning at 3:00 pm on Saturday,
July 15. Attendees will be given a Conference ID key
card (which also serves as a meal card) and room
keys at that time. The front desk will be attended until
12:00 midnight. After 12:00 midnight, there will be
a building supervisor on call to check in any late-arriving guests.
Attendees may pick up the meeting program and
packet and badge in the Stone Lobby of the George
Sherman Union from 3:00 pm to 6:00 pm on Saturday,
July 15 or from 8:00 am to 11:00 am on Sunday, July
16. If you must pick up your badge and the meeting
program and packet after these scheduled times, please
contact an onsite ASCB staff person or visit the onsite
ASCB office on the East Balcony of the George Sherman Union.
Housing check-out time is by noon on Tuesday, July
18. Limited luggage storage is available at 10 Buick
Street during the last session, but luggage needs to be
picked up by 3:00 pm. Buses departing for the airport
will leave from the front of 10 Buick Street Residence
Hall immediately following the last morning session.
Computer Lab
There are no accessible student computer labs on campus. However, in the basement of the George Sherman
Union there are computer terminals where guests can
check email, etc. Residents of 10 Buick Street can have
Internet access in their rooms with their own laptops.
Please bring an Ethernet card and cable. There is no
charge for this service. Printing is available at the Fedex
Kinko’s at 115 Cummington Street. See Business
Center section.
Dining Hall and Campus Dining
Breakfast and dinner will be located at the West Campus Dining Hall near the 10 Buick Street housing.
Lunch will be located in the Warren Towers Dining
Hall across Commonwealth Avenue from the George
Sherman Union. Your Conference Identification key
card is your access to the dining halls for your meals.
Daily Dining Hours for the meeting are:
Breakfast:
7:00 am – 9:00 am
Lunch:
11:30 am – 1:15 pm
Dinner:
5:30 pm – 7:00 pm
The Market Street Café and convenience store, located at the entrance to the 10 Buick Street, offers quick
meals, coffee, and some staples. Hours of operation are
available from the 10 Buick Street front desk.
Drinking Policy
The ASCB and BU encourage responsible drinking
for those drinking alcohol. Nonalcoholic beer, wine
and soft drinks will be offered in addition to beer and
wine at the buffet dinner. Alcohol will not be served
to anyone under the age of 21; identification may be
needed. Any guest of legal drinking age is allowed to
consume alcohol in his or her suite. The use of alcoholic
beverages in common areas of the residence hall is
strictly prohibited.
Driving Directions
From the West:
Take the Massachusetts Turnpike (I-90) East to Exit
18, Brighton/Cambridge. Exit left. Follow signs to
Cambridge to the second set of lights. Turn right at the
lights onto Soldiers Field Road/Storrow Drive. Exit
Storrow Drive at the Kenmore exit. Follow directions
from Kenmore Square below.
From the North or South:
Take I-93 Route 3 (Southeast Expressway) North to
Boston. Exit onto Storrow Drive. Continue on Storrow
Drive to the Kenmore Square exit. Follow directions
From Logan International Airport:
Take Route 1-A through the Sumner Tunnel. Bear
right onto the ramp to Storrow Drive. Continue on
Storrow Drive to the Kenmore exit. Follow directions
From Kenmore Square:
At the first set of traffic lights, turn right onto Beacon
Street. At this point, the road forks. The right fork is
Bay State Road. Take Bay State Road for the Office
of Admissions, the Towers Residence Hall, and Upper
and Lower Bay State Road Area Residence Halls.
The left fork takes you into Kenmore Square (the large
building on the right is the Myles Standish Residence
Hall). Bear right at the far end of Kenmore Square onto
Commonwealth Avenue. The George Sherman Union
will be on your right after you pass Granby Street but
immediately before you hit University Road. The
residence hall, 10 Buick Street, will be on your right
past George Sherman Union at Buick Street.
Housing
All attendees will be housed in the air-conditioned,
suite-style campus residence at 10 Buick Street. Guests
will be assigned to single-occupancy rooms within a
four-bedroom suite. Couples requiring housing will
be assigned to a suite with another couple on a space
available basis. Suites are set up with four single occupancy bedrooms, semi-private bathrooms (two per
suite), kitchenette and living room area. Each guest
room is furnished with a bed, dresser, closet, desk,
desk chair, and garbage can. Beds are made upon arrival with appropriate linen, and towels are provided.
Guests will be charged for any linen and towels not
accounted for upon departure. Hotel-sized amenity
packs are provided to all guests, as well as a drinking
cup and bath mat. Individuals must bring their own
toiletries. Please note that the accommodations do not
have telephones.
Coin-operated laundry facilities are located in the
basement of 10 Buick Street.
Keys
All attendees who have arranged for sleeping accommodations are responsible for their room key and
Conference ID key card, which allows you to access the
building and serves as your meal card. The replacement
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Boston University
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cost for a lost room key is $100 per key, and the replacement cost for a lost Conference ID key card is $25.
Library
Conference guests are allowed access into the campus
libraries with a Conference ID key card. However, items
may not be checked out.
Lost and Found
Please turn in found items to the ASCB staff onsite.
Meals
The following meals are included with registration:
July 15
Dinner immediately following
Keynote Session in the George
Sher man Union Ziskind
Lounge
July 16–17*
Breakfast, lunch, and dinner
at campus dining facility – see
Dining Hall and Campus Dining, p. 7, for specific locations.
July 18*
Breakfast at campus dining facility. Boxed lunches provided at
the George Sherman Union.
*Your Conference Identification key card is your access
to the dining halls for your meals.
Message Center
A message board will be located in the Stone Lobby
outside of Metcalf Hall in the George Sherman Union,
where participants may leave messages for other meeting attendees.
Parking
Overnight parking, as well as daily parking, is available
at the 808 Commonwealth Avenue parking lot (Lot
F on the inbound side of Commonwealth Avenue).
Upon arrival, guests will need to ask for a temporary
parking permit from the 10 Buick Residence front
desk. This permit will allow you to park in the lot on
both Saturday and Sunday. Monday morning you will
need to go to Parking Services located in the GSU (just
next to Stone Lobby and Metcalf Hall) and pay for
the weekend permit(s) and any remaining days you will
park on campus. The rate is $10/car/day. Violators of
this policy will be towed at the owner’s expense.
Photo Release
Photographs will be taken at the 2006 ASCB Summer
Meeting. By registering for this meeting, you agree to
allow ASCB to use your photo in any ASCB-related
publications or website.
Posters—Metcalf Hall
Posters may be set up between 12:00 noon and 8:00
pm on Saturday, July 15. The poster room will be open
from 8:00 am until 11:00 pm on Sunday, July 16, and
Monday, July 17.
Posters must be taken down by 11:00 pm on
Monday, July 17.
Poster Presentation times are:
Sunday, July 16
7:15 pm – 8:45 pm
Odd number posters
Monday, July 17
7:15 pm – 8:45 pm
Even number posters
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Recreational Facilities
Guests can use the University’s Fitness and Recreation Center during their stay at the residence hall.
Summer hours of operation, fees, and availability are
available at the 10 Buick Street Front Desk. Daily
rates start at $10.00. Summer Hours:
Monday–Thursday 6:00 am–9:30 pm
Friday
6:00 am–8:00 pm
Saturday
8:00 am–6:00 pm
Sunday
8:00 am–8:00 pm
All hours are subject to change.
Safety and Security
The safety and security of attendees and staff is the
ASCB’s first priority. Working closely with Boston
University campus security and local authorities; we
are committed to make every effort to ensure a safe
and productive event for everyone. Please be aware
of your surroundings at all times and note the closest emergency exit in all facilities. Residential Safety
staff are available in the residence hall 24 hours a day,
seven days a week. Campus police are also available
at any time.
Emergency:
Should you have or be aware of an emergency, please
be prepared to contact Campus Police at 3-2121 from
any campus phone and provide:
• the nature of the emergency
• exact location of the emergency
General Safety:
• Keep a separate list of important credit cards and
identification.
• Carry with you the name, address, and phone
number of an individual to contact in case of an
emergency.
• Do not carry a large amount of cash.
• Do not reveal your room number around strangers
and avoid walking alone.
Pedestrian Safety:
We ask that everyone obey pedestrian crosswalks in
order to safely access the George Sherman Union for
the sessions from residential housing and other sections of the campus.
Session Room, Metcalf Hall
All sessions will take place at Metcalf Hall in the
George Sherman Union. Please refer to the Program
for session topics, speakers, and times.
Smoking Policy
For the comfort and health of all attendees, smoking
is not permitted in buildings or within 20 feet of any
building’s exterior.
Speaker Information
All speakers should report to the Metcalf Hall at least
30 minutes prior to their scheduled talk with their laptop, cables, and presentation.
Telephones
Public telephones are available for use throughout the
campus. No telephone will be provided in the sleeping
rooms. Long distance calls must be made with use of
a credit card or prepaid phone card.
The main desk phone number in the 10 Buick
Street Residence is (617) 358-0657.
What to Bring
Because you will be walking on campus, attendees
should consider bringing comfortable walking shoes,
a hat, umbrella or rain jacket, sunscreen, sunglasses,
a telephone if you would like to make calls from your
room, and an alarm clock.
What to Wear
Attire is casual. Temperatures during the day in
Boston in July average 80°F. Evenings cool off to the
mid-70s.
Coffee Shops/Bakeries
Angora Cafe
1024 Commonwealth Avenue
(617) 232-1757
Bruegger’s Bagel Bakery
644 Beacon Street
(617) 262-7939
Dunkin Donuts
(617) 232-1975
Espresso Royale
(617) 277-8737
Lollicup Coffee and Tea
(617) 437-9955
Starbucks Coffee
(617) 734-3691
Cafés and Restaurants
Ankara Café (Deli/Sandwiches)
(617) 437-0404
Beijing Café (Chinese)
(617) 536-1616
Boston Pizza Express (Pizza/Italian)
1026A Commonwealth Avenue
(617) 734-7777
Campo de’ Fiori (Pizza/Italian)
(617) 236-2066
Cookin’ Café & Grille (Deli/Sandwiches)
(617) 566-4144
Cornwall’s Restaurant Oyster Bar (Pub)
644 Beacon Street
(617) 262-3749
July 15–18, 2006
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Boston University
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ascbinfo@ascb.org
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www.ascb.org
Dugout Café (Pub)
(617) 247-8656
Eastern Standard (Pub)
(617) 532-9100
Great Bay (Seafood)
(617) 532-5300
India Quality Restaurant (Indian)
(617) 267-4499
Maluken Japanese Restaurant (Japanese/Sushi)
645 Beacon Street
(617) 859-8828
Min Sok Korean Bistro (Korean)
(617) 783-8702
Noodle Street (Thai)
(617) 536-3100
Nud Pub Restaurant (Thai)
(617) 536-8676
Quan’s Kitchen (Chinese)
(617) 232-7617
Sunset Grille (Deli/Sandwiches)
(617) 731-8646
T. Anthony’s Pizzeria Restaurant (Pizza/Italian)
(617) 734-7708
The American Society for Cell Biology
Congratulates the
2006 Summer Meeting
Student/Postdoctoral Travel Awardees
Nathalie Brouard
Allison Falender
Karen Renee Groot
Martin Judex
Marcin Jurga
Catherine Kolf
Shihuan Kuang
Jungwoo Lee
Geeta Mehta
Jesung Moon
Helicia Paz
Thomas Schreiber
Marco Seandel
Nicole Anne Siddall
Justin Voog
Yukiko Yamashita
Peter MacCallum Cancer Center, Australia
Baylor College of Medicine
University College London, United Kingdom
Kinikum Rechts der Isar, Germany
Polish Academy of Sciences, Poland
Johns Hopkins University
Ottawa Health Research Institute, Canada
Purdue University
University of California, Los Angeles School
of Medicine
University of Tuebingen, Germany
Memorial Sloan-Kettering Cancer Center
University of Melbourne, Australia
The Salk Institute for Biological Studies
Stanford University
These awards were enabled by grants from:
National Heart, Lung, and Blood Institute/NIH
National Institute of Neurological Disorders and Stroke/NIH
National Institute of Child Health and Human Development/NIH
Boston University Campus
T’s Pub (Pub)
(617) 254-0807
Shopping
There are shops on Commonwealth Avenue.
For information about
tourism in Boston,
visit www.bostonusa.com
or see a Boston Guide
available at both housing
and meeting check-in
locations.
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July 15–18, 2006
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Boston University
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ascbinfo@ascb.org
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www.ascb.org
Stem Cell Niches—Program Schedule
Saturday, July 15
3:00 pm – 6:00 pm
Arrival and Meeting Registration*
George Sherman Union Stone Lobby
*Please note: If you registered for housing, you must check-in at 10 Buick Street Residence Hall to receive
your room key and Conference ID key card.
6:15 pm – 7:15 pm
Keynote Address
6:15 pm – 6:30 pm
6:30 pm – 7:15 pm
George Sherman Union Metcalf Hall
K1
7:30 pm – 9:00 pm
Introductory Remarks
Analyzing Stem Cell Biology Using Drosophila. Allan Spradling, Carnegie Institution/HHMI
Welcome Dinner Buffet/Mixer
Ziskind Lounge
Sunday, July 16
8:00 am – 11:00 pm
Posters Open
7:00 am – 9:00 am
Breakfast
West Campus Dining Hall
9:00 am – 11:40 am
Session I—Germline Stem Cells
Session Chair: Sean Morrison, University of Michigan/HHMI
Germline stem cells from model organisms including C. elegans and Drosophila have made powerful contributions to our understanding of the mechanisms
that regulate the maintenance of stem cells, and that balance self-renewal with differentiation. The ability to precisely identify stem cells and image their
behavior in these systems sets a high standard for the field.
9:00 am – 9:05 am
9:05 am – 9:35 am
S1
9:35 am – 9:55 am
S2
9:55 am – 10:25 am
S3
10:25 am – 10:50 am
10:50 am – 11:10 am
S4
11:10 am – 11:40 am
S5
11:40 am – 1:15 pm
Introduction
Specification and Function of a Stem Cell Niche: Lessons from the C. elegans Distal Tip Cell. Judith
Kimble, University of Wisconsin
A Soma-Germ Line Feedback Mechanism Controls the Number of Germ Line Stem Cell Precursors in the
Drosophila Gonad. Lilach Gilboa, New York University School of Medicine
Aging-related Changes to Stem Cells and the Stem Cell Niche. Leanne Jones, The Salk Institute for
Biological Studies
BREAK
Differential Segregation of Mother and Daughter Centrosomes during Asymmetric Stem Cell Division.
Yukiko Yamashita, Stanford University
Making Stem Cells in the Drosophila Spermatogonial Stem Cell Niche. Erika Matunis, Johns Hopkins
University
Lunch
Warren Towers Dining Hall
1:45 pm – 4:45 pm
Session II—Neural/Melanocytic Stem Cell Niches
Session Chair: Judith Kimble, University of Wisconsin
Advances related to the identification of neural stem cells and melanocytic stem cells have provided insights into the environments that regulate the
function of these cells.
1:45 pm – 1:50 pm
Introduction
1:50 pm – 2:20 pm
S6
Vascular Development by a Non-Angiogenic Mechanism: Evidence for Adult Neural Stem Cell Plasticity.
Andrew Wurmser, University of California, Berkeley
2:20 pm – 2:50 pm
S7
HSC Biology: Implications of HSC Traffic for HSC Niches in Leukemia Spread. Irving Weissman, Stanford
University School of Medicine
2:50 pm – 3:20 pm
S8
Stem Cells and Their Niche in the Adult Mammalian Brain. Fiona Doetsch, Columbia University
3:20 pm – 3:45 pm
BREAK
3:45 pm – 4:15 pm
S9
The MITF Transcription Factor: Master Regulator of Melanocyte Development. David Fisher, Harvard
Medical School
4:15 pm – 4:45 pm
S10
How to Define Stem Cells, by Quiescence or Proliferative Ability? Shin-ichi Nishikawa, RIKEN, Japan
5:30 pm – 7:00 pm
Dinner
7:15 pm – 8:45 pm
Evening Poster Session—Odd Poster Numbers
Cash Bar and light refreshments will be served from 7:15 pm – 9:00 pm.
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Boston University
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Monday, July 17
8:00 am – 11:00 pm
Posters Open
7:00 am – 9:00 am
Breakfast
9:00 am – 11:40 am
Session III—Hematopoietic Stem Cell Niches
Session Chair: Allan Spradling, Carnegie Institution/HHMI
Recent advances have provided insights into the mechanisms that regulate the maintenance of hematopoietic stem cells, as well as markers that can be
used to localize these stem cells in vivo. A better understanding of the mechanisms that regulate hematopoietic stem cell self-renewal and differentiation
could influence the clinical use of stem cells as well as our understanding of how mammalian tissue homeostasis is maintained.
9:00 am – 9:05 am
9:05 am – 9:35 am
S11
9:35 am – 9:55 am
S12
9:55 am – 10:25 am
10:25 am – 10:50 am
10:50 am – 11:10 am
S13
11:10 am – 11:40 am
S15
S14
11:40 am – 1:15 pm
Introduction
PTEN Controls Both Proliferation and the Niche Interaction of Hematopoietic Stem Cells. Linheng Li,
Stowers Institute for Medical Research
Expression and Function of ADAMs (Disintegrin Metalloproteinases) on Human Hematopoietic Stem Cells.
Thomas Schreiber, University of Tuebingen, Germany
Niche Regulation of Quiescent Stem Cells by ROS. Toshio Suda, Keio University, Japan
BREAK
Angiopoietin-like Proteins Expressed by a Novel Stromal Cell Population Stimulate Ex Vivo Expansion of
Hematopoietic Stem Cells. Chengcheng Zhang, Whitehead Institute for Biomedical Research
Most Hematopoietic Stem Cells Appear to Reside in Vascular Niches within the Bone Marrow and
Extramedullary Tissues. Sean Morrison, University of Michigan/HHMI
Lunch
Warren Towers Dining Hall
1:45 pm – 4:25 pm
Session IV—Hematopoietic Stem Cell Niches (Continued)
Session Chair: Fiona Doetsch, Columbia University
1:45 pm – 1:50 pm
1:50 pm – 2:20 pm
2:20 pm – 2:40 pm
S16
S17
2:40 pm – 3:10 pm
S18
3:10 pm – 3:35 pm
3:35 pm – 3:55 pm
S19
3:55 pm – 4:25 pm
S20
Introduction
Modifying the Hematopoietic Stem Cell Niche. David Scadden, Massachusetts General Hospital
HIF-2α Regulates Oct-4: Effects of Hypoxia on Stem Cell Function, Embryonic Development, and Tumor
Growth. Brian Keith, University of Pennsylvania
Regulation of Stem Cell Differentiation and Trafficking by Organ-specific Vascular Niches. Shahin Rafii,
Cornell University
BREAK
Notch Signaling from the Germ Line Stem Cells Induces Stem Cell Niche in the Drosophila Ovary. Ellen
Ward, University of Washington
Mouse Models to Manipulate and Visualize Hematopoietic Stem Cells in Their Niche. Kateri Moore,
Princeton University
5:30 pm – 7:00 pm
Dinner
7:15 pm – 8:45 pm
Evening Poster Session
Even Poster Numbers
Cash Bar and light refreshments will be served from 7:15 pm – 9:00 pm.
Tuesday, July 18
7:00 am – 9:00 am
Breakfast
9:00 am – 11:05 am
Session V—New Niches for Stem Cells
Session Chair: David Scadden, Massachusetts General Hospital
The identification of new niches for stem cells offers the possibility of discovering new mechanisms that regulate stem cell maintenance and function, as
well as insight into the evolutionary significance of these mechanisms.
11:05 am
9:00 am – 9:05 am
9:05 am – 9:35 am
9:35 am – 9:55 am
S21
S22
9:55 am – 10:15 am
10:15 am – 10:35 am
S23
10:35 am – 11:05 am
S24
Introductions
Hematopoietic Stem Cell Niches in the Placenta. Hanna Mikkola, University of California, Los Angeles
smedpten Genes Prevent Abnormal Stem Cell Proliferation by Using the TOR Pathway in Planarians. Nestor
Oviedo, Harvard Medical School
BREAK
The RNA-binding Protein Musashi is Required for Stem Cell Maintenance in the Drosophila Testis. Nicole A.
Siddall, University of Melbourne
Neural Influence in the Stem Cell Niche. Paul Frenette, Mount Sinai School of Medicine
Boxed Lunches
George Sherman Union Stone Lobby
Buses will depart from 10 Buick Street Residence Hall for Logan International Airport immediately following boxed lunch pickup.
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Boston University
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Speaker Abstracts
K1
Analyzing Stem Cell Biology Using
Drosophila
A. Spradling, B. Ohlstein, T. Nystul, M. Buszczak,
R. Cox, T. Tootle, L. Morris; Embryology,
Carnegie Institution/Howard Hughes Medical
Institute, Baltimore, MD
Special microenvironments known as niches
maintain adult tissue stem cells and stimulate
them to divide. By identifying and studying
several types of Drosophila stem cells and
niches, we are uncovering general principles
and genetic pathways governing stem cell
behavior. Stromal niches, such as those that
sustain germline stem cells (GSCs) in the testis
and ovary, are built by specific tissue cells.
Differentiated cap cells in the female or hub
cells in the male, along with partner epithelial
stem cells, adhere to GSCs and provide them
with local signals that control their growth and
development. In contrast, adult intestinal stem
cells (ISCs) do not appear to require analogous
stromal cells, but do closely contact the underlying basement membrane that separates
them from gut circular muscle. ISCs generate
multiple cell types that strongly resemble those
generated in the mammalian intestinal crypt.
Notch signaling controls the choice between
absorptive and secretory lineages in both
Drosophila and mammals. Moreover, in the
midgut, ISC daughters termed enteroblasts
(EBs) require Notch signaling to differentiate,
a role that has yet to be tested in vertebrate
ISCs. EBs bearing Notch pathway mutants fail
to stop growing and form intestinal tumors. We
have identified Delta as the ligand that signals
to EBs and causes them to exit the niche and
differentiate normally.
S1
Specification and Function of a Stem Cell
Niche: Lessons from the C. elegans Distal
Tip Cell
J. Kimble 1,2; 1Biochemistry, University of
Wisconsin-Madison, Madison, WI, 2Howard
Hughes Medical Institute, Madison, WI
In the nematode C. elegans, a single mesenchymal cell, called the distal tip cell (DTC),
provides a niche for germline stem cells (GSC).
The DTC provides a particularly well-defined
and experimentally accessible model for studies of niche development and function. Our
work has focused on three questions central to
niche biology: how is the niche specified, how
does the niche maintain stem cells in an undifferentiated state, and how does it anchor stem
cells within the niche. The Wnt/MAPK pathway
controls DTC specification and formation of the
niche. The DTC and the GSC niche are missing
in mutants lacking key Wnt/MAPK signaling
components (e.g. the POP-1/TCF DNA-binding protein, the SYS-1/β-catenin transcriptional
co-activator); by contrast, extra DTCs and extra
niches are generated upon overexpression of
SYS-1/β-catenin. In addition, we have identified the ceh-22 gene as a direct transcriptional
target of Wnt/MAPK signaling. Indeed, the
CEH-22/Nkx2.5 homeodomain transcription
factor is both necessary and sufficient for DTC
specification and formation of the niche. The
DTC maintains GSC by Notch signaling. One
direct Notch target is fbf-2, which encodes
a PUF (for Pumilio and FBF) RNA-binding
protein. Another direct target is lip-1, which
encodes a conserved phosphatase. FBF-2
maintains GSC by repression of mRNAs
promoting differentiaition, and LIP-1 does so
by downregulating MPK-1, a C. elegans Ras/
Mitogen-activated protein kinase. Specialized
junctions between the DTC and GSC have not
been observed; however, the DTC extends
short processes that nearly encircle GSC. We
suggest that these DTC processes anchor
GSC within the niche.
S2
A Soma-Germ Line Feedback Mechanism
Controls the Number of Germ Line Stem
Cell Precursors in the Drosophila Gonad
L. Gilboa, R. Lehmann; Developmental
Genetics, New York University School of
Medicine, Howard Hughes Medical Institute,
New York, NY
Tight control over the number of stem cells
an organ contains is important for its normal
development and function. Here we describe a
feedback mechanism between primordial germ
cells (PGCs), which are the precursor cells for
germ line stem cells (GSCs), and somatic cells
in the Drosophila ovary, which senses the number of PGCs and ensures that their number is
sufficient to occupy all the stem cell niches that
form during ovarian development. Oogenesis
in Drosophila depends on GSCs that reside
within somatic niches. These niches form at
the end of larval development. PGCs proliferate during larval development and by the end
of that time, enough PGCs exist to occupy all
the newly formed somatic niches. We show
that when fewer PGC are incorporated into the
larval ovary, they divide faster, to compensate
for their initial low numbers, and by the end of
larval development their numbers are sufficient
to occupy all the somatic niches. We discovered
that the control of PGC numbers during larval
development is achieved via a feedback loop
between PGCs and intermingled cells (IC)—a
special somatic population of cells that contacts PGCs. The feedback loop is composed
of a positive and a negative signal. PGCs
express the Epidermal Growth Factor (EGF)
ligand Spitz, which is required for IC survival.
In return, ICs inhibit PGC proliferation. We suggest that during larval development the sensing
of PGC numbers (via Spitz-mediated survival
of ICs) is coupled to a correction mechanism
(a signal from ICs inhibiting PGC proliferation).
When fewer PGC are incorporated into the
ovary, fewer ICs survive. Consequently, less
inhibition of PGC proliferation allows for rapid
PGC divisions and compensation for the initial
low numbers. The properties of this feedback
loop make it ideal to coordinate organ growth
and control homeostasis.
S3
Aging-related Changes to Stem Cells and
the Stem Cell Niche
C. Wong, M. Boyle, M. Rocha, L. Jones;
Laboratory of Genetics, Salk Institute for
Biological Studies, La Jolla, CA
Aging is characterized by compromised
organ and tissue function. Adult stem cell
populations maintain highly differentiated
but short-lived tissues such as blood, skin,
and sperm throughout the lifetime of an
individual; therefore, many consequences
of aging may be due to loss of stem cell
function. The transparency of the Drosophila
testis, the spatiotemporal organization of
germ cells, and the availability of markers
for identifying various germ cell stages
allow precise identification of germ line
stem cells (GSCs) in vivo. GSCs and their
associated somatic stem cells, known as
cyst progenitor cells (CPCs), surround a
cluster of post-mitotic somatic cells called
the apical hub. Hub cells secrete a ligand,
Unpaired (Upd), which activates JAK-STAT
signaling in the adjacent GSCs, thereby
specifying stem cell fate. Thus, the hub is
a critical component of the somatic niche
that supports the maintenance of the stem
cell population. We are using the Drosophila
male germ line as a model system to analyze how the process of organismal aging
affects stem cell behavior. Testes from 50
day-old males contain fewer differentiating
germ cells, when compared to testes from
newly eclosed males. The decline in spermatogenesis during aging is due in part to
a decrease in the number of GSCs. Agerelated changes are also observed within
the niche, as altered morphology and gene
expression changes are observed within
hub cells in aged testes. Our data suggest
that aging-related changes within stem cell
niches may be a significant contributing
factor to reduced tissue homeostasis and
regeneration in older individuals.
S4
Differential Segregation of Mother and
Daughter Centrosomes during Asymmetric
Stem Cell Division
Y. M. Yamashita,1 A. P. Mahowald,2 J. R. Perlin,1
M. T. Fuller1; 1Developmental Biology, Stanford
University, Stanford, CA, 2Molecular Genetics
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Boston University
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and Cell Biology, University of Chicago,
Chicago, IL
Adult stem cells produce highly differentiated
but short-lived cells throughout life. Through
a critical balance of cell fate choice, daughter
cells produced by stem cell divisions either selfrenew stem cell identity to maintain the stem
cell population or initiate the differentiation.
The asymmetric outcome of stem cell divisions
can be specified by local self-renewing signals
from niche supporting cells, combined with a
stereotypical orientation of mitotic spindle in
the dividing stem cell. The Drosophila male
germ line stem cells (GSCs) divide always
asymmetrically, giving rise to one stem cell and
one differentiating cell, thereby keeping both
cell populations. This asymmetry is controlled
by asymmetric placement of daughter cells of
GSC divisions inside and outside of stem cell
niche, which specify stem cell identity. Male
GSCs orient their mitotic spindle perpendicularly to their niche (hub cells), such that one
daughter cell of GSC division stays within the
niche, while the other is displaced away from
the niche. This spindle orientation is set up by
the stereotyped positioning of centrosomes
during interphase. Here we show that developmentally programmed asymmetric behavior
and inheritance of centrosomes underlies the
stereotyped spindle orientation and asymmetric outcome of stem cell division in Drosophila
male GSCs: mother centrosome remains to
be anchored to the hub-GSC interface and is
always inherited to the GSC, whereas daughter centrosome moves away from the hub
and is inherited to the cell that commits to the
differentiation. This is the first demonstration
that mother and daughter centrosomes are
distinguished in the developmental context,
achieving asymmetric cell division.
S5
Making Stem Cells in the Drosophila
Spermatogonial Stem Cell Niche
C. Brawley, M. Issigonis, R. Sheng, M. De
Cuevas, C. Cherry, E. Matunis; Department of
Cell Biology, Johns Hopkins University School
of Medicine, Baltimore, MD
Stem cells regenerate tissue by dividing asymmetrically, producing both new stem cells
(self-renewal) and daughters that differentiate.
Although differentiation is usually considered
irreversible, there is increasing evidence that
the rules of irreversibility can be broken in
response to injury or aging. The conversion
of a differentiated cell to a less differentiated
cell type, or dedifferentiation, endows certain
organisms with remarkable regenerative
properties. Despite centuries of investigation,
however, dedifferentiation is not understood
molecularly. We use Drosophila spermatogenesis as a model stem cell system, since
it parallels mammalian spermatogenesis, yet
we can precisely locate the sperm-producing
spermatogonial stem cells and manipulate
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their microenvironment (niche) genetically. In
this niche, we have found that conditionally
manipulating either the stem cell maintenance
factor Signal Transducer and Activator of
Transcription (STAT), or the differentiation
factor Bam (Bag-of-marbles) induces differentiating spermatogonia to reverse their path
and dedifferentiate into spermatogonial stem
cells. This observation raises the possibility
that spermatogonia may also dedifferentiate
into stem cells in a normal testis, and that
dedifferentiation may be a general feature of
many stem cell systems.
S6
Vascular Development by a Non-Angiogenic
Mechanism: Evidence for Adult Neural
Stem Cell Plasticity
A. E. Wurmser,1 V. A. Sciorra,1 F. H. Gage2;
1
Department of Molecular and Cell Biology,
University of California, Berkeley, Berkeley,
CA, 2Laboratory of Genetics, The Salk Institute
for Biological Studies, San Diego, CA
Adult neural stem cells (NSCs) are brain-specific, somatic stem cells that were originally
proposed to differentiate only to the neuronal
and glial lineages. Bromo-deoxyuridine (Brd
U)-labeling of actively dividing cells in the
postnatal CNS indicated that NSCs are concentrated at interfaces with the vasculature
within proliferative clusters comprised of 40%
neuronal and glial cells, 37% endothelial cells
(ECs) and approximately 1% smooth muscle
cells (SMCs). These newly-formed cells and
vascular structures define the extracellular
environment or “niche” of the NSC (Palmer
et al., J Comp Neurol., 2000). The traditional
view suggests that Brd U-labeled ECs within
the stem cell niche arise by angiogenesis,
a process whereby endothelial cells of preexisting blood vessels proliferate to form new
vascular branches directed at oxygen- and
nutrient-deprived tissue. Unexpectedly, we
found that instructive, intercellular signals
originating from vascular ECs divert NSCs
from the ectodermal neural lineages, instead
inducing NSCs to become mesodermal ECs
and SMCs. The mechanism by which NSCs
convert to ECs and SMCs is referred to as
stem cell plasticity, a poorly understood phenomenon whereby tissue-specific stem cells
broaden their differentiation repertoires and
differentiate to cells of another lineage. Thus,
NSC plasticity rather than angiogenesis may
account for a significant proportion of blood
vessel generation in brain, expanding the
importance of the adult NSC in maintaining
the cellular composition and function of the
CNS.
S7
HSC Biology: Implications of HSC Traffic
for HSC Niches and Leukemia Spread
I. Weissman; Department of Pathology,
Stanford University School of Medicine,
Stanford, CA
Abstract unavailable at time of publication.
S8
Stem Cells and Their Niche in the Adult
Mammalian Brain
M. Tavazoie, M. Louissaint, L. Colonna, F.
Doetsch; Departments of Pathology, Neurology
and the Center for Neurobiology and Behavior,
Columbia University, New York, NY
Neural stem cells persist in specialized niches
in the adult mammalian brain where they continuously generate large numbers of neurons
that become functionally integrated into neural
circuits. The subventricular zone (SVZ) is an
extensive germinal layer along the length of
the lateral wall of the lateral ventricles, which
generates olfactory bulb interneurons. The
stem cells for in vivo adult neurogenesis are
a subset of astrocytes, glial cells classically
associated with support functions in the brain.
Stem cell SVZ astrocytes divide to generate
neuroblasts via an intermediate rapidly dividing
transit-amplifying cell. The neuroblasts then
migrate as chains along a network of pathways
to join the rostral migratory stream that leads
to the olfactory bulb where they differentiate
into inhibitory neurons. The SVZ is therefore
a region in which stem cell self-renewal and
differentiation as well as extensive migration
are tightly coupled. In addition to their role
as stem cells, SVZ astrocytes are important
contributors to the adult neurogenic niche.
We have characterized morphologically distinct subpopulations of SVZ astrocytes, which
differ in their proliferation characteristics, and
expression of growth factor receptors and
transcription factors. Ependymal cells, multiciliated cells that line the ventricles, are also
a key component of the SVZ niche, causing
flow of the cerebrospinal fluid, and secreting
factors that support neurogenesis in the SVZ.
The SVZ stem cell niche is also enriched in
blood vessels and has a specialized basal
lamina. We find dividing SVZ cells closely associated with blood vessels. Thus, signals from
the vasculature as well as cell-cell interactions
play key regulatory roles in the adult neural
stem cell niche.
S9
The MITF Transcription Factor: Master
Regulator of Melanocyte Development
D. E. Fisher; Melanoma Program in Medical
Oncology, Division of Pediatric Hematology/
Oncology, Dana-Farber Cancer Institute,
Children’s Hospital Boston, Harvard Medical
School, Boston, MA
Numerous coat color mutants have collectively
shed important light on the genetic regulators
of pigmentation. Among the genes affecting
pigmentation, MITF is in the group whose
mutation affects melanocyte viability, rather
than simply melanin production. Despite this,
the MITF gene was discovered to encode a
bHLHzip transcription factor which regulates
July 15–18, 2006
expression of numerous genes thought to
participate in melanin biosynthesis. In addition, MITF was found to be a major target of
the signaling pathway initiated by Melanocyte
Stimulating Hormone, a major hormonal
regulator of pigmentation. Separately, however,
MITF’s role as a viability factor has been investigated through strategies designed to identify
additional transcriptional targets (independent
of pigmentation genes). Several such target
genes which may contribute to lineage survival
include Bcl2, CDK2, and the c-MET receptor.
The concept that MITF may modulate key
aspects of lineage survival has also spurred
investigation into whether MITF over-activity
may be related to malignancy. Indeed gene
amplifications of MITF have been discovered
in a fraction of human melanomas, and MITF
or its close relatives TFEB and TFE3 have also
been implicated as oncogenes in a variety of
human sarcomas or kidney neoplasms. MITF’s
role in stem cell maintenance has also been
investigated in hair follicle dynamics. Utilizing
transgenic mice with LacZ-tagged melanocytes, the bulge melanocyte population (the
stem cell niche) could be followed during aging,
in several models of age-related hair graying.
Melanocyte stem cell loss was seen to precede
graying in multiple models. Age-related graying
in genetically wildtype mice and humans was
associated with melanocyte stem cell attrition
via a process which appears to involve in
situ differentiation within the niche, a process
which is accelerated by mild loss-of-function
mutation of MITF.
S10
How to Define Stem Cells, by Quiescence
or Proliferative Ability?
S. Nishikawa; RIKEN, Kobe, Japan
It is difficult to find the common definition of
stem cells (SC) valid for all SC systems. One
reason for this is the presence of two clearly
distinct ways in defining SC; one concerns
quiescence whereas the other proliferative
ability for the definition. The typical example for
the former is melanocyte, whose SC is defined
by quiescence and the localization, whereas a
latter example is mesenchymal stem cell that
has been defined by its proliferative ability in
culture. The two SC systems appear as though
markedly contrasting each other, but there may
be some common features. For instance, as it
is possible to support sustained growth of purified melancytes in culture, in vitro proliferative
activity can be applied also to the definition of
melanocyte SC. Intriguingly, with respect to the
melanocyte lineage, only the resting compartment in the tissue can proliferate in vitro. As
such, the quiescent SC in the tissue and the SC
with the in vitro proliferative activity are identical
in some SC systems. In fact, satellite cells for
muscle cells and oval cells for hepatocytes are
known to be quiescent in the tissues but proliferative in the culture. Hence, it is conceivable
that mesenchymal SC also forms a quiescent
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Boston University
compartment in the tissue. To investigate this
possibility, it is essential to identify mesenchymal SC in the tissues. In this symposium, we
will present our recent studies concerning the
SC of these two lineages with an expectation
that comparison of two SC systems will reveal
the common molecular mechanisms regulating
quiescence and proliferation of SC.
S11
PTEN Controls Both Proliferation and the
Niche Interaction of Hematopoietic Stem
Cells
L. Li; Stowers Institute for Medical Research,
Kansas City, MO
The stem cell niche plays an essential role in
regulation of stem cells through generating
key extrinsic signals. These external signals
coordinate with internal signals to maintain a
homeostatic balance between self-renewal and
differentiation of stem cells. PTEN, an internal
signal, through control of the PI3K-Akt pathway
regulates a variety of cellular events including
proliferation and migration. Conditional-inactivation of PTEN leads to myeloproliferative
disorder and leukemia. In addition, further
characterization has revealed that PTEN,
through regulation of PI3K/Akt activity, controls
the quiescent and active states of stem cells
and also regulates the interaction between
stem cells and their niche. Lack of PTEN
control leads to enhanced stem cell activation
and uncontrolled proliferation, as well as loss
of the ability of the niche to retain stem cells
in the hematopoietic system.
S12
Expression and Function of ADAMs
(Disintegrin Metalloproteinases) on Human
Hematopoietic Stem Cells
T. D. Schreiber, 1 J. T. Wessels, 2 G. Klein1;
1
Center for Medical Research, Section for
Transplantation Immunology, University of
Tuebingen, Medical Clinic II, Tuebingen,
Germany, 2 Depar tment of Nephrology,
University of Goettingen, Goettingen,
Germany
Despite of the widespread use of hematopoietic stem cells for cellular therapies, neither the
mechanism of stem cell egress out of the bone
marrow nor the precise definition of the bone
marrow niches are fully understood. Since matrix metalloproteinases are well established as
part of the induced stem cell mobilization process, we started to investigate the expression
and function of the disintegrin metalloproteinases on human hematopoietic progenitor cells.
The ADAM (a disintegrin and metalloproteinase) family includes structurally and functionally related membrane-bound proteases that
have essential roles in different physiological
processes such as cell differentiation, cell-cell
interaction and protein ectodomain shedding.
These processes are also relevant in the bone
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marrow niches. However, little is known about
the expression and function of ADAMs on human hematopoietic stem cells. Using highly
enriched isolated CD133+ or CD34+ cell populations, we could show by RT-PCR analysis
that several members of the ADAM family are
transcribed in hematopoietic stem cells. Flow
cytometric analysis and immunofluorescence
staining of isolated CD34+ cells revealed that
ADAM8, 10, 15, 17, and 19 can be detected
on the cell surface of hematopoietic progenitor
cells. Immunoblotting of ADAM8 and ADAM10
clearly showed that processed forms of
ADAMs without the prodomain are present in
highly purified CD34+ cells. Functionally, antiADAM10 antibodies had a strong influence on
hematopoietic progenitor cell differentiation as
analyzed by colony-forming unit (CFU) assay.
Incubation of isolated CD34+ cells with antiADAM10 antibodies drastically diminished
the numbers of developed colonies after 16
days of incubation, whereas incubation with
other anti-ADAM antibodies did not significantly
change the number of CFUs, in comparison to
the controls. Taken together, our results provide
strong evidence that distinct members of the
ADAM family are expressed on the cell surface
of hematopoietic stem cells and that ADAM10
is functionally involved in the developmental
fate of progenitor cells.
S13
Niche Regulation of Quiescent Stem Cells
by ROS
T. N. N. Suda; Cell Differentiation, Keio
University, Tokyo, Japan
The quiescent state is thought to be required
for maintenance of hematopoietic stem cells
(HSCs). Interaction of HSCs with their particular microenvironments, known as stem
cell niches, is critical for adult hematopoiesis
in the bone marrow (BM). We previously demonstrated that HSCs expressing the receptor
tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP)
of HSCs that adheres to osteoblasts (OBs) in
the BM niche. The interaction of Tie2 with its
ligand Angiopoietin-1 (Ang-1) induces cobblestone formation of HSCs in vitro and maintains
long-term repopulating activity of HSCs in vivo.
Since Ang-1 enhances expression of N-cadherin in HSCs and induces cell adhesion to
bone, we examined the role of N-cadherin in
vivo. When HSC cells were transfected with a
dominant negative form N-cadherin lacking the
extracellular domain, transfected cells could
reach the bone marrow but could not enter
the osteoblastic niche, resulting in defects in
hematopoietic reconstitution. These data suggest that the Tie2/Ang-1 and the N-cadherin
signaling pathways play a critical role in the
maintenance of HSCs in a quiescent state in
the BM niche. Maintenance of the quiescence
is critical for protection of HSCs. When the BM
is ablated during BM transplantation or after
treatment with myelosuppressive agents, qui-
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escent HSCs enter the cell cycle and proliferate
to supply progenitors of committed hematopoietic cells. We observed that the ATM-deficient
mouse shows progressive bone marrow failure
due to a defect in HSC function associated
with elevated reactive oxygen species (ROS).
Similarly, ROS was elevated after serial BM
transplantation, and up-regulation of MAPK
p38 and INK4a was detected only in HSCs. In
vivo treatment of recipient mice with anti-oxidative agents or a p38 MAPK inhibitor restored
the reconstitution capacity of ATM-deficient
HSCs. I will discuss how HSCs in a stem cell
niche can be regulated by external signals.
S14
Angiopoietin-like Proteins Expressed by
a Novel Stromal Cell Population Stimulate
Ex Vivo Expansion of Hematopoietic Stem
Cells
C. Zhang, M. Kaba, H. Lodish; Whitehead
Institute for Biomedical Research, Cambridge,
MA
Recently we identified a novel cell population
that supports HSC expansion—CD3+Ter119cells isolated from Embryonic Day 15 (E15)
mouse fetal livers. DNA array experiments
showed that, among other proteins, insulin-like
growth factor 2 (IGF-2) and angiopoietin-like 2
and 3 (Angptl2 and Angptl3) are expressed in
these cells but not in several other cell types
that do not support HSC expansion in culture.
I then developed a simple culture system for
bone marrow HSCs using low levels of SCF,
TPO, IGF-2, FGF-1, and Angptl2 or 3 in serum-free medium. As measured by competitive
repopulation analyses, there was a 24–30-fold
increase in numbers of long-term repopulating
HSCs (LT-HSC) after 10 days of culture. I further identified several homologs of Angptl2 that
are also new growth factors of HSCs. Ongoing
studies will define the precise role Angptls play
in hematopoietic stem cell biology.
S15
Most Hematopoietic Stem Cells Appear to
Reside in Vascular Niches within the Bone
Marrow and Extramedullary Tissues
S. J. Morrison; HHMI/Department of Internal
Medicine, University of Michigan, Ann Arbor,
MI
The identification of SLAM family markers allowed us to systematically examine the localization of hematopoietic stem cells (HSCs) in
the bone marrow and other hematopoietic tissues. This is the first time that it has been possible to examine the localization of HSCs using
markers that were functionally validated to give
very high levels of HSC purity. This revealed
that over 60% of CD150+CD48-CD41- HSCs
localized to the surface of sinusoidal endothelial cells in the bone marrow and in the spleen
(Cell 121:1109). In bone marrow, 14% of such
HSCs localized to the endosteum, while the
remaining HSCs were not in contact with
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recognizable structures. Differences in HSC
localization did not correlate with differences
in HSC activation as virtually all CD150+CD48CD41- cells were quiescent, with only 3% in
S/G2/M phase of the cell cycle. These results
appear to contrast with previously published
results that identified osteoblastic HSC niches
based on the localization of N-cadherin+ BrdU
label-retaining cells. However, we could find
no evidence that HSCs express N-cadherin or
retain BrdU, raising the possibility that osteoblasts affect HSCs indirectly or via diffusible
factors. To test this we administered PTH to
mice, which was previously shown to increase
osteoblast numbers and HSCs. Consistent with
the possibility of indirect effects, PTH caused
many changes in the bone marrow beyond increasing osteoblast numbers, including changes in vascularity. To further test the functional
significance of osteoblasts we examined mice
that are deficient for the extracellular matrix
proteoglycan, biglycan. These mice develop
osteoporosis as adults as a result of reductions
in osteoblast numbers. However, we could not
detect any defect in hematopoiesis or in HSC
maintenance within these mice. These data
demonstrate that not all changes in osteoblast
numbers affect HSCs, raising the possibility
that only a subset of osteoblasts secrete factors
that affect HSC function.
S16
Modifying the Hematopoietic Stem Cell
Niche
D. Scadden; AIDS Research Center,
Massachusetts General Hospital, Charlestown,
MA
Abstract unavailable at time of publication.
S17
HIF-2α Regulates Oct-4: Effects of Hypoxia
on Stem Cell Function, Embr yonic
Development, and Tumor Growth
K. L. Covello,1 J. Kehler,2 M. C. Simon,3 B. Keith4;
1
Department of Cell and Developmental Biology,
University of Pennsylvania, Philadelphia, PA,
2
Center for Animal Transgenesis and Germ
Cell Research, New Bolton Center, University
of Pennsylvania, Philadelphia, PA, 3Howard
Hughes Medical Institute, University of
Pennsylvania, Philadelphia, PA, 4Abramson
Family Cancer Research Institute, University
of Pennsylvania, Philadelphia, PA
The division, differentiation, and function of
stem cells and multipotent progenitors are
influenced by complex signals in the microenvironment, including oxygen availability. Previous
reports from multiple laboratories (including
ours) have described the effects of physiological hypoxia on the function and differentiation
of hematopoietic and neural stem cells. In
addition, a recent connection has been made
between Notch signaling and hypoxia, in which
intracellular Notch activity is potentiated by the
oxygen regulated transcription factor HIF-1α,
leading to inhibition of myogenic and neural
precursor cell differentiation (Gustaffson et
al. [2005] Dev. Cell 9: 617–628). In this report,
we identify a different mechanism whereby
hypoxia regulates stem cell and progenitor
function. Using a genetic “knock-in” strategy,
we replaced HIF-1α expression with HIF-2�
in murine ES cells and mice (Covello et al.
[2006] Genes & Dev. 20: 557–570). This genetic switch results in expanded expression
of HIF-2α specific targets including Oct-4, a
transcription factor essential for maintaining
stem cell pluripotency. We show that HIF-2α,
but not HIF-1α, binds to the Oct-4 promoter and
induces Oct-4 expression and transcriptional
activity, thereby contributing to impaired development in homozygous Hif-2α KI/KI embryos,
defective hematopoietic stem cell differentiation in embryoid bodies, and large ES-derived
tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2α severely
reduces the number of embryonic primordial
germ cells, which require Oct-4 expression for
survival and/or maintenance. These results
identify Oct-4 as a HIF-2α specific target
gene, and indicate that HIF-2α can regulate
stem cell function and/or differentiation through
activation of Oct-4, which in turn contributes to
HIF-2α’s tumor promoting activity.
S18
Regulation of Stem Cell Differentiation
and Trafficking by Organ-specific Vascular
Niches
S. Rafii; Department of Genetic Medicine,
Cornell University Medical College, New
York, NY
Emerging evidence indicate the organ specific stem cells, including hematopoietic stem
cells, reside in close proximity to sinusoidal
endothelial cells. As such close interaction
of endothelial cells with stem cells may not
only be critical for trafficking of stem cells but
also proliferation and survival of these cells.
Indeed, marrow’s vascular niche comprise of
a unique microenvironment, which supports
lineage-specific differentiation of stem and
progenitor cells. Angiogenic factors released
by the stem and progenitor cells including
Vascular Endothelial Growth Factor (VEGFA) as well as FGF-2 contribute to the maintenance and establishment of the vascular
niche. In turn, endothelial cells by deployment
of stem cell active cytokines, including soluble
and membrane kit-ligand as well as SDF-1
support survival of the stem cells. In addition,
VEGF-A through interaction with its tyrosine
kinase receptor, VEGFR1, expressed on the
hematopoietic stem cells support the localization of stem cells from the osteoblastic niche
to the vascular niche. Activation of VEGFR1
results in the release of proteases including,
MMP-9 which support the release of soluble
kit-ligand thereby positioning stem cells within
the vascular niche. Targeted disruption of the
vascular niche results in severe impairment in
July 15–18, 2006
hematopoietic reconstitution. SDF-1/CXCR4
signaling pathway is the molecular hub
regulating the reconstruction of the vascular
niche as chronic inhibition of CXCR4 blocks
VEGF-A induced recruitment of vascular cells
and retards regeneration of the hematopoietic
stem cells. As such, SDF-1 and VEGF-A may
be used to stimulate hematopoiesis in part
through accelerating the reconstruction of the
marrow’s vascular niche.
S19
Notch Signaling from the Germ Line Stem
Cells Induces Stem Cell Niche in the
Drosophila Ovary
E. J. Ward, H. R. Shcherbata, S. H. Reynolds,
K. A. Fischer, S. D. Hatfield, H. Ruohola-Baker;
Biochemistry, University of Washington,
Seattle, WA
Stem cells are maintained and retain their
capacity to continue dividing due to the influence of a microenvironment, termed a niche.
While niches are important to maintain ‘stemness’ in a wide variety of tissues (e.g. skin,
gut, blood), the mechanism by which niches
are established or maintained is unknown.
The Drosophila Germ line Stem Cells (GSCs)
reside in a somatic cap cell niche. We show
that Notch activation can induce a niche
even in an adult fly: Delta over-expression in
the germ line, or expression of constitutively
active Notch in the somatic cells, generates
somatic niche cells, up to ten fold over the
normal number. In turn, these ectopic niche
cells induce ectopic GSCs. Conversely, when
GCSs do not produce functional Delta ligand,
the TGF-β pathway is not activated in the
GSCs and the GSCs are subsequently lost
from the niche. Importantly, Notch activity is
not required in GSCs. Rather, clonal analysis
reveals that the receiving end of the Notch
pathway is required in the cap cells. These
data show that a feedback loop exists between
the stem cells and niche cells: Delta from the
GSC can activate Notch in the somatic cells
inducing a functional niche, that in turn controls GSC maintenance. Demonstration that
stem cells can contribute to niche formation
has far-reaching consequences for stem cell
therapies, and may provide insight into how
cancer can spread throughout an organism
via populations of cancer stem cells.
S20
Mouse Models to Manipulate and Visualize
Hematopoietic Stem Cells in Their Niche
K. Moore; Department of Molecular Biology,
Princeton University, Princeton, NJ
We are using functional genomics approaches
to dissect the molecular mechanisms at play
in hematopoietic stem cell (HSC) niches.
The Wnt signaling pathway is prominently
represented in our studies, suggesting a role
in the extrinsic control of HSC. We elected to
over-express Wnt Inhibitory Factor-1 (Wif-1)
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Boston University
in a stem cell supporting stromal cell line
(AFT024). Wif-1 expressing AFT024 cells
exhibited a 50% reduction in stem cell support. These data prompted us to express
Wif-1 specifically in osteoblasts via the 2.3
Col1a1 promoter in a transgenic mouse (pOBcol2.3/Wif-1/GFP). Highly purified stem cells
from these mice (LSK/CD34-/lo) appear to be
present at the same frequency compared to
control pOBcol2.3/GFP mice. Similar frequencies were also observed for immediate in vitro
progenitors. But, after 4 weeks in culture on
AFT024 cells we observed an increase in more
primitive progenitors. These data, although
very preliminary, suggest that a Wnt-inhibited
niche may increase the self-renewal capability
of their resident stem cells. This hypothesis is
being tested in a transplantation model. We
are also developing a mouse model to visualize rarely dividing, quiescent HSC within their
niches. We have crossed a transgenic mouse
with a tetracycline (Tet)-responsive expression
system under control of the human CD34 regulatory elements (hCD34tTA) with a transgenic
mouse that expresses a histone H2B-GFP
fusion protein controlled by a Tet-responsive
promoter (Tet-O-H2B-GFP). These mice will
continually label H2B histones with GFP in
cells that are controlled by hCD34tTA when
not given Dox. At 8 weeks of age they were
fed Dox for an additional 8+ weeks to allow
progressive dilution of GFP in dividing cells.
FACS analyses revealed a very discrete LSK/
CD34-/lo GFP high population of marrow cells
that was decreased when resident stem cells
were induced to divide after 5FU treatment.
Bone sections are being processed to see
where these high GFP label-retaining cells are
located within the marrow cavity. We anticipate
that this will be a useful model for visualizing
stem cells within their native niches both when
perturbed and during normal homeostasis.
S21
Hematopoietic Stem Cell Niches in the
Placenta
Y. Wang,1 C. Francis,1 E. Hamalainen,1 H.
Helgadott,2 S. Orkin,2 H. Mikkola1; 1Molecular,
Cell and Developmental Biology, Institute for
Stem Cell Biology and Medicine, Jonsson
Comprehensive Cancer Center, UCLA, Los
Angeles, CA, 2Pediatric Oncology, DanaFarber Cancer Institute, Boston, MA
Development of hematopoietic stem cells
(HSC) is a complex process that involves
several anatomical sites. Murine placenta
harbors a large pool of HSCs during midgestation, yet, the origin of placental HSCs and
the physical niches where they reside have
not been defined. We utilized the runx1LacZ/+
mouse strain where developing HSCs are
marked by LacZ expression to localize placental HSCs. Interestingly, LacZ+ cells were
found in chorioallantoic mesenchyme around
the large fetal blood vessels from E9.5 onwards. Additionally, LacZ+ cells appeared
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in fetal capillaries in the labyrinth, often as
pairs or small clusters, suggesting that they
had divided within labyrinthine vessels. As
heterozygosity for runx1 may affect kinetics of
HSC development in the placenta, location of
hematopoietic cells in wild type placentas was
also analyzed by immunohistochemistry and
confocal microscopy. Indeed, CD34+, CD41+
and/or CD45+ expressing cells were found in
the wall of the large chorionic vessels, adjacent
mesenchyme, and in the labyrinth, verifying
the presence of multiple hematopoietic niches
in the placenta. Interestingly, LacZ expressing
cells could be found in placental mesenchyme
also in runx1Lac Z/null embryos, which are
unable to generate definitive hematopoietic
cells, although cells with active runx1-locus
accumulate in sites where de novo hematopoiesis normally occurs. At E12.5, the age when
runx1LacZ/null embryos die, the frequency and
intensity of LacZ expressing cells in placental
mesenchyme and yolk sac of the mutants had
increased dramatically, possibly reflecting inability of runx1-deficient cells to develop into
definitive hematopoietic cells that can leave
their site of origin. As expected, no LacZ+
cells appeared in the labyrinth or in the fetal
liver in the mutants. These results support the
hypothesis that the chorioallantoic mesoderm
in the placenta is another independent source
of HSCs, and pinpoint labyrinthine vessels as
putative niches where hematopoietic progenitors/HSCs mature and expand before seeding
intra-embryonic hematopoietic sites.
S22
smedpten Genes Prevent Abnormal Stem
Cell Proliferation by Using the TOR Pathway
in Planarians
N. J. Oviedo,1 M. Levin,1 A. Sanchez Alvarado2;
1
Center for Regenerative & Developmental
Biology, Forsyth Institute/Harvard Medical
School, Boston, MA, 2 Depar tment of
Neurobiology and Anatomy, Howard Hughes
Medical Institute, School of Medicine,
University of Utah, Salt Lake City, UT
Planarian stem cells (also known as neoblasts)
are capable of responding to a variety of
instructive signals that regulate their proliferation, migration and differentiation. As such,
neoblasts are instrumental in the processes
of tissue homeostasis and regeneration in
planarians. However, the signals, attendant
molecular pathways, and cellular mechanisms
regulating neoblast activities are unknown.
PTEN has been found to be mutated in many
malignancies related to cancer and implicated in both cell migration and chemotaxis.
Here, we report on the identification, gene
expression and functional characterization
of two novel invertebrate pten-related genes
(smedpten-1 and -2) from the freshwater planarian Schmidtea mediterranea. We found that
smedpten-related genes are highly conserved
when compared with their deuterostome and
ecdysozoan counterparts, and that they are
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expressed at low copy number and mostly by
differentiated tissues. Functional perturbation
using RNA-interference of smedpten genes
induced significant increases in cellular proliferation and the appearance of abnormal
outgrowths in the parenchyma. Interestingly,
neoblast elimination by γ-irradiation prevented
the smedpten phenotype. Moreover, rapamycin
treatment in smedpten(RNAi) worms selectively disrupted abnormal proliferation but did
not affect proliferation during physiological turnover or regeneration. Additional analyses of the
RNAi-induced defects also uncovered a role
for smedpten-related genes during neoblast
differentiation. Altogether, our data indicate
that PTEN plays an instructive role in regulating neoblasts, and that the smedpten(RNAi)
phenotype is mediated through the TOR
(Target Of Rapamycin) pathway, establishing an important difference between normal
and abnormal proliferative neoblasts. Thus,
planarians provide a novel context in which
to investigate the roles PTEN plays in tissue
homeostasis and study mechanisms used by
abnormal, proliferative adult stem cells.
S23
The RNA-binding Protein Musashi Is
Required for Stem Cell Maintenance in the
Drosophila Testis
N. A. Siddall,1,2 E. A. McLaughlin,3,2 N. L.
Marriner,1,2 G. R. Hime1,2; 1Anatomy and Cell
Biology, University of Melbourne, Parkville,
Australia, 2ARC Centre of Excellence in
ascbinfo@ascb.org
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Biotechnology and Development, Callaghan,
Australia, 3School of Environmental and Life
Science, University of Newcastle, Callaghan,
Australia
Katayama,1 S. A. Thomas,2 A. Hidalgo,1 A.
J. Peired1; 1Mount Sinai School of Medicine,
New York, NY, 2University of Pennsylvania,
Philadelphia, PA
Stem cell populations respond to a variety of
regulatory cues that control rates of cell survival, regeneration and differentiation in order
to balance the renewal of the stem cell pool
with a requirement for tissue replacement. In
this study, we have identified a cell autonomous
requirement for the RNA-binding protein and
translational repressor, Musashi (Msi) for maintaining germline stem cells in the Drosophila
testis. We show that Msi is expressed in
germline stem cells and in the somatic cells of
the stem cell niche. We found that loss of Msi
function disrupts the balance between GSC
renewal and differentiation, resulting in the premature differentiation of GSCs. Furthermore,
we show that although Msi is expressed in both
somatic and germ cells, Msi function is required
intrinsically in stem cells for maintenance of
stem cell identity. In this study, we also show a
requirement for Msi later in spermatogenesis,
as msi mutants exhibit non-disjunction and
failure of cytokinesis. Murine Msi homologues
are also expressed in both germline and somatic testicular cells suggesting evolutionary
conservation of Msi function.
Hematopoietic stem and progenitor cells
(HSPCs) reside in specific niches that regulate
their survival, proliferation, self-renewal or differentiation in the bone marrow (BM). Stem
cells, attracted by the chemokine CXCL12,
reside in apposition to osteoblasts and endothelial cells in the BM. HSPC migration out of
the BM is a critical process that underlies modern clinical stem cell transplantation. We have
recently found that enforced HSPC egress from
BM niches depends critically on the nervous
system. UDP-galactose ceramide galactosyltransferase (Cgt) is required for the synthesis of
galactolipids in oligodendrocytes and Schwann
cells. Cgt-/- mice exhibit aberrant nerve conduction and display virtually no HSPC egress from
BM following granulocyte colony-stimulating
factor (G-CSF) or fucoidan administration. In
steady-state C57BL/6 mice, CXCL12 is expressed at high levels in bone tissues. G-CSF
dramatically suppresses osteoblast function
and downregulates bone CXCL12 in wild-type
mice. In contrast, Cgt-/- mice have constitutive
deficits in osteoblast function and resistance to
G-CSF-induced bone CXCL12 downregulation.
We present evidence suggesting that signals
from the sympathetic nervous system regulate
osteoblast function and the attraction of stem
cells to their niche.
S24
Neural Influence in the Stem Cell Niche
P. S. Frenette, 1 M. Battista, 1 W. Kao, 1 Y.
Things to Do in the Boston Area
18
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Historic Faneuil Hall Marketplace. A five-building complex that includes Quincy Market, the historic marketplace
has more than 100 places to eat, shop, and drink. French merchant Peter Faneuil (pronounced FAN-you-wull) gave the
hall where the marketplace is located to his adopted home of Boston in 1742. It has been called the Cradle of Liberty
because of the number of revolutionaries and abolitionists who delivered important speeches here. Free tours on the halfhour from 9:00 am-5:00 pm daily.
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John F. Kennedy Presidential Library and Museum. The John F. Kennedy Presidential Library and Museum is dedicated to the memory of the U.S.’s thirty-fifth president and to all those who through the art of politics seek a new and
better world. Admission is $10 for Adults, $8 for Seniors and Students (with valid college ID), $7 for ages 13–17, and
free for children 12 and under.
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Freedom Trail. Most of the Boston National Historical Park sites are connected by the Freedom Trail. Recognized as a
National Recreation Trail, the three-mile trail is a walking tour of 16 sites and structures of historic importance in downtown Boston and Charlestown. Ninety-minute tours begin at the Visitor Center at 15 State Street and cover the heart of
the Freedom Trail from the Old South Meeting House to the Old North Church. Walking tours are $12 for adults, $10
for seniors, and $6 for students (12 and under).
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Museum of Fine Arts. With approximately 450,000 objects in the collection, there’s always something new on view. The
MFA also offers an ongoing schedule of special exhibitions and daily activities including gallery talks, films, concerts,
artist lectures, and family programs. Stay as long as you like and see just what you want—from an Impressionist painting
to a 5,000-year-old mummy to a current film. Admission is $15 for adults, $13 for Seniors and Students 18 and over, and
free for 17 and under.
July 15–18, 2006
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Boston University
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Poster Abstracts
1
Human Squamous Cell Carcinoma Cell Lines
Contain a Selectable Side Population (SP) that
Is Cell Cycle Dependent and Has Cancer Stem
Cell-like Properties
K. R. Groot,1 C. Simpson,2 L. Prichard,1 D. Davies,2
S. M. Janes1; 1Centre of Respiratory Research,
University College London, London, United
Kingdom, 2Flow Cytometry Laboratory, Cancer
Research UK, London, United Kingdom
“Side population” (SP) cells are present in a number of adult tissues. These cells have a unique
capacity to efflux lipophillic DNA binding dyes
such as Hoechst 33342. In bone marrow and
muscle, SP cells have been demonstrated to have
stem cell characteristics; being able to self renew
and have the capacity to form differentiated cells.
The SP cell’s high efflux capacity correlates with
the elevated expression of drug-transporter proteins, including ABCG2. Hence these cells may
be intrinsically resistant to a number of cytotoxic
drugs and thereby contribute to tumour resistance
and disease relapse. An SP has been identified
in neuroblastomas and it has been suggested
that they are a cancer stem cell population.
Aim: We examined a squamous cell carcinoma
cell line (H357) for the presence of an SP cell
fraction and proceeded to see if it demonstrated
cancer stem cell characteristics. Methods: H357
cells were incubated with Hoechst 33342, FACS
sorted into SP and non-SP populations and
subsequently used in proliferation, colony forming and drug resistance assays. Results: SP
cells can be repeatedly FACS sorted leading to a
higher SP fraction in subsequent sorts. We show
that the SP fraction is not a strict sub-population
of cells but in fact Hoechst efflux is dependent
on the cell cycle. SP cells show a higher rate of
proliferation and colony formation than non-SP
cells. SP cells are more resistant than non-SP
cells to chemotoxic drugs such as mitotraxone.
Treatment with drug transporter inhibitors such as
verapamil reduces the survival advantage of SP
cells in the presence of mitotraxone. Conclusion:
Squamous cell cancers contain an SP population
that is selectable. SP cells are more proliferative,
have higher colony formation efficiency and have
greater resistance to chemotoxic drugs than
non-SP cells and hence have some properties
of cancer stem-like cells.
2
A Key Physical Feature of Microenvironment
That Drives MSC Differentiation
A. J. Engler, S. Sen, H. L. Sweeney, D. E. Discher;
Pennsylvania Muscle Institute and Molecular &
Cell Biophysics Lab, University of Pennsylvania,
Philadelphia, PA
Microenvironments and niches appear important
in stem cell lineage specification but can be
difficult to adequately characterize or control with
soft tissues. While it is known that pre-committed
cell types such as myoblasts will ‘feel’ the softness
of their matrix through contractile mechanisms
that influence adhesions and cytoskeleton, the
impact—if any—of matrix elasticity on naive
stem cells is unknown and key to understanding
microenvironments. Mesenchymal stem cells
are shown here to specify lineage and commit
to phenotypes with extreme sensitivity to tissuelevel elasticity, contrasting with the elasticityinsensitive commitment of differentiated cell
types. We find soft matrices that mimic brain
appear neurogenic, stiffer matrices that mimic
muscle are myogenic, and comparatively rigid
matrices that mimic collagenous bone prove
osteogenic. Myosin-II inhibition blocks this
elasticity-directed lineage specification, while
inhibition of the Rho-GTPase ROCK selectively
blocks osteogenesis. Elasticity-dependent
expression profiles indeed correlate Ras family
activators with lineage-specific transcription
factors and also highlight the adhesion-contractile
balance across lineages. The results have
significant implications for understanding physical
effects of the in vivo microenvironment.
3
WITHDRAWN
4
Creating In Vitro Niches for Hematopoietic
Stem Cells
G. Mehta,1 J. Lee,1 N. Kotov,2 J. J. Linderman,3
S. Takayama 4 ; 1 Biomedical Engineering,
University of Michigan, Ann Arbor, MI, 2Chemical
Engineering, University of Michigan, Ann
Arbor, MI, 3Chemical Engineering, Biomedical
Engineering, University of Michigan, Ann Arbor,
MI, 4Biomedical Engineering, Macromolecular
Science and Engineering, University of Michigan,
Ann Arbor, MI
Our goal is to create in vitro HSC niche to allow
basic biological studies of the roles of various
cells and growth factors in the marrow niche. One
of the first steps towards creating such a microenvironment is to successfully culture primary bone
marrow HSC supporting cells in ex vivo cultures.
We create these niches in poly(dimethyl siloxane)
(PDMS) microbioreactors. In these bioreactors,
the 3-D microenvironments of the marrow sinusoids can be simulated as there is a higher density of cells and relatively small volumes of fluids
in the vicinity. Our previous efforts to grow bone
marrow cells onto PDMS substrate have not been
successful. In this study we have used microfluidic perfusion systems to develop nanocoatings
made from electrostatic self assembly of PDDA
[poly(diallyldimethyl ammonium chloride)], clay,
type IV collagen and fibronectin to attach primary
murine bone marrow onto PDMS bioreactors.
Our microfluidic nanocoating process has many
advantages over popular traditional layer-by-layer
methods: Computerized micropumps and valves
afford greater flow control, automatic processing is possible with programmable software,
customizable nanopatterns with different coating
compositions can be created in different microchannels at the same time, and lesser amounts
of polymers and protein solutions are needed. We
have cultured primary bone marrow cells in bio-
reactors with no coatings (negative control) and
bioreactors with four different nanocoatings. The
adherent cells of marrow attached and spread on
nanocoated PDMS microchannels for five days.
We are working on developing long term culture
of the supporting stroma in PDMS bioreactors.
Once a stromal niche is established inside the
PDMS bioreactor, HSCs can be cultured and
studied in them.
5
In Vivo Inhibition of Wnt through Dkk1
Overexpression in the Endosteal Niche
Suppresses Hematopoietic Stem Cell
Function
H. E. Fleming, 1,2 V. Janzen, 1,2 J. Guo, 3 H.
M. Kronenberg, 3 D. T. Scadden 1,2 ; 1 Center
for Regenerative Medicine, Massachusetts
General Hospital, Boston, MA, 2Harvard Stem
Cell Insititute, Harvard University, Cambridge,
MA, 3Endocrine Unit, Massachusetts General
Hospital, Boston, MA
Wingless (Wnt) is a potent morphogen demonstrated in multiple cell lineages to promote
the expansion and maintenance of stem and
progenitor cell populations. Exposure to Wnt proteins or manipulations to activate its downstream
signaling cascade promote the ex vivo expansion
of hematopoietic stem cells (HSC) and improve
their engraftment potential in a transplant setting.
However, the impact of Wnt signaling in vivo has
yet to be fully explored. Dickkopf1 (Dkk1) is a
natural inhibitor of the Wnt cascade, as it binds
to the Wnt coreceptor LRP5/6 and promotes its
internalization. We have assessed the role of Wnt
signals on HSCs in vivo by transgenic expression of Dkk1 targeted to the hematopoietic niche
using an osteoblastic promoter. Assessment
of the transgenic mice revealed no significant
differences in white blood cell counts, total BM
cellularity or representation of mature cell lineages in the steady state. In contrast, analysis
of the primitive hematopoietic compartment
demonstrated that transgenic mice harbored a
2-fold reduced LKS-CD34lo (cKit+,Sca1+,Lineage,CD34lo) population, known to contain long-term
HSCs. Primitive progenitor cells were also found
to be reduced based on a lower frequency of in
vitro colony forming cells (CFCs). Stem cells
from transgenic or control mice were assessed
by competitive repopulation of irradiated wildtype
hosts. Bone marrow cells isolated from a Dkk1expressing microenvironment were less competitive (11 wk peripheral reconstitution = 25.4%),
relative to littermate controls (56.4%; p<0.025).
Interestingly, initial reconstitution by transgenicderived cells was also reduced (4 wk peripheral
reconstitution = 27.0%), compared to wildtype
(47.8%; p<0.002), indicating that both short and
long-term repopulating cells were decreased in
number in the setting of Dkk1 overexpression
by osteoblasts. These results suggest that Wnt
proteins may be active in vivo, and inhibiting Wnts
in the endosteal niche by Dkk1 decreases the
quality and/or quantity of HSCs.
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6
A Stem Cell Niche for Muscle Satellite Cells
S. Kuang, K. Kuroda, M. A. Rudnicki; Molecular
Medicine, Ottawa Health Research Institute,
Ottawa, ON, Canada
Muscle satellite cells are adult stem cells responsible for postnatal growth, repair and maintenance of skeletal muscles. However, the stem cell
niche and mechanism of satellite cell self-renewal
have not been elucidated. Here, we report two
important stem cell characteristics of satellite
cells: the existence of hierarchal subpopulations
of phenotypical, behavioral and functional distinct
satellite cells within the satellite cell niche, and the
asymmetric self-renewal of satellite cells in vivo.
The homeodomain transcription factor Pax7 and
bHLH family myogenic regulatory factor Myf5 are
specific satellite cell markers. Genetic analysis
of satellite cell lineage revealed that satellite cell
compartment contains 88% Pax7+/Myf5+ and
12% Pax7+/Myf5- cells. In vitro and in vivo clonal
assays demonstrated that the Pax7+/Myf5- subpopulation represent a stem cell reservoir that
self-renew and give rise to Pax7+/Myf5+ satellite
cells through asymmetric division. The fate of
daughter cells is determined by their relative position within the satellite cell niche. Furthermore, we
established parameters to perspectively isolate
Pax7+/Myf5+ and Pax7+/Myf5- satellite cells with
FACS and confirmed the distinct gene expression
profiles of these subpopulations. Finally, the stem
cell function of the Pax7+/Myf5- subpopulation is
demonstrated by their capability to occupy satellite cell niche in host muscle after transplantation
and by their improved efficiency in the restoration
of dystrophin expression in MDX mice, a mouse
model for Duchenne’s muscular dystrophy. These
results provide novel insights into the biology of
satellite cells and open new avenues for therapeutic treatment of muscular diseases.
7
3D Nanostructured Microenvironments for
In Vitro Replication of Hematopoietic Stem
Cells Niches
J. Lee,1 J. Bahng,1 M. Cuddihy,2 N. Kotov1,2,3;
1
Biomedical Engineering, University of Michigan,
Ann Arbor, MI, 2Chemical Engineering, University
of Michigan, Ann Arbor, MI, 3Material Science
& Engineering, University of Michigan, Ann
Arbor, MI
Multiple studies indicate that an engineered 3D
environment can control cellular function and
development. The aim of our study is to develop
fairly universal methods for in vitro replication
of the differentiation niches of HSCs by using
research tools from nanotechnology. As model
systems we are aiming at replication of 3D bone
marrow and thymus niches for HSCs differentiation into B-cells and CD4 T cells. For this purpose,
inverted colloidal crystal (ICC) 3D scaffolds,
which consist of interconnected spherical cavities
arranged in an ordered hexagonal crystal lattice,
were constructed from hydrogel. Highly-interconnected cavities facilitate nutrient transport and
provide large surface area for cell adhesion.
Additionally, the spherical chambers force extensive cell-cell/-matrix interactions by partially
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restricting free movement of floating cells. To
mediate the well-known problem of poor cell
adhesion characteristics for hydrogel materials,
the ICC scaffold surface was modified utilizing
layer-by-layer (LBL) assembly. LBL results in the
formation of a nanostructured clay-polyelectrolyte
composite, providing a biocompatible coating
suitable for adhesion of epithelial, stromal, and
other cells. The nanocomposite is transparent,
has strong mechanical coupling with hydrogel,
and offers nanoscale surface roughness and
sufficient Youngs modulus for cell adhesion.
Additionally, layers of bioactive proteins defining the niche functionalities can be uniformly
deposited on the scaffolds with minimal loss of
bioactivity. Five bilayers of clay and poly(diallyld
imethylammonium chloride) and one bilayer of
Delta-like-1(DL-1) Notch ligand (provided from
Prof. Irwin Bernstein at Fred Hutchinson Cancer
Research Center), one of the essential insoluble
signaling factors determining HSC developmental fate, were coated on ICC scaffolds. Human
CD34+ HSCs were successfully cultured in the
system. Surface marker characterization under
a confocal microscope supports that LBL coated
DL-1 ligand layer guided HSC differentiation
pathway into CD4 T cell lineage. The results of
this study show that one can potentially simulate
differentiation niches for different components of
hematopoietic system.
8
Development of In Vitro Human Bone Marrow
Niches Using 3D Inverted Colloidal Crystal
Scaffolds
J. Lee,1 S. Wang,2 J. A. Niles,3 J. Bahng,1 S.
Warner,4,5 R. Mlcak,4,5 E. H. Fleming,3 J. Cortiella,4
J. E. Nichols,3,4,6 N. A. Kotov1,7,8; 1Biomedical
Engineering, University of Michigan, Ann Arbor,
MI, 2Nomadics Inc, Stillwater, OK, 3Internal
Medicine, University of Texas Medical Branch,
Galveston, TX, 4Anesthesiology Laboratory of
Tissue Engineering and Organ Regeneration,
University of Texas Medical Branch, Galveston,
TX, 5Shriner Burns Hospital, University of Texas
Medical Branch, Galveston, TX, 6Microbiology
and Immunology, University of Texas Medical
Branch, Galveston, TX, 7Chemical Engineering,
University of Michigan, Ann Arbor, MI, 8Material
Science & Engineering, University of Michigan,
Ann Arbor, MI
Numerous experimental results prove that a 3D
cell culture system offers a more realistic microand local-environment where the functional properties of cells can be observed and manipulated.
The objective of this study is to develop in vitro
human bone marrow niches for HSC self-renewal
and differentiation into B-lymphocytes. Human
bone marrow consists of an extensive network
of large sinusoids lined by stromal cells and surrounded by reticular fibers. To mimic this 3D cellular environment, we are using a 3D scaffold with
inverted colloidal crystal (ICC) geometry lined by
HS-5 human bone marrow stromal cells. The open
geometry of the ICC, along with the exceptionally
high porosity (74%) and large surface area and
extensive interconnectivity is similar to the topology of an actual bone marrow matrix. Additionally,
the spherical shape of the cavities in ICC creates
a supportive microenvironment which helps to
nurture and support the HSCs residing within
them. Silicate and hydrogel ICC scaffolds were
used. To improve stromal cell adhesion on hydrophilic hydrogel scaffolds, the surface was coated
with a thin layer of clay-polyelectrolyte composite
through a layer-by-layer method. Stromal cells
were allowed to form a dense layer of cells on
the ICC scaffold surface, strongly reminiscent of
the reticular network existing in the bone marrow,
prior to the seeding of HSCs. CD34+HSCs were
cultured in 3D ICC scaffolds and 2D well plates
for up to 40 days. Their self-renewal and B cell
differentiation were characterized by using lineage
specific markers and by detection of secreted immunoglobulin. 3D cultures consistently showed a
significantly higher presence of CD34+/CD150+
cells, and CD40+ cells with secreted immunoglobulin compared to 2D cultures. Our in vitro
artificial bone marrow niches will contribute to the
understanding of the complex mechanism of HSC
self-renewal and B cell differentiation not only in
2D but also in 3D.
9
cAMP-PKA Signaling Involvement in
Regulation of Adipogenesis, Osteogenesis
and Osteoclast-inducing Activity of Human
Mesenchymal Stem Cells
S. Hung, D. Yang; Medical Research and
Education, Veterans General Hospital-Taipei,
Taipei, Taiwan
Bone remodeling has long been considered to
be controlled by osteoclastic bone resorption and
osteoblastic bone formation. It is possible that
other cells are involved in bone remodeling. Bone
marrow mesenchymal stem cells (MSCs) have the
capacity of self-renewal and can differentiate into
several distinct cell types, including osteoblasts
and adipocytes. Human MSCs underwent adipogenesis in adipocyte induction medium (AIM) with
IBMX, a phosphodiesterase inhibitor, which was
reported to stimulate cAMP-dependent protein
kinase activity (cAMP-PKA). In current study,
we further demonstrated that PKA stimulators
when added in AIM enhanced the differentiation
of KP-hMSCs into adipocytes with an increase
in Oil-Red O staining and mRNA expression of
markers for adipocyte (PPARγ2 and LPL). On the
other hand, PKA inhibitors when added in AIM
resulted in an increase in mRNA expression of
markers for osteoblast (Runx2 and osteopontin).
PKA modulators also caused a change in mRNA
expression of receptor activator of NF-B ligand
(RANKL) and osteoprotegerin (OPG), which
coordinate together to play a critical role in the
regulation of osteoclast formation. We further
found that the secretion of leptin and the mRNA
expression of leptin by KP-hMSCs decreased as
the addition of PKA stimulators and increased
as the addition of PKA inhibitors. Interestingly,
exogenous leptin blocked forskolin effects on
adipogenesis and osteogenesis in a dose-dependent manner, suggesting an important role
of leptin in mediating the effects of cAMP-PKA
in regulating MSC differentiation. Transfection of
Kp-hMSCs with DN (dominant negative) CREB
further blocked the adipogenic effect of forskolin
in AIM, but stimulated the mRNA expression of
July 15–18, 2006
leptin and Runx2. From these results, we propose
a new bone metabolic unit which highlights the
role of MSCs, upon which cAMP-PKA signaling
determines the constitution of the cellular components by regulating adipogenesis, osteogenesis
and osteoclast-inducing activity via controlling the
release of leptin from MSCs.
10
Vascularization of Developing NSC Niches
A. E. Falender,1 M. K. Moore,1 M. D. Garcia,2 I. V.
Larina,2 R. Lovell-Badge,3 M. E. Dickinson,2 K. K.
Hirschi1; 1Center for Cell and Gene Therapy, Baylor
College of Medicine, Houston, TX, 2Department
of Molecular Physiology and Biophysics, Baylor
College of Medicine, Houston, TX, 3Division of
Developmental Genetics, MRC National Institute
for Medical Research, London, United Kingdom
Objective: To define spatial relationships, and
signaling pathways, between vascular cells of
the perineural vascular plexus (PNVP) and neural
stem cells (NSC) within embryonic NSC niches.
Background: During development, the vascular
and neural systems are patterned in parallel; thus,
it is not surprising that NSC are in close proximity to
vascular endothelial cells (EC). The blood vessels
associated with the central nervous system (CNS)
arise from the PNVP that forms around the neural
tube E8.5-10.5. Initially, CNS microvasculature
is composed of EC-lined tubes; by E12.0, mural
cells are recruited to form the surrounding vessel
wall (Nakao et al. 1988). Smooth muscle α actin
(SMAA)-expressing mural cell precursors are
recruited by EC via platelet-derived growth
factor (PDGF)-BB signaling (Hirschi et al. 1998),
and reciprocally via mural precursor-derived
angiopoietin-1 (Suri et al. 1996). Methods: To
define the spatial relationships between vascular
cells of the PNVP and NSC during development,
E8.0-14.5 embryos were immunostained for
markers of EC (CD31), mural cells/precursors
(SMAA) and NSC (Sox2). Stained tissues were
analyzed via confocal microscopy and 3D image
reconstruction. To verify gene expression within
NSC, RNA was isolated from neurospheres
derived from E14.5 tissues, and analyzed via
RT-PCR. Results: At E8.5, the developing
neural tube and brain were not vascularized and
contained Sox2+ cells that co-expressed SMAA.
At E9.5, EC tubes had invaginated the spinal cord
and brain; interestingly, the Sox2+ cells closest
to EC no longer expressed SMAA. Similarly, at
E14.5, after the NSC niche is established, all
Sox2+ cells except those closest to EC expressed
SMAA. E14.5 NSC in vitro expressed SMAA, as
well as Ang-1 and PDGFRβ, but not EC markers
CD31 or VE-cadherin. Conclusions: Prior to
vascularization, NSC are phenotypically similar
to mural cell precursors, and may interact with
invading EC via analogous signaling pathways
to establish NSC niches.
11
WITHDRAWN
12
Biological Activity of Stem Cell Factor on
Zebrafish ES and EG Cell Cultures and
Targeted Insertion of Plasmid DNA into the
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Boston University
Cells by Homologous Recombination
J. Moon, P. Collodi; Animal Sciences, Purdue
University, West Lafayette, IN
Stem cell factor (SCF) has diverse biological
activities on a variety of cell types in culture. In
vivo studies have shown that mutant strains of
mice that lack SCF or its receptor exhibit defects
in hematopoiesis, gametogenesis and melanogenesis. Zebrafish possess two SCFs that are
encoded by different genes, however very little
information is known about their biological activities. In this study, we examined the biological
activity of each zebrafish SCF isoform (A and B)
on in vitro cultures of pluripotent embryonic stem
(ES) cells that were initiated from early-stage
zebrafish embryos and embryonic germ (EG)
cells derived from vasa::RFP transgenic fish.
The results showed that feeder cells expressing
SCF- a and b both stimulated ES cell growth
in culture compared to non-transfected feeder
cells. Also, the results of in situ hybridization and
RT-PCR analysis revealed that zebrafish ES in
the presence of SCF-a express germ cell-specific genes including nanos, vasa, cxcr4b for a
longer period of time in culture. To examine the
gene targeting potential of our cultures, targeting
vectors were designed to insert into and disrupt
the zebrafish myostatin I gene in the ES cell
cultures. The targeting vector was introduced
into the cultures by electroporation and colonies
of homologous recombinants were isolated using
two approaches. Postive-negative selection was
conducted using both the neomycin resistance
(neo) and the diphtheria toxin (dt) genes together
on the vector. Six colonies were isolated from
neomycin resistant colonies and confirmed to be
homologous recombinants by PCR and Southern
blot analysis. In conclusion, these results demonstrate that SCF can be used to optimize the
culture conditions for zebrafish ES and EG cells.
The ES cells possess the capacity to undergo
homologous recombination at a frequency that
will make them suitable for use in a gene targeting approach.
13
Differential Hematopoietic Supportive Activity
of OP9-derived Osteoblastic and Adipocytic
Stroma
O. Naveiras,1,2 V. Nardi,1 P. Sharma,3 P. Hauschka,3
G. Q. Daley 1,4 ; 1 Division of Hematology/
Oncology, Children’s Hospital, Boston, MA,
2
Graduate Program of Biological and Biomedical
Sciences, Harvard Medical School, Boston,
MA, 3 Or thopaedic Surger y and Oral and
Developmental Biology, Children’s Hospital,
Boston, MA, 4Department of Biological Chemistry
and Molecular Pharmacology, Harvard Medical
School, Boston, MA
Adult hematopoietic stem cells (HSCs) reside
in the bone marrow, where they exhibit the
unique ability to either self-renew or differentiate
into more committed progenitors. The extent to
which this cell fate decision is cell-autonomous
or determined by the HSC microenvironment
is still an open question. In the bone marrow,
osteoblasts constitute a functional niche providing signals for HSC self-renewal: Jagged,
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N-cadherin, Angiopoietin-1 and possibly BMPs.
HSC interactions with the endothelium have also
been described, although the significance of this
alternative niche is still unknown. In addition to
hematopoietic cells, endothelial cells and osteoblasts, adult bone marrow contains numerous
adipocytes. It is well known that the amount of
bone marrow fat is indirectly proportional to the
hematopoietic activity of the marrow. Whether
adipocytes act as mere space-fillers in this context or whether they have a direct effect on HSCs
or other downstream hematopoietic progenitors
has not been explored. In order to determine
the potential role of bone marrow adipocytes in
hematopoiesis, bone marrow-derived OP9 cells
were induced to differentiate into either osteoblastic or adipocytic stroma, serving as surrogate
in vitro HSC niches for progenitor-enriched bone
marrow cell preparations. The proliferation, CFUactivity, and long term repopulation ability of adult
HSCs cultured in this stroma was measured,
suggesting that in these conditions the presence
of bone marrow-derived adipocytes suppresses
the expansion of short-term HSCs but does not
interfere with long-term HSC support. Ongoing
experiments will determine whether this model
holds true when the bone marrow adipocyte
compartment is manipulated in vivo.
14
Cellular Analyses of the Mitotic Region and
Its Distal Tip Cell Niche in the C. elegans Adult
Germ Line
S. L. Crittenden,1 D. T. Byrd,2 K. A. Leonhard,2 J.
Kimble1; 1Biochemistry, University of Wisconsin–
Madison and HHMI, Madison, WI, 2Biochemistry,
University of Wisconsin–Madison, Madison, WI
The C. elegans germ line provides a model for
understanding how signaling from a stem cell
niche promotes continued mitotic divisions at the
expense of differentiation. Here we report cellular analyses designed to investigate the niche
and to identify germline stem cells within the
germline mitotic region of adult hermaphrodites.
Our results support several conclusions. First, all
germ cells within the mitotic region are actively
cycling, as visualized by BrdU labeling. No quiescent cells were found. Second, germ cells in
the mitotic region lose BrdU label uniformly, either
by movement of labeled cells into the meiotic
region or by dilution, probably due to replication.
No label-retaining cells were found in the mitotic
region. Third, the distal tip cell niche extends
processes that nearly encircle adjacent germ
cells, a phenomenon that is likely to anchor the
distal-most germ cells within the niche. Fourth,
germline mitoses are not oriented reproducibly,
even within the immediate confines of the niche.
We propose that germ cells in the distal-most
rows of the mitotic region serve as stem cells and
more proximal germ cells embark on the path to
differentiation. We also propose that C. elegans
adult germline stem cells are maintained by
proximity to the niche rather than by programmed
asymmetric divisions.
15
Osteoblasts as Regulators of Adult Human
Mesenchymal Stem Cell Osteogenesis
21
July 15–18, 2006
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Boston University
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C. M. Kolf,1,2 L. Song,2 R. S. Tuan2; 1Department
of Biology, Johns Hopkins University, Baltimore,
MD, 2Cartilage Biology and Orthopaedics Branch,
National Institute of Arthritis and Musculoskeletal
and Skin Diseases, Bethesda, MD
Objective: To determine the role of osteoblasts
(OBs) in mesenchymal stem cell (MSC) osteogenesis. Mesenchymal stem cells are located
within a mostly uncharacterized niche in the bone
marrow (BM). Understanding how this niche acts
to regulate MSCs could help us harness their
therapeutic potential. Osteoblasts may be crucial
to the niche since they are a principal constituent
of the BM and are important in maintaining the
“stemness” of the other stem cells that reside
there—the hematopoietic stem cells (HSCs).
Methods: To analyze how OBs may regulate
MSCs, we monitored MSC osteogenesis in the
presence of OBs. Human BM-derived MSCs and
OBs were cocultured in a 1:1 ratio for 12 days.
Promoter-reporter assays and real-time PCR
were used to analyze the osteogenic progression of the MSCs. Cells were either cultured in
direct contact with each other or separated by
Transwell inserts that allowed cellular communication through protein permeable membranes
but prevented direct cell contact. In the mixed
cultures, MSCs were pre-labeled with the fluorescent dye, DiI, to allow their fractionation by
FACS post-coculture. Results: When OBs were
in direct contact with MSCs, the MSCs tended to
express higher amounts of osteogenic markers
while undergoing osteogenesis as compared
to the MSC-only controls. When OBs were only
allowed to communicate with the MSCs via secreted factors, MSCs expressed lower amounts
of osteogenic markers. Conclusion: Our data
show that cell contact is a key regulator of the
interaction between OBs and MSCs. OBs seem
to normally act from a distance to maintain MSCs
in their naïve state but occasionally they may act
as ports to which the MSCs must be docked in
order to undergo a robust maturation into OBs.
Future tests to identify the factors involved will be
key to understanding the regulation of stem cells
by their microenvironments.
16
Who Are the Players in the Neighborhood:
Signaling Pathways in the Hematopoietic
Stem Cell Niche
H. Paz,1 H. Shafizadeh,2 M. Lynch,2 U. Ganapati,2
J. C. Gasson3; 1Molecular Biology IDP, University
of California, Los Angeles School of Medicine, Los
Angeles, CA, 2Division of Hematology-Oncology,
University of California, Los Angeles School
of Medicine, Los Angeles, CA, 3Department of
Biological Chemistry, University of California, Los
Angeles School of Medicine, Los Angeles, CA
The ability of hematopoietic stem cells (HSC)
to either self-renew or differentiate depends on
both intrinsic cues and extrinsic cues from the
neighboring microenvironment or bone marrow
(BM) niche. The niche is composed of a heterogeneous population of cells including fibroblasts,
adipocytes, endothelial cells, and osteoblasts.
The Notch gene family has been implicated in
the self-renewal and commitment decisions of
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the HSC and recent literature suggests that HSC
adhere to osteoblasts via the Notch receptor and
Jagged ligand interaction. To explore the function
of the Notch in hematopoiesis, we used a co-culture system wherein murine E14Tg2a embryonic
stem cells, which express an inducible ligand-independent, constitutively active form of Notch1,
were grown on a confluent layer of OP9 stromal
cells (Era and Witte, PNAS, 97; 1737–1742,
2000). The OP9 cells provide a favorable microenvironment such that they induce the embryonic
stem cells to differentiate into hemangioblasts
and subsequently into hematopoietic precursors.
Previous studies indicate that activation of Notch
signaling in the Flk+ hemangioblast results in the
maintenance of hematopoietic progenitors in an
immature state. In order to further elucidate the
intrinsic and extrinsic molecular mechanisms
associated with Notch mediated self-renewal,
parallel microarray experiments were performed
on co-cultured hematopoietic precursors and
OP9 cells. Our studies indicate that activation of
Notch signaling in the hemangioblast resulted
in a reciprocal increase in Jagged expression
in the OP-9 cells. Moreover, the downstream
targets of Notch, Hey 1, Hey 2, and Dltx1 were
also upregulated in the OP-9 cells, indicating
there is cross-talk between the hematopoietic
precursors and the stroma. Finally, OP9 cells
exhibited an upregulation in members of the Wnt
and TGF-superfamilies upon Notch1 activation.
Overall, these studies yield new insights into the
signaling pathways associated with the stem cell
niche. In the future, this information might aid out
understanding of cancer stem cells.
17
Prospective Isolation of the Cellular
Component of the Hematopoietic Stem Cell
Niche in the Murine Foetal Liver
N. Brouard, D. Blashki, M. Liu, P. J. Simmons;
Stem Cell Laboratory, Peter MacCallum Cancer
Centre, East Melbourne, Australia
A well-documented feature of the hematopoietic
system in mammals is the sequential migration of
hematopoietic stem cells (HSC) to anatomically
distinct sites during ontogeny: from the paraaortic
splanchnopleura region, to the foetal liver (FL),
to the bone marrow. Underlying this ontogeny
related migration is the development of HSC in
discrete stem cell niches at each anatomical location. Currently the best characterized HSC niche
is that present within the endosteal region of the
adult bone marrow. In contrast little is known about
the cellular composition or location of the HSC
niche in foetal liver. We hypothesized that a variation of the methodology we previously described
to prospectively isolate the cellular components
of the HSC niche from adult mouse bone tissue
could be applied to isolation of the putative HSC
niche in the foetal liver. E13.5 foetal livers were
disaggregated by enzymatic digestion, depleted
of leukocytes, erythroid and vascular endothelial
cells and positively selected using CD51 and
CD106 antibodies. CD45-CD31-TER119-CD51+
CD106+ cells exhibited a fibroblast-colony forming cells (CFU-F) frequency of 1/300 while the
CD45-CD31-TER119-CD51+CD106- subpopulation exhibited a CFU-F incidence of 1/40. To
investigate the capacity of these 2 populations
to support HSC in vitro, stromal layers derived
from the CD45-CD31-TER119-CD51+CD106and CD45-CD31-TER119-CD51+CD106+ sorted
cells were co-cultured with Lin-Sca1+Kit+ (LSK)
HSC under serum-free conditions. Despite
lower CFU-F frequency and proliferative potential, stromal cell layers derived from the LinCD51+CD106+ fraction were significantly more
efficient in supporting hematopoiesis in vitro
in presence or absence of exogenous growth
factors (SCF, Flt3L, IL-6, IL-11). In addition, the
Lin-CD51+CD106- fraction markedly suppressed
the proliferation of LSK cells in response to this
combination of cytokines. In conclusion, we have
identified two phenotypically and functionally
distinct populations of stromal elements in mouse
foetal liver which we hypothesize represent cellular constituents of the HSC niche at this stage
of hematopoietic ontogeny.
18
Analysis of the Hematopoietic Stem Cell
Niche of the Aorta-Gonads-Mesonephros
Region
M. Judex,1 J. Renstroem,1 R. Lang,2 J. Mages,2
C. Peschel,1 R. A. J. Oostendorp1; 1Department
of Internal Medicine, Klinikum rechts der Isar,
München, Germany, 2Department of Microbiology
and Immunology, Klinikum rechts der Isar,
München, Germany
During mouse development, definitive hematopoiesis emerges from the embryonic aorta-gonads-mesonephros (AGM) region. The molecular
mechanisms governing emergence, maintenance
and expansion of hematopoetic stem cells (HSC)
from the AGM region are still unclear. We recently
described the generation of a number of cell lines
from subregions of the midgestation AGM region
and investigated their ability to maintain HSC in
culture. From this analysis, two cell lines, EL081D2 and UG26-1B6, were found to maintain highly
enriched repopulating cells. What sets these cell
lines apart from cell lines from other embryonic
or adult tissues is that, apparently, the midgestation niche does not require direct contact with the
HSC in order to transmit maintenance signals. To
identify molecules involved in this maintenance,
we performed a detailed analysis of the two cell
lines and compared these with four midgestation
embryonic-derived cell lines which do not maintain
HSC in culture. Our analyses show that the six cell
lines share 98% of the present calls in the affymetrix 430 array. In total, only 250 tags (1.8% of total)
were in fact expressed in the two supportive cell
lines, but not in the four non-supportive cell lines.
These tags included only 8 secreted molecules,
which included two chemokines, two members of
the TGFβ family, a protease, an interleukin, and a
tyrosine kinase receptor ligand. We are currently
knocking down the expression of several differentially expressed genes to investigate their role
in the support of HSC maintenance. In addition,
we are identifying interaction partners of these
stromal molecules using a yeast two-hybrid library
constructed from Lin-Kit+ marrow cells. This
preliminary analysis has, so far, revealed three
interaction partners which are involved in cell
signalling, protein sorting and cell division. Using
July 15–18, 2006
this approach, we hope to identify stroma-HSC
interaction partners which are involved in HSC
maintenance on AGM-derived cell lines.
19
Self-Renewal and Differentiation of Adult
Stem Cells
J. Kang; Bioproduct Technical Support Division,
Korean Food & Drug Administration, Seoul,
Republic of Korea
Adult stem cells (ASCs) are defined functionally
as cells that have the capacity to self-renew
as well as the ability to generate differentiated
cells. Self-renewal of stem cells is achieved by
symmetrical cell division while maintaining pluripotency. In order not to exhaust ASCs throughout
the lifespan of the organism, most ASCs remain
quiescent and only a limited number enter the
cell cycle. In this study, the cell cycle was crucially
regulated by chromatin modification drugs such
as histone deacetylase (HDAC) inhibitor valproic
acid and DNA methylation inhibitor 5-azacytidine.
In addition, intrinsic transcription factor Bmi-1
controled their growth through their effect on
gene transcription. In terms of the particular roles
in regulation of the cell-cycle, the cyclin-dependent kinase inhibitor (CDKI), p21cip1/waf1, p27kip1,
p16INK4A, and p19ARF were shown to maintain the
quiescence of ASCs, thereby governing their
available pool sizes. These results make evident
that appropriate cell cycle control, particularly
at the early stage of stem/progenitor cells, is required for maintaining normal stem cell kinetics.
20
Effector T Cells (ETC) against Normal Tissue
Induce Transdifferentiation of MSC to Different
Cell Lineage Related with the Specificity of
the ETC
G. A. Moviglia,1,2 G. Varela,1 J. Saslavsky,1 C.
Gaeta,1 M. T. Moviglia-Brandolino,1 C. F. Bastos1;
1
Regina Mater Foundation, Ciudad de Buenos
Aires, Argentina, 2Universidad de Maimónides,
Buenos Aires, Argentina
Background: Continuing the work presented at
ISCT 2005 where we proved that effector T cells
(ETC) against CNS induce transdifferentiation
of MSC to NSC; we studied the influence of
ETC against different organs to induce MSC
transdifferentiation to different progenitor cell
lines. Methods: Circulating ETC against CNS,
pancreas and heart of patients with chronic
damage caused by brain hypoxia were in vitro
isolated and cocultured with autologous MSC
for 2 to 15 days. The process for obtaining MSC
and autoreactive lymphocytes was previously described: (Cytotherapy 2005, 7 [sup1]:180). MSC
were isolated from the patient’s bone marrow
and divided into 4 different groups. These groups
were used to perform 4 types of cell culture. MSC
were cocultured:
Group 1: with ETC against pancreas.
Group 2: with ETC against heart.
Group 3: with ETC against CNS.
Group 4: with unselected lymphocytes kept as
control.
Results: After 48 hours: Group 1 MSC presented
a transformation to cubical alignment of cells
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Boston University
that resembled glandular adenomera. Their cytoplasm showed several secretory granules, but
no exocytosis. Two cell types were observed in
group 2. The first presented capillary endothelial
shape. The second showed a typical myocardial
structure, intercalated disks and myocardial fibrillar differentiation. A few of these cells showed
spontaneous contraction as observed at phase
control microscope. Group 3 MSC had adopted a
spindle shape. They presented clear polarization
of their cytoplasm and important nucleus, intense
stained chromatin and one or two larger nucleolus only. Group 4 MSC remained unchanged.
Histochemical and immunohistochemical stains
were performed on each set to corroborate the
morphological findings. Conclusion: These
experiments suggest that ETC against normal
tissue have direct involvement in the physiological
transdifferentiation of MSC to different cell lineage
related with the specificity of the ETC. These
results may be the basis for further therapeutic
approaches.
21
WITHDRAWN
22
Neurospheres of Human Umbilical Cord
Blood—A Prototype of Neural Stem Cell
Niche In Vitro
M. Jurga, A. Sarnowska, A. Habich, K. DomanskaJanik; NeuroRepair, Medical Research Institute;
Polish Academy of Sciences, Warsaw, Poland
Neural stem cells (NSC) can form neurospheres
when they are isolated from fetal or adult neural
tissue. Here we described alternatively neurospheres derived from non-transformed neural
stem cell line of human umbilical cord blood
(N-HUCB). Single free-floating neurosphere
comprises heterogeneous population of neuraltype cells at various stages of their differentiation. These cells are organized in two different
regions: the inner core consisted of dormant NSC
(Nestin+GFAP-Ki67-) and the surface coat of
repopulating neuroblasts (Nestin-GFAP+Ki67+).
The aim of our study was to investigate the
mechanisms regulating growth and differentiation
of NSC in N-HUCB proposed here as a model
of NSC niche in vitro. The influence of cell-cell
interactions, extracellular matrix, mitogenes and
endothelium signaling has been addressed in
vitro and after N-HUCB transplantation on rat hippocampal organotypic culture (HOC). Results:
After serum-induced adhesion of N-HUCB
only outer cells began produce beta-tubulinIII
and MAP2 within 16h, while inner cells stay
undifferentiated even up to 3 weeks. Addition of
mitogenes (EGF or bFGF) promoted extensive
migration and proliferation of neuroblasts from
surface region of N-HUCB. This process was
inhibited by laminin and fibronectin or during coculture with endothelium-derived cells (t-END). To
verify whether this behavior would be preserved
after transplantation on recipient tissue, we examined co-cultures of neurospheres seeded on
different regions of rat hippocampus. We found
that neuroblasts from the surface of N-HUCB
integrated well with HOC neuronal network.
Noteworthy, implanted neurospheres prevented
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growth of recipient astrocytes but promoted appearance of Doublecortin+ neuroblasts originated
both from donor and recipient cells. Induced
streams of neuroblasts migrating out from implant
exhibited strong regional dependency and never
occurred in non-physiological directions (ex. CA
towards DG). Conclusion: A unique N-HUCB,
behaving in several aspects like surrogate NSC
niche, may provide novel, promising material for
experimental cell therapy. Supported by MEiN
grant no: 2P05A17729.
23
Little Evidence That Osteoblasts Interact
Directly with Hematopoietic Stem Cells
M. J. Kiel, S. J. Morrison; Cell and Developmental
Biology, University of Michigan, Ann Arbor, MI
Objective: To test whether hematopoietic stem
cells (HSCs) reside in direct contact with osteoblasts. Results: It has been proposed that
most bone marrow HSCs, identified based on
BrdU label-retention and/or N-cadherin expression localize to an “osteoblastic niche” in direct
contact with osteoblasts. However, it has not yet
been tested whether N-cadherin expressing bone
marrow cells are enriched for HSC activity, nor
whether HSCs retain BrdU. Indeed, based on the
rate at which adult HSCs enter cell cycle (4 to 8%
per day) they should not be enriched among BrdU
label-retaining cells. Although CD150+CD48CD41- cells contain essentially all of the HSC
activity within adult bone marrow and 45% of
single cells from this population give long-term
multilineage reconstitution in irradiated mice (Cell
121:1109), we have thus far found no evidence for
N-cadherin expression by these cells at the RNA
or protein levels. Moreover, bone marrow cells
that stained with the anti-N-cadherin antibody that
has been used in prior studies failed to give any
HSC activity upon transplantation into irradiated
mice. Likewise, when BrdU was administered
to mice for 10 days followed by a 70 day wash
out period (as done in prior studies), only 0.46%
of CD150+CD48-CD41- cells retained BrdU as
determined by flow cytometry. Similar results
were obtained with cells identified using other
HSC phenotypes. Conclusions: These data do
not support the idea that HSCs can be identified
based on N-cadherin expression or BrdU label
retention. This calls into question the identity of
the N-cadherin+BrdU label-retaining cells that
have been imaged interacting with osteoblasts.
These results raise the possibility that osteoblasts
influence HSC frequency via diffusible factors or
by indirect mechanisms (such as by effects on
other cells). These possibilities would be consistent with our observation that most bone marrow
CD150+CD48-CD41- cells appear to localize to
vascular niches (Cell 121:1109).
24
Defining the Hypoxic Stem Cell Niche in Bone
Marrow: Possible Role of Reoxygenation in
Homeostatic Control of Hematopoiesis after
Chemotherapy
K. Parmar,1 M. Umphrey,1 J. Clyne,1 P. Mauch,2
J. D. Down 3 ; 1 Radiation Oncology, DanaFarber Cancer Institute, Boston, MA, 2Radiation
Oncology, Dana-Farber Cancer Institute and
23
July 15–18, 2006
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Brigham and Women’s Hospital, Boston, MA,
3
Genetix Pharmaceuticals, Inc., Cambridge, MA
It is well known that most bone marrow hematopoietic stem cells (HSCs) in the adult mouse are
non-cycling under normal steady-state conditions.
Our previous experiments using Hoechst (Ho) dye
perfusion and the pimonidazole hypoxic marker
provide evidence for sequestration of HSCs to
regions of low oxygen tension in the bone marrow.
We hypothesize that the existence of HSCs in a
hypoxic microenvironment is a key determinant
in maintaining their quiescent state and confers
resistance to certain cytotoxic agents. In particular, the effects of 5-fluorouracil (5-FU) have been
extensively studied as an agent that selectively
depletes cycling progenitor populations in the
bone marrow but spares primitive HSCs. More importantly, HSCs can subsequently respond to the
initial loss of progenitors by a transition to active
proliferation. This phenomenon has encouraged
us to measure in vivo perfusion of the Ho dye to
simulate changes in oxygenation of the bone marrow after 5-FU treatment. Flow cytometric analysis
of bone marrow cells harvested 10 min after i.v.
Ho dye injection showed a wide distribution of Ho
fluorescence in untreated mice with a high concentration of primitive HSCs in the hypoperfused
regions of the marrow. The Ho gradient became
considerably shortened with increased overall
perfusion over a 6-day period that returned to
normal 14 days after 5-FU. These changes corresponded to the decrease and recovery of bone
marrow cellularity. The increased fluorescence and
shortening of the Ho gradient after 5-FU treatment
is suggestive of improved oxygenation of HSCs.
We speculate that prior 5-FU treatment has the
effect of destroying oxygen-consuming and metabolically active progenitors allowing reoxygenation
of the HSC niche. Thus an increase in the oxygen
tension in the bone marrow microenvironment
during perturbed hematopoiesis may play an
essential role in controlling HSC proliferation in
response to injury.
25
Extrinsic Regulation of Human Retinal
Progenitor Cell Proliferation by Retinal
Pigmented Epithelium (RPE) Secreted
Factors
L. S. Wright, R. L. Shearer, J. N. Melvan, C. N.
Svendsen, D. M. Gamm; Waisman Center Stem
Cell Research Program, University of Wisconsin,
Madison, WI
Objective: To examine the effect of factors secreted by human prenatal RPE on the proliferation
and long-term growth of human prenatal retinal
progenitor cells (hRPCs). Methods: RPE and
hRPCs were isolated separately from human
prenatal retinas (54-120 days post-conception).
Confluent monolayer RPE cultures were maintained in serum-free media supplemented with
2% B27. hRPC were grown and passaged as
retinal neurosphere suspensions in media supplemented with 2% B27, EGF, FGF2 and heparin ±
conditioned RPE media (RPE CM). Growth and
proliferation rates were determined by measuring
changes in sphere volume, BrdU incorporation
and Ki67 immunostaining. CREB phosphoryla-
24
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tion studies utilized long-term hRPC cultures that
were starved for mitogens for 24 hr and then rechallenged for 7 min prior to fixation and probing
with phospho-CREB antibody. Centricon filtration
devices with various kDa cut-offs were used to
fractionate RPE CM components. Results: hRPE
CM increased hRPC neurosphere growth and
proliferation, but demonstrated no growth-promoting effect on neurospheres generated from other
neural tissues. hRPC grown long-term in RPE
CM expressed nestin and sox2 and displayed a
predicted, progressive change in cell fate potential over time. RPE CM challenge following 24 hr
mitogen starvation demonstrated that CM could
lead to CREB activation, and therefore, may exert
its effects through the EGF and/or FGF2 signaling
pathways. A protein fraction with growth-promoting
effects on hRPC neurospheres was then partially
purified from RPE CM. Conclusions: 1. Addition
of RPE CM selectively enhances hRPC proliferation without alteration of cell fate. 2. The effect of
RPE CM appears to be mediated by secreted
protein factor(s) that activate the EGF and/or FGF
signaling cascades. 3. Thus, RPE may influence
the proliferative capacity of adjacent embryonic
hRPCs via a paracrine mechanism.
26
The Influence of Pum2 RNA-binding Protein
on HSC Maintenance, Activation Status,
Differentiation and CD34 Expression
J. Yang, K. DiVito, R. Nachtman, R. Jurecic;
Microbiology and Immunology, University of Miami
School of Medicine, Miami, FL
Over-expression of RNA-binding protein Pum2
leads to increased maintenance and attenuated
mutilineage differentiation of murine hematopoietic stem cell (HSC) line EML. To analyze further
the biology of EML cells over-expressing Pum2
(Pum2-EML cells), we examined the expression
of c-kit, Sca-1, Flk-2 and CD34 HSC markers on
EML and Pum2-EML cells. Almost all EML and
Pum2-EML cells are Sca-1+ c-kit+ Flk2-, but exhibit
heterogeneous expression of CD34, with ~80% of
EML cells being CD34+. In contrast, Pum2-EML
cells exhibited a 3-fold increase in the frequency
of CD34- cells (~60%). Functionally, the CD34+
population of EML and Pum2-EML cells contains
the majority of cells capable of mutilineage differentiation in response to cytokines, whereas
the CD34- population differentiates poorly or not
at all. The CD34+ and CD34- EML cells differed
in their morphology as well, with the majority of
CD34+ cells having irregular oval shape with short
pseudopodia, and the majority of CD34- cells being
round. Several studies have shown that mouse
CD34- HSC differentiate poorly, and become
CD34+ after mobilization with G-CSF or treatment
with 5-fluorouracil. However, after transplantation, these “activated” CD34+ HSC revert back to
CD34- “inactive” state. In view of these findings our
results suggest (a) that CD34- EML cells resemble
quiescent HSC that differentiate poorly or not at all,
whereas CD34+ cells could represent “activated”
stage at which they can undergo differentiation,
and (b) that Pum2 over-expression leads to an
increase of the CD34- EML cell population. Since
the CD34 is involved in HSC migration and adhesion, and primary CD34+ cells are known to form
pseudopodia, different morphology of CD34+
and CD34- EML cells could reflect their different
migratory capacity and differential interaction with
hematopoietic niches. Our results contribute further to the notion about the putative link between
HSC activation status, readiness to differentiate
and CD34 expression.
27
Comparative Global Histone Methylation
Profile of Membrane Kit Ligand-deficient (Sld/
Sld) and Wild Type Adult Male Mouse Germline
Stem Cells
M. Seandel,1,2 S. Rafii2; 1Medicine, Memorial Sloan
Kettering Cancer Center, New York, NY, 2Genetic
Medicine, Weill Cornell Medical College, New
York, NY
Epigenetic signals are thought to play a major role
in stem cell fate decisions. However, the epigenetic
profiles of specific adult mammalian stem cell
populations in vivo are poorly characterized.
Histone methylation has been implicated as major
component in context-dependent transcriptional
activation and repression. Objectives: We sought
to characterize the global histone methylation
profile of adult mammalian spermatogonial stem
cells both in vivo using histologic means and in vitro,
using a long term culture method. We hypothesize
that adult germline stem cells display a global
epigenetic signature distinct from differentiated
spermatogenic cells. Methods: Adult wild type or
homozygous Steel Dickie (Sld/Sld) mouse testes
were harvested for paraffin embedding. Long term
cultures of adult spermatogonial stem cell-like cells
were derived from 2 to 7 month old mouse testes
using enzymatic dissociation of tubular tissues
and propagated on mitomycin-treated feeder
cells. Immunohistochemistry was performed using
rabbit polyclonal antibodies against specific diand tri-methylated histone lysines and arginines
(H3K9Me2, MeH3K9Me3, H4R3Me2, H3R17Me2,
H3K4Me2, H4K20Me2, H4K20Me3) and selected
arginine methyltransferases (PRMT-1 and PRMT5). The profile of histone methyl modifications
was similarly characterized for the cultured
stem cell-like cells. Results: Certain histone
methyl marks appear to preferentially delineate
spermatogonia in vivo (e.g., H3K9Me2), whereas
the same cells display relatively lower levels of
other marks (e.g., H4R17Me2). Adult Sld/Sld testes
display residual undifferentiated spermatogonia
and spermatogonial stem cells, revealing an
altered profile of histone modifications. Cultured
spermatogonial stem cell-like cells in vitro may
recapitulate some of the same histone modification
patters seen in vivo. Conclusions: Elucidation
of the repertoire of histone methyl modifications
unique to adult mammalian germline stem cells
may enable identification of the epigenetic and
genetic signals that control self renewal vs.
differentiation in this cell type.
28
Integration of Erythropoietin and Integrin
Mediated Signals in Erythroid Proliferation
S. Eshghi,1 M. G. Vogelezang,2 R. O. Hynes,2 L.
G. Griffith,3 H. F. Lodish4; 1Biological Engineering,
Massachusetts Institute of Technology/Whitehead
Institute, Cambridge, MA, 2Center for Cancer
July 15–18, 2006
Research, Massachusetts Institute of Technology,
Cambridge, MA, 3 Biological Engineering,
Massachusetts Institute of Technology,
Cambridge, MA, 4 Biology, Massachusetts
Institute of Technology/Whitehead Institute,
Cambridge, MA
Intracellular signaling initiated by binding of erythropoietin to its receptor has long been understood
to be the major pathway governing erythroid
development. The role of signals originating from
the extracellular matrix in this system has been
less rigorously studied. Here we present evidence
that signaling initiated by binding of fibronectin
to α4β1 integrins on the surface of erythroid
progenitor cells is necessary for proper erythroid
differentiation and proliferation. Furthermore our
data suggest a two-stage model for erythroid
development, where an early Epo-dependent,
integrin-independent phase is followed by an
Epo-independent, integrin-dependent phase.
Using flow cytometry, fetal liver erythroid progenitors were separated at four distinct stages of
development based on expression of CD71 and
Ter119. This system allowed us to show that α4β1
integrins are developmentally regulated during
erythropoiesis and that these integrins mediate
adhesion to specific domains of fibronectin. An
in vitro differentiation assay indicated that α4β1
integrins are necessary for terminal erythroid
proliferation but only after an early Epo-dependent phase. This work not only supports the view
that normal erythroid development is achieved
through signaling pathways initiated by α4β1
integrin in addition to the erythropoietin receptor
but also provides new insight to the more general
integration of signals from growth factor and
environmental cues in development.
29
A Role for escargot in Stem Cell Niche
Maintenance
J. Voog,1 G. Hime,2 M. Rocha,1 M. Boyle,1 M.
Fuller, 3 L. Jones 1; 1Laboratory of Genetics,
The Salk Institute for Biological Studies, La
Jolla, CA, 2Department of Anatomy and Cell
Biology and ARC Centre of Excellence in
Biotechnology and Development, University of
Melbourne, Melbourne, Australia, 3Departments
of Developmental Biology and Genetics, Stanford
University School of Medicine, Stanford, CA
The somatic apical hub at the tip of the Drosophila
testis is a primary component of the male germline stem cell niche. Hub cells secrete the ligand
Unpaired (Upd), which activates the JAK-STAT
pathway in adjacent germline stem cells (GSCs)
to specify stem cell self-renewal. This study’s
objective is to characterize shutoff (shof), an
allele of escargot (esg), a member of the Snail
family of transcription factors. In adult esgshof
males we observe a loss of GSCs and somatic
stem cells, also known as cyst progenitor cells
(CPCs). Immunofluorescence analysis of testes
from shof adults revealed a loss of hub markers,
including FasIII, DE-cadherin, DN-cadherin,
and Center-divider (Cdi). Furthermore, loss of
expression of upd and shotgun (shg), the gene
that encodes DE-cadherin, was observed in shof
testes from larval (L3) males. These findings sug-
■
Boston University
gest that hub cells may be lost and niche function
compromised during development in shof males.
Adherens junctions, composed of homotypic
Cadherin dimers, anchor GSCs to hub cells and
are concentrated between hub cells. We hypothesize that stem cell loss in esgshof mutants is due
to loss of adherens junctions between hub cells
and between hub cells and stem cells. Germline
stem cell clones homozygous mutant for shg
were not capable of long-term self-renewal, supporting the notion that DE-cadherin expression is
necessary for stem cell maintenance. Mammalian
homologs of esg have been shown to directly
regulate E-cadherin, an essential component of
adherens junctions. Furthermore, in Drosophila,
esg has been shown to act genetically upstream
of shg in the trachea. Therefore, we propose that
escargot acts to regulate shg expression within
hub cells. Characterization of genetic programs
that regulate stem cell-niche cell adhesion will
facilitate the establishment of in vitro stem cell
niche models and the propagation and manipulation of stem cells in culture.
30
A Novel Method to Image the Effect of RNAi
in Non-Adherent, Differentiating Erythroid
Cells
S. E. Haigh,1 I. Biran,2 N. Zurgil,3 M. Deutsch,3
O. S. Shirihai1; 1Pharmacology, Tufts University,
Boston, MA, 2Molecular Cytomics, Boston, MA,
3
Physics, Bar-Ilan University, Ramat Gan, Israel
Objective: To develop a method to image the
effects of mitochondrial protein targeted RNAi
knockdown on heme biosynthesis in non-adherent, differentiating erythroid cells. Background:
To study cellular functions in heterogeneous
populations of cells, such as stem cells and
tissue-derived cells, there is a need to continuously monitor functional parameters at a single
cell resolution in a large number of cells. This is
becoming a unique challenge when the cells of
interest are non-adherent, as in the case of blood
progenitors or bone marrow samples. A major
challenge of using RNAi with heterogeneous
cell populations, such as differentiating erythroid
cells, is to correlate between the level of RNAi
mediated gene disruption and physiological and
morphological effects in the same cell. Method:
To overcome this limitation, we used the Optical
LiveCell Array, a disposable slide-based device
containing a densely packed array of transparent
micron-sized wells. We used a lentivirus delivered shRNA construct harboring a GFP marker,
targeted against specific mitochondrial proteins.
Results: Using the cell array we were able to
identify a sub-population of mature erythroid
cells (stained with TRITC-labeled Glycophorin
A antibody) which also expressed high levels
of RNAi (based on GFP fluorescence intensity).
To measure heme content in this subpopulation, benzidine was subsequently applied and
light absorbance quantized at the resolution
of the individual cell. Heme content was then
cross-correlated with the RNAi expression and
cell maturation data. Conclusion: By imaging a
large population of individual differentiating cells
we were able to observe the effect of RNAi on
heme biosynthesis function.
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31
Striatal Neural Precursor Plasticity Evaluated
by Transfection of the Transcription Factor
Nurr1
C. Soldati, G. Tocco; Department of Cell and
Developmental Biology, Università degli Studi di
Roma La Sapienza, Rome, Italy
Recently work provides insights into the roles of
transcription factors in regulating the generation
of neural precursors, regionalization of the nervous system, and subsequent differentiation of
specific cell types within defined regions. Neural
stem cells (NSC) are capable of differentiating
toward neuronal and glial lineages, depending
on their spatial location within the nervous system (CNS); however the molecular mechanisms
underlying lineage commitment in NSCs are just
beginning to be understood. Furthermore, it is still
not clear how specification of a cell lineage results
in the suppression of alternative pathways in the
NSCs. Thus a thorough understanding of various
signal transduction cascades activated via growth
factors and of the role of different CNS microenvironments are critically required to determine the
full potential of NSCs. We investigated the possible role of transcription factor, Nurr1 on ST14A
cell line, which are representative of successive
stages of neuronal differentiation. In the midbrain
a critical role in dopaminergic differentiation is
performed by the neurospecifc transcription factor
Nurr1. Nurr1 controls the expression of key genes
involved in DA neurotransmission.We used neural
precursors cell line ST14A to teste the plasticity
of ST14A cells and in particular whether cells
derived from the striatum could be directed to
differentiate as dopaminergic neurons, by forcing
the expression of the transcription factor Nurr-1.
Levels of Nurr-1 comparable to midbrain can be
obtained in ST14A cells, but do not appear compatible with cell propagation. Futhermore there
this result can be related to a “committed” state
of the cell line or to its immortalized state cannot
be established and requires further experiments.
It is however interesting to recall that Nurr-1
transfection in embryonic stem cells has yielded
dopaminergic neurons, thus suggesting that the
committed state of ST14A may be responsible
for their inability to stable expressing of high
level of Nurr-1.
32
Extrinsic Regulation of Hematopoietic Stem
Cell Function
S. R. Mayack, A. J. Wagers; Developmental
and Stem Cell Biology, Joslin Diabetes Center,
Boston, MA
Hematopoietic stem cells (HSC) are the rare
precursors found predominantly in the bone
marrow (BM) that function to seed daily hematopoiesis. HSC number and function are at least
in part regulated by cell-to-cell contacts made
within the BM niche. Specifically, osteoblastlineage cells appear to play a role in regulating
HSC expansion, since certain mutant mice with
elevated numbers of osteoblasts also display
an increase in HSCs. HSCs in the BM can be
dramatically expanded in vivo, and their migration to peripheral organs induced, through the
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July 15–18, 2006
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Boston University
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administration of particular cytokines and growth
factors (called HSC “mobilization”); however, the
role of HSC-supporting niche cells in this process
has not been well-defined. To better understand
the extrinsic regulators of HSC function at steady
state and in the context of HSC mobilzation,
and to further investigate the precise role of
BM-derived osteogenic cells in supporting HSC
function, we have studied microenvironmental
changes in the BM and peripheral organs associated with cytokine-induced HSC mobilization. Upon cytokine induced HSC mobilization,
we demonstrate that osteoblasts within the BM
niche become activated and expand. Additionally,
the frequency of a subset of putative osteoblast
cells increases in both the peripheral blood and
spleen of cytokine-mobilized mice. Interestingly,
we also find that alkaline phosphatase (ALP)
activity, which correlates to osteoblast activation
and developmental status, actually decreases
in OPN+ cells from cytokine treated compared
to untreated mice. Since, ALP expression and
activity has been demonstrated to change both
during osteogenesis and mineralization this may
suggest that the OPN+ population of cells in cytokine treated mice represents the expansion of
a developmental or functionally distinct stage of
bone-derived cells. Finally, in vivo transplantation
studies using osteoblasts isolated from cytokine
treated mice results in an overall increase in the
frequency of HSC after transplantation. These
data suggest the intriguing possibility that HSC
expansion and migration requires a concomitant expansion and migration of niche-forming
osteoblasts, that these cells may be functionally
and/or developmentally distinct from OPN+ cells
in non-cytokine treated mice, and importantly that
these newly expanded osteo-lineage cells may
directly effect HSC function.
33
Fetal Human Articular Mesenchymal Cell
Chondrocytic Differentiation Requiring Both
Sox9 and Rho Gene Expression
H. Hao, R. A. Teitge, P. H. Wooley; Orthopaedic
Surgery, Wayne State University, Detroit, MI
Objective: Chondrocytic transplantation is a
potential cell therapeutic method that has been
used to treat the cartilage damage and chronic
cartilage degenerative disorders. Although
mesenchymal cell (MC) has been recognized to
give rise to chondrocytes and other cells including osteocytes, synovial cells, endothelial cells,
muscular cells and fibroblast cells, the precise
mechanism of MC-chondrocytic differentiation
remains to be elicited. Methods: Human fetal
articular MC primary culture were developed using fresh fetal knee tissue. MC isolation was performed using SH2/SH3/CD29/CD140 antibody
conjugated magnet beads. Based on our previous
results, TGF-β 3 and BMP2 were used to promote
the chondrocytic differentiation. Anti-Sox 9/Rho
mRNA nucleotides were designed and used for
the gene suppression studies. MC-chondrocytic
differentiation signals and morphological alterations were determined by immunocytochemistry
and protein chemistry analyses. The cell biological
characterizations were also estimated using myocytic and fibroblast specific antibodies. Results:
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Using this in vitro model, we demonstrated (1)
chondrocytic-like cells can be directly derived
from TGF-β3/BMP2 treated MC, (2) Sox 9 and
RhoA were up-regulated (5 to 7 folds) following
TGF-β3/BMP2 treatment, (3) MC lost the capacity
of chondrocytic differentiation after the cultures
were treated with anti-Sox9 and RhoA mRNA
antisenses, (4) However, suppression of Sox9
and RhoA did not affect the MyoD and myogenin,
as well as Thy-1 expression. Conclusions: SH2/
SH3/CD29/CD140 positive human fetal articular
MCs are able to generate chondrocytic-like and
myocytic-like cells. Both Sox9 and RhoA gene expression were increased in MC culture following
TGF-β3 and BMP2 treatment. Inhibition of Sox9
and Rho/RAK expression reduced the possibility
of MC cultures to express both collagen II and
proteoglycan genes permanently after cytokine
exposure. However, MC-myocytic differentiation
was not suppressed because the increased
MyoD and myogenin muscular transcription factor expression did not influenced by Sox9/RhoA
antisense. These results suggest that the Sox9
and RhoA/RAK signaling pathway are critical for
MC-chondrocytic differentiation.
34
Molecular Regulation of the Bladder Epithelial
Stem Cell Niche
I. U. Mysorekar,1 J. C. Mills,2 S. J. Hultgren1;
1
Depar tment of Molecular Microbiology,
Washington University School of Medicine,
St. Louis, MO, 2Department of Pathology and
Immunology, Washington University School of
Medicine, St. Louis, MO
The urinary bladder is lined by a transitional urothelium with normally slow turnover but extraordinary injury-inducible regenerative capacity. The
urothelial stem cell (USC) niche comprises basal
stem cells and underlying mesenchyme. Little is
known about the regulatory mechanisms governing USC niche activation. To determine how
otherwise quiescent USCs cells are activated, we
have developed a mouse model where urothelial
injury is induced by infection with uropathogenic
Escherichia coli (UPEC). We found that colonization of superficial cells by UPEC leads to rapid
exfoliation and compensatory proliferation and
differentiation of underlying immature cells such
that the superficial layer is renewed within 48h.
Following infection, 14% of basal cells are in
S-phase (i.e., are BrDu+) vs. <1% in controls,
suggesting that UPEC infection activates the
USC niche to fuel the regeneration cascade.
Global gene expression analysis of infected vs.
control bladders showed marked differences in
Bone morphogenetic protein 4 (Bmp4) and Sonic
hedgehog (Shh) expression, and follow-up immunofluorescence and in situ hybridization studies
revealed that Bmp4 and Shh were expressed in
the USC niche. To determine whether USC niche
activation was a general feature of injury, we
treated bladders with the basic cationic protein
protamine sulfate (PS). While this chemical, noninfectious injury, also led to facet cell exfoliation
and regeneration (<48hrs); in these bladders
S-phase (BrDu+) cells were now observed only
within the suprabasal layer and Bmp4/Hh pathways were not activated. Together, our findings
suggest that the bladder has both stem cell-dependent and independent molecular and cellular
mechanisms of regeneration that may depend
on type of injury. Moreover, our results elucidate
signaling pathways important for regulation of the
heretofore poorly characterized USC niche and,
specifically, establish a foundation to analyze
roles for Bmp4/Hh signaling in maintenance and
regulation of this niche in normal development
and in urothelial tumorigenesis.
35
Cell Instructive Polymers for Muscle
Regeneration
H. Storrie,1 E. A. Silva,1 E. Hill,1 J. C. Tapia,2
J. W. Lichtman, 2 D. J. Mooney 1; 1Division of
Engineering and Applied Sciences, Harvard
University, Cambridge, MA, 2Department of
Molecular and Cell Biology, Harvard University,
Cambridge, MA
Spatially and temporally regulated signaling
between and within cell populations and the
extracellular matrix regulate tissue homeostasis, pathology and regeneration, and polymeric
materials that can mimic or enhance this communication have the potential to intervene in
these processes in a therapeutic manner. These
cell instructive polymer systems provide insoluble
signaling molecules and cues (e.g., adhesion
peptides) or soluble signaling molecules (e.g.,
growth factors) alone or in specific combinations
to either host tissue cells or to transplanted cells
to regulate their activation, multiplication and
differentiation. Multiple aspects of regeneration
must be considered to design effective approaches to enable functional tissue replacement, and
this issue has been addressed in the context of
skeletal muscle regeneration. Myoblast transplantation has been explored for regeneration of the
parenchyma, while specific growth factor delivery
has been used to promote tissue vascularization
and reinnervation of neuromuscular junctions in
in vivo and in vitro models.
Disclaimer
Each individual attending the ASCB
Stem Cell Niches meeting assumes
all risks associated with his or her
attendance and participation in onand off-campus activities. Each individual attendee agrees to indemnify
and hold harmless the ASCB and its
governing bodies, officers, directors,
employees, and/or agents from all
loss, damage, liability, injury, and/or
death arising out of or related to his
or her attendance at the ASCB Stem
Cell Niches meeting.
The ASCB 46th Annual Meeting
December 9-13, San Diego, CA
Mary Beckerle, President ■ Anthony Bretscher, Program Chair ■ Arshad Desai, Local Arrangements Chair
KEYNOTE SYMPOSIUM
Saturday, December 9
Frontiers in Cell Biology—6:00 pm
Thomas R. Cech, Howard Hughes Medical Institute,
1989 Nobel Laureate
Susan Hockfield, Massachusetts Institute of Technology
SYMPOSIA
Sunday, December 10
Coordination of Adhesion and Migration—
8:00 am
Denise Montell, Johns Hopkins Medical School
Clare Waterman-Storer, The Scripps Research Institute
Kenneth Yamada, National Institute of Dental &
Craniofacial Research/NIH
Deciphering Evolution—10:30 am
Sean Carroll, University of Wisconsin–Madison/HHMI
Eric Jarvis, Duke University Medical Center
David Kingsley, Stanford University School of
Medicine/HHMI
Monday, December 11
Mechanisms in Mitosis—8:00 am
Rebecca Heald, University of California, Berkeley
Lucille Shapiro, Stanford University School of Medicine
Ronald D. Vale, University of California, San Francisco/
HHMI
Developmental Decisions—10:30 am
Hans Clevers, Netherlands Institute for Developmental
Biology
Elliot Meyerowitz, California Institute of Technology
Susan Strome, Indiana University
Tuesday, December 12
Membrane Assembly and Dynamics—8:00 am
Gillian Griffiths, University of Oxford
Janet Shaw, University of Utah
Marino Zerial, Max Planck Institute of Molecular Cell
Biology & Genetics
From Cellular Mechanisms to Therapeutic
Intervention—10:30 am
Susan Lindquist, Whitehead Institute for Biomedical
Research
Christine Seidman, Harvard Medical School/HHMI
Xiaodong Wang, University of Texas
Southwestern Medical Center/HHMI
Wednesday, December 13
Functional Networks—8:00 am
Susan Mango, University of Utah
Kevan Shokat, University of California,
San Francisco
Tian Xu, Yale University School of Medicine/HHMI
Stem Cell Biology—10:30 am
George Q. Daley, Children’s Hospital Boston
Elaine Fuchs, Rockefeller University/HHMI
Margaret Fuller, Stanford University School of Medicine
MINISYMPOSIA
Apoptosis
Eileen White, Rutgers University
Junying Yuan, Harvard Medical School
Applications of Biosensors
Atsushi Miyawaki, RIKEN Brain Science Institute
Alice Ting, Massachusetts Institute of Technology
Cancer Mechanisms
Lisa Maria Coussens, University of California, San Francisco
Mary J. C. Hendrix, Children’s Memorial Research Center/
Northwestern University Feinberg School of Medicine
Cell Cycle
Mary Dasso, National Institute of Child Health & Human
Development/NIH
Jonathon Pines, The Wellcome Trust/Cancer Research UK
Cell Migration
Diane L. Barber, University of California, San Francisco
Gregg G. Gundersen, Columbia University College of
Physicians & Surgeons
Computational Applications in Cell Biology
Douglas A. Lauffenburger, Massachusetts Institute of Technology
Alex Mogilner, University of California, Davis
Cytoskeleton, Adhesion and Disease
Kathleen J. Green, Northwestern University Feinberg
School of Medicine
Alpha S.K. Yap, University of Queensland
ECM and Cell Signaling
Jean E. Schwarzbauer, Princeton University
Christopher Turner, SUNY Upstate Medical University
Endo- and Exocytosis
Todd Graham, Vanderbilt University
Margaret Scott Robinson, CIMB/The Wellcome Trust
Epigenetics and Chromatin Remodeling
Peggy Farnham, University of California, Davis
Andrew Feinberg, Johns Hopkins University School of Medicine
Epithelial Organization and Morphogenesis
Andrea I. McClatchey, Massachusetts General Hospital
Ulrich Tepass, University of Toronto
GTPases in Cellular Traffic
Francis Barr, Max Planck Institute of Biochemistry
Shou-ou Shan, California Institute of Technology
Host Pathogen Interactions
Jorge Galan, Yale University School of Medicine
Francoise Gisou van der Goot, University of Geneva Medical School
Kinetochores and Centrosomes
Michel L. F. Bornens, Institute Curie, Paris
Peter Todd Stukenberg, University of Virginia School of
Medicine
Life at the Microtubule Plus End
Anna Akhmanova, Erasmus University
Kevin Vaughan, University of Notre Dame
Mechanisms of Actin Dynamics
Bruce Lane Goode, Brandeis University
Dorit Hanein, The Burnham Institute
Mechanisms of Cell Polarity
Patrick Brennwald, University of North Carolina at Chapel Hill
Chris Q. Doe, University of Oregon/HHMI
Membrane Traffic in Disease
Esteban Carlos Dell’Angelica, University of California, Los Angeles
School of Medicine
Daniel Klionsky, University of Michigan
Microtubule Motors
Erika L. F. Holzbaur, University of Pennsylvania
Claire E. Walczak, Indiana University
Motile and Sensory Cilia
Kathryn Anderson, Memorial Sloan-Kettering Cancer Center
Elizabeth F. Smith, Dartmouth College
Myosin-based Movement
Folma Buss, Cambridge University
Arturo DeLozanne, University of Texas
Neural Degeneration and Regeneration
Zhigang He, Harvard University
Stephen Strittmatter, Yale University School of Medicine
Nuclear Pore and Traffic
Michael P. Rout, Rockefeller University
Katharine S. Ullman, University of Utah
Organelle Inheritance and Maintenance
Liza A. Pon, Columbia University College of Physicians & Surgeons
Michael Schrader, University of Marburg
Regulation of the Cytoskeleton
Keith W. T. Burridge, University of North Carolina at Chapel Hill
Anne J. Ridley, Ludwig Institute for Cancer Research
RNA and Development
Oliver Hobert, Columbia University College of Physicians &
Surgeons/HHMI
Roy Parker, University of Arizona/HHMI
Imaging
J. Richard McIntosh, University of Colorado
Eva Nogales, University of California, Berkeley/HHMI
Signaling in Development
Marcos González-Gaitán, Max Planck Institute of Molecular
Cell Biology & Genetics
Alexandra Joyner, New York University School of Medicine/HHMI
Immune Cell Adhesion and Recognition
Andrey Shaw, Washington University School of Medicine
Colin Watts, University of Dundee
Stem Cells
M. Kathryn Barton, Carnegie Institution of Washington
Linheng Li, Stowers Institute of Medical Research
Intermediate Filaments and Disease
Don W. Cleveland, University of California, San Diego
Colin Stewart, National Cancer Institute–Frederick
Synapse Assembly and Plasticity
Ann Marie Craig, University of British Columbia
Nancy Y. Ip, Hong Kong University of Science & Technology
For more information, contact the ASCB at (301) 347-9300,
ascbinfo@ascb.org or www.ascb.org.
July 15–18, 2006
■
Boston University
■
ascbinfo@ascb.org
■
www.ascb.org
Author Index
Bahng, J. ........................... 7, 8
Bastos, C. F. ....................... 20
Battista, M. .......................S24
Biran, I. .............................. 30
Blashki, D. ......................... 17
Boyle, M. .....................29, S3
Brawley, C. .........................S5
Brouard, N. ........................ 17
Buszczak, M. ..................... K1
Byrd, D. T. ......................... 14
Cherry, C. ..........................S5
Clyne, J. .............................. 24
Collodi, P. ........................... 12
Colonna, L. ........................S8
Cortiella, J. ............................ 8
Covello, K. L. ...................S17
Cox, R. ............................. K1
Crittenden, S. L. ................. 14
Cuddihy, M. ......................... 7
Daley, G. Q. ....................... 13
Davies, D. ............................. 1
De Cuevas, M. ....................S5
Deutsch, M. ........................ 30
Dickinson, M. E. ................ 10
Discher, D. E. ....................... 2
DiVito, K. .......................... 26
Doetsch, F. ..........................S8
Domanska-Janik, K. ............ 22
Down, J. D. ........................ 24
Engler, A. J. ......................... 2
Eshghi, S. ........................... 28
Falender, A. E. ................... 10
Fischer, K. A. ...................S19
Fisher, D. E. .......................S9
Fleming, E. H. ...................... 8
Fleming, H. E. ...................... 5
Francis, C. ........................S21
Frenette, P. S. ....................S24
Fuller, M. T. .................S4, 29
Gaeta, C. ............................ 20
Gage, F. H. ........................S6
Gamm, D. M. ..................... 25
Ganapati, U. ...................... 16
Garcia, M. D. ..................... 10
Gasson, J. C. ....................... 16
Gilboa, L. ...........................S2
Griffith, L. G. ..................... 28
Groot, K. R. ......................... 1
Guo, J. .................................. 5
28
Habich, A. ......................... 22
Haigh, S. E. ....................... 30
Hamalainen, E. .................S21
Hao, H. .............................. 33
Hatfield, S. D. ..................S19
Hauschka, P. ....................... 13
Helgadott, H. ...................S21
Hidalgo, A. ......................S24
Hill, E. ............................... 35
Hime, G. R. ...............S23, 29
Hirschi, K. K. ..................... 10
Hultgren, S. J. .................... 34
Hung, S. .............................. 9
Hynes, R. O. ...................... 28
Issigonis, M. .......................S5
Janes, S. M. .......................... 1
Janzen, V. ............................. 5
Jones, L. .......................29, S3
Judex, M. ............................ 18
Jurecic, R. ........................... 26
Jurga, M. ............................ 22
Kaba, M. ..........................S14
Kang, J. .............................. 19
Kao, W. ............................S24
Katayama, Y. ....................S24
Kehler, J. ..........................S17
Keith, B. ..........................S17
Kiel, M. J. .......................... 23
Kimble, J. .....................14, S1
Klein, G. ..........................S12
Kolf, C. M. ......................... 15
Kotov, N. A. ................ 4, 7, 8
Kronenberg, H. M. ............... 5
Kuang, S. ............................. 6
Kuroda, K. ........................... 6
Lang, R. ............................. 18
Larina, I. V. ........................ 10
Lee, J. ........................... 4, 7, 8
Lehmann, R. ......................S2
Leonhard, K. A. ................. 14
Levin, M. .........................S22
Li, L. ...............................S11
Lichtman, J. W. ................... 35
Linderman, J. J. .................... 4
Liu, M. .............................. 17
Lodish, H. F. ..............S14, 28
Louissaint, M. .....................S8
Lovell-Badge, R. ................. 10
Lynch, M. ........................... 16
Mages, J. ............................ 18
Mahowald, A. P. .................S4
Marriner, N. L. .................S23
Matunis, E. .........................S5
Mauch, P. ........................... 24
Mayack, S. R. .................... 32
McLaughlin, E. A. ...........S23
Mehta, G. ............................. 4
Melvan, J. N. ...................... 25
Mikkola, H. ......................S21
Mills, J. C. .......................... 34
Mlcak, R. ............................. 8
Moon, J. ............................. 12
Mooney, D. J. ..................... 35
Moore, K. .........................S20
Moore, M. K. ..................... 10
Morris, L. .......................... K1
Morrison, S. J. ............23, S15
Moviglia, G. A. .................. 20
Moviglia-Brandolino, M. T. . 20
Mysorekar, I. U. .................. 34
Nachtman, R. ..................... 26
Nardi, V. ............................ 13
Naveiras, O. ....................... 13
Nichols, J. E. ........................ 8
Niles, J. A. ........................... 8
Nishikawa, S. ...................S10
Nystul, T. .......................... K1
Ohlstein, B. ....................... K1
Oostendorp, R. A. J. .......... 18
Orkin, S. ..........................S21
Oviedo, N. J. ....................S22
Parmar, K. .......................... 24
Paz, H. ............................... 16
Peired, A. J. ......................S24
Perlin, J. R. .........................S4
Peschel, C. .......................... 18
Prichard, L. .......................... 1
Rafii, S. ......................27, S18
Renstroem, J. ...................... 18
Reynolds, S. H. ................S19
Rocha, M. ....................S3, 29
Rudnicki, M. A. ................... 6
Ruohola-Baker, H. ............S19
Sanchez Alvarado, A. .......S22
Sarnowska, A. .................... 22
Saslavsky, J. ........................ 20
Scadden, D. T. .............5, S16
Schreiber, T. D. ................S12
Sciorra, V. A. .....................S6
Seandel, M. ........................ 27
Sen, S. ................................. 2
Shafizadeh, H. .................... 16
Sharma, P. .......................... 13
Shcherbata, H. R. .............S19
Shearer, R. L. ..................... 25
Sheng, R. ...........................S5
Shirihai, O. S. .................... 30
Siddall, N. A. ...................S23
Silva, E. A. ........................ 35
Simmons, P. J. ..................... 17
Simon, M. Celeste .............S17
Simpson, C. .......................... 1
Soldati, C. .......................... 31
Song, L. ............................. 15
Spradling, A. ..................... K1
Storrie, H. .......................... 35
Suda, T. N. N. .................S13
Svendsen, C. N. .................. 25
Sweeney, H. Lee ................... 2
Takayama, S. ........................ 4
Tapia, J. Carlos .................... 35
Tavazoie, M. .......................S8
Teitge, R. A. ...................... 33
Thomas, S. A. ..................S24
Tocco, G. ............................ 31
Tootle, T. ........................... K1
Tuan, R. S. ......................... 15
Umphrey, M. ...................... 24
Varela, G. ........................... 20
Vogelezang, M. G. .............. 28
Voog, J. ............................... 29
Wagers, A. J. ...................... 32
Wang, S. ............................... 8
Wang, Y. ...........................S21
Ward, E. J. ........................S19
Warner, S. ............................ 8
Weissman, I. ........................S7
Wessels, J. T. .....................S12
Wong, C. ............................S3
Wooley, P. H. ...................... 33
Wright, L. S. ...................... 25
Wurmser, A. E. ..................S6
Yamashita, Y. M. ................S4
Yang, D. ............................... 9
Yang, J. ............................... 26
Zhang, C. .........................S14
Zurgil, N. ........................... 30

2006 ASCB Summer Meeting Program - Boston, MA

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