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www.coriell.org | ccr.coriell.org

Coriell Institute For Medical Research

Ex loreCoriell

2008

In this IssueCoriell Personalized Medicine Collaborative:Evidence-Based Research 2

Te CPMC Web Portal EectivelyCommunicating Complex Genetic Inormationin a User-Friendly Format 4

Genotyping and Microarray Center A New High-Troughput Resource 6

Genotyping o an Arican-American Cohort 7

Eects o Ex Vivo Expansion on MesenchymalStem Cell Phenotype 8

Regenerative Potential o Human Cord BloodMesenchymal Stem Cells 10

Recent Innovations in Stem Cell echnology 12

Characterization oHerpesvirus saimiriransormed -lymphocytes 13

Using Copy Number Analysis as a ool or

Cytogenetics 16

NINDS Repository or Neurological Disease 19

Collaborative For Discovery For HuntingtonDisease Markers: Call or Participants 21

Finding a Needle in the Haystacks 22

Adipose Stromal Cells or Adult Stem CellResearch 24

Methylation Patterns at the Prader-Willi Locusin Culture Lymphoblasts 26

2007 2008: New Opportunities, Challengesand Achievements 27


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several years. Additionally, we were in atten-

dance at the invitation-only 2008 Personal-

ized Health Care Summit, which brought

together top executives and leaders rom the

public, private and academic sectors to create

strategies to accelerate the integration o per-

sonalized healthcare into clinical practice and

healthcare delivery.

Te Coriell Personalized Medicine Collabora-

tive (CPMC) is a research study that employs

an evidence-based approach to determine the

utility o using personal genome inormation

in health management and clinical decision-

making. Te CPMC also aims to build a

cohort with rich genotypic and phenotypic

data with which to discover genetic variants

that aect drug toxicity and ecacy, as well as

to discover presently unknown gene variants

that elevate a persons risk o cancer and other

complex diseases.

Tis orward-looking, collaborative eort in-volves physicians, scientists, ethicists, genetic

counselors, volunteer study participants and

inormation technology experts. Its goal is to

better understand the impact o personalized,

or genome-inormed, medicine and guide its

ethical, legal and responsible implementation.

Coriell understands the importance o engag-

ing medical proessionals to develop successul

strategies or integrating complex genetic in-

ormation into the current medical paradigm.

Coriell also appreciates the commonality o

cancer in society and the enormous poten-tial or cancer research and cancer care to be

impacted by personalized medicine. Tus, we

have established relationships with neighbor-

ing healthcare partners or the CPMC study

and are engaging these individuals in the

CPMC both as collaborators and partici-

pants.

Far exceeding our initial goal o enrolling

1,000 participants into the study by the end

o the summer, the CPMC currently has more

than 3,000 participants and dozens o upcom-

ing enrollment opportunities scheduled. Te

study aims to enroll 10,000 individuals by the

end o 2009 with an ultimate goal o 100,000

participants. Tere is no charge to study par-

ticipants.

Te study has received a great deal o media

coverage and has had such gures as U.S.

Senator Robert Menendez (D-NJ), Represen-

tative Robert E. Andrews (D-NJ), Assembly-

man Louis D. Greenwald (D-NJ), and New

Jersey Senator Diane Allen (R-NJ) enroll into

the study as examples to the Delaware Valleycommunity.

In January 2008, Coriell hosted top-ranking

ocials rom the Federal Department o

Health and Human Services (HHS) to dis-

cuss the CPMC. Billed as the Challenges

and Solutions in Personalized Medicine, the

meeting brought together HHS leaders with

the specic responsibility or and oversight o

personalized medicine with our scientists and

executives, as well as individuals rom our hos-

pital partners.

Following the HHS visit, Coriell was com-

missioned by Secretary Leavitts Personalized

Health Care Initiative to prepare a white

paper describing important aspects o our

research study. Tis paper is included in the

HHS end-o-year report entitled Pioneers,

Pathways, and Partnerships: owards a Per-

sonalized Health Care System, which ocuses

on the challenges o integrating personalized

approaches into healthcare during the next

Coriell Personalized Medicine Collaborative: Evidence-Based Research

Michael F. Christman, Ph.D., is the President and Chie Executive Ocer o the Coriell Institute orMedical Research. Dr. Christman is an expert in genetics and genomics and most recently served as proessor

and ounding chair o the Department o Genetics and Genomics or Boston University School o Medicine.

In that position, he led an international team o scientists in one o the rst genome-wide scans associated withhuman genetic variation in disease using samples rom the Framingham Heart Study. Dr. Christman received

his bachelors degree in chemistry with honors rom the University o North Carolina, Chapel Hill, in 1981 and

a doctorate in biochemistry rom the University o Caliornia, Berkeley, in 1985. He was awarded a Jane Con

Childs postdoctoral ellow at the Massachusetts Institute o echnology in 1986. Dr. Christman is a member o

the Genetics Society o America, the New Jersey echnology Council Board o Directors, and the NIH Drug

Discovery and Experimental Pharmacology Study Section.

Michael F. Christman, Ph.D.President and CEO, Coriell Institute or Medical Research

From top to bottom: Senator Robert Menendez (D-NJ),

Counselor Richard Campanelli, Representative Robert E.

Andrews (D-NJ), each with Coriell personnel.


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Erynn Gordon, MS, CGC, is a board-certied genetic counselor recruited rom the Uni-versity o Maryland School o Medicine to join the Coriell Institutes Coriell Personalized Medicine

Collaborative as the Senior Genetic Counselor. Ms. Gordon earned a Master o Science degree in

genetic counseling rom the University o Pittsburgh in 2001 and was certied by the American

Board o Genetic Counseling in 2002. Ms. Gordon previously worked as the genetic counselor or

the Muscular Dystrophy Association Clinic at Childrens National Medical Center in Washing-

ton, D.C., providing genetic counseling to amilies aected by neuromuscular diseases. While at

Childrens, Ms. Gordon also served as the laboratory genetic counselor or the Research Center or

Genetic Medicine, conducting research on the psychosocial impact o non-disease genetic testing.

She then moved to the University O Maryland School o Medicine, where she was the genetic

counselor or the Huntingtons disease clinic and the adult genetics clinic. While at the University

o Maryland, Ms. Gordon also provided cancer genetic counseling services at Baltimore Washington

Medical Center.

Erynn Gordon, MS, CGCSenior Genetic Counselor, Coriell Personalized Medicine Collaborative

ara Schmidlen, MS, CGC, earned a Bachelor o Science degree in biobehavioral healthrom Pennsylvania State University in 2002, a Master o Science degree in genetic counseling rom

Arcadia University in 2006, and certication by the American Board o Genetic Counseling in

2007. Prior to joining the Coriell Personalized Medicine Collaborative, Ms. Schmidlen worked as

a laboratory genetic counselor providing telephone-based genetic counseling or common complex

diseases at Kimball Genetics in Denver, Colorado, and as a genetic coordinator acilitating testing

and case management or reproductive and pharmacogenetic testing at Genzyme Genetics in West-

borough, Massachusetts.

ara Schmidlen, MS, CGCGenetic Counselor, Coriell Personalized Medicine Collaborative

Te CPMC will deliver genetic results to study participants via the se-

cure web portal in the coming months. While this is an exciting step

orward in our research study aimed at assessing the impact o person-

alized genetic results on participant health and behavior, the task o

delivering complex results in a clear and understandable ashion is a

challenging one.

Earlier this year, we hired two board-certied genetic counselors, Erynn

Gordon, MS, CGC, and ara Schmidlen, MS, CGC, to develop edu-

cational materials or study participants and health proessionals and to

provide ree genetic counseling to study participants and their physi-

cians by phone, email and/or in person.

Te CPMC genetic counselors will be available to participants or both

pre-results and post-results genetic counseling. Genetic counselors can

help participants understand complex genetic inormation and the im-

plications o the CPMC results or themselves and their amily mem-

bers, and can work with participants to incorporate amily and medi-

cal history inormation to estimate the risk o developing a particular

disease. In addition, the CPMC genetic counselors may also provide

participants with inormation about testing, treatment and prevention

options that may be available or diseases or which they are at risk.

Genetic counselors provide emotional support or people having a hard

time adjusting to their risk or getting a disease or to their potential to

pass on a genetic risk o disease to their children. Genetic counselors

are not physicians and will not prescribe a treatment plan or tell study

participants what to do about their genetic risk; instead, the CPMC ge-

netic counselors will work to help participants understand their genetic

results and inorm them o options that are available or addressing that

risk. However, based on the outcome o the risk assessment, the CPMC

genetic counselors may recommend that participants see their primary

care physician or urther evaluation.

Te CPMC leadership was quick to recognize the need or genetic

counselors as medical proessionals who will be instrumental in the e-

ective communication o genomic inormation to study participants.

Genetic Counseling and the CPMC


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Josena Nash isDirector o Coriells Inormation Systems Department.As such, Ms. Nash manages thesotware development, databases and overall Inormation echnology (I) inrastructure o Coriell. She serves as

the I liaison to Coriells Principal Investigators or its various National Institutes o Health contracts, as well as

a liaison to external I collaborators such as dbSNP, dbGAP and Harvard Partners Center or Genetics and Ge-

nomics. Her department is currently ocusing on such tasks as the development and maintenance o the Coriell

Personalized Medicine Collaboratives (CPMC) web portal, the QUEUE Repository Inormation Management

System and the Coriell Cell Repositories web catalog. Ms. Nash joined the Coriell Institute or Medical Research

in May 1997 rom BIOSIS, a non-prot organization in Philadelphia that produced an indexed, searchable bib-

liographic database o biological scientic literature. At BIOSIS, Ms. Nash served as a supervisor in the indexing

department and worked on the development o a bibliographic index database system.

Josena NashDirector, Inormation Systems, Coriell Institute or Medical Research

Personalized medicine oers a new paradigm

or healthcare and the practice o medicine.

Eective implementation o personalized

medicine into day-to-day medical care, how-

ever, will require robust decision support

systems built upon electronic health records

that contain not merely physiological data,

such as a patients blood pressure, temperature

and blood chemistry results, but also the pa-

tients own genetic and genomic data. By un-

derstanding illnesses on the molecular level,

including gene variations linked to disease or

drug response, doctors may be able to make

more precise diagnoses and tailor treatment

decisions. Similarly, drug makers can work

to develop more targeted treatment therapies

and identiy potential clinical trial participants

more eectively.

Te Coriell Personalized Medicine Collab-

orative (CPMC) is an evidence-based research

study designed to determine the utility o us-

ing genome inormation in clinical decision-

making. A comprehensive inormation systemhas been developed to advance the objec-

tives o the CPMC and to bring together the

various constituents that will interact in the

study. Te system brings together, in a web

portal, participant inormation obtained rom

medical history questionnaires and medical

records, genetic results and a genetic variant

knowledge base. As the central inormation

hub o the study, the web portal contains tools

or participants to update their consent op-

tions, share their data with physicians, request

complete this during several sessions, track-

ing their progress toward completion with

a graphic or each portion o the question-

naire. Completion o these questionnaires is

mandatory and a prerequisite to the release

o personal genetic variant inormation. Par-

ticipants will be asked to review and update

their medical and amily history inormation

annually and will be able to update contact

inormation at anytime through their account

manager. Te longitudinal nature o the study

will capture medical and amily history data

over time, allowing tracking o changes in

both health and wellness criteria.

appointments with the CPMC genetic coun-

selors and complete questionnaires that assess

the utility o the genetic inormation that is

presented to them. Te web portal also pro-

vides extensive educational material available

to study participants, medical proessionals

and the general public.

Te web portal will contain a secure My

Account area that includes account man-

agement, health inormation management

and genetic results-reporting interaces. Par-

ticipants will be required to complete medical

history, amily history and liestyle question-

naires. Te portal will allow participants to

Te CPMC Web Portal Eectively CommunicatingComplex Genetic Inormation in a User-Friendly Format


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Norman Gerry, Ph.D., is a genetics scientist recruited rom the Department o Genetics and Genomics at

Boston University to join Dr. Christman in the Coriell Personalized Medicine Collaborative. Dr. Gerry, ormerdirector o Boston Universitys Genotyping and Microarray Resource, established and directs Coriells Genotyp-

ing and Microarray Center the acility that perorms the genome analyses or the CPMC. He has been involved

in microarray and DNA testing or several years and has successully processed thousands o single nucleotide

polymorphism (SNP) and gene expression arrays, including all o the genotyping rom the rst genome scan o

the Framingham Heart Study [Herbert et al. (2006), Science; Herbert et al. (2007), Nature Genetics]. He has

also authored or co-authored numerous peer-reviewed scientic publications.

Norman P. Gerry, Ph.D.Director, Coriell Genotyping and Microarray Center

Associate Proessor, Coriell Institute or Medical Research

Genotyping and Microarray Center A High-Troughput Resourcebioinormatics, is also available.

During the past year, the GMAC obtained

CLIA certication or processing samples on

the Aymetrix SNP 6.0 array. In addition,

GMAC researchers have processed samples or

several research projects, including:

Genotyping 2,000 participants of the

Howard University Family Study (HUFS) or

a genome-wide association study o cardiac-

related phenotypes.

Genotyping 400 samples from the Hap-

Map Diversity Panels or distribution to the

research community.

Genotyping 600 Samples from the General

Medicine Repository or CNV and molecular

karyotyping analysis.

preparation and data analysis or Aymetrix

expression and SNP proling GeneChips. Te

second is assistance in the design and analysis

o arrays or non-model organism expression

proling or unique applications such as ChIP

on chip or resequencing. For these experi-

ments, the CPMC, along with Aymetrix or

other third-party vendors, will work with in-

vestigators to produce an array as well as pre-

pare samples or hybridization.

Te GMAC is a high-capacity acility, consist-

ing o twelve FS450 Aymetrix uidics stations

and three state-o-the-art GCS3000 scanners.

Each scanner is equipped with an autoloader.

Te acility can process up to 3,200 DNA or

RNA samples per month. Optional assistance

with data interpretation, both statistical and

Te Coriell Institute Genotyping and Mi-

croarray Center (GMAC) was established in

August 2007. Te mission o our acility is to

provide investigators with the tools they need

to ulll their research goals. Te widespread

use o microarrays has led to a paradigm shit

in detecting sequence variations and gene ex-

pression on a genomic scale. Tis is just the

tip o the iceberg in terms o possibilities.

Tere are continuing developments in the ar-

eas o pathogen detection, mapping o DNA

protein binding sites (ChIP on chip), micro-

and non-coding RNA expression, analysis o

recombination, and determination o copy

number variations (CNVs).

Genotyping and Microarray services all

into two main categories. Te rst is sample


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Genotyping o an Arican-American Cohort

Early this year the Coriell Institute or Medi-

cal Research received a major grant rom the

W.W. Smith Charitable rust to und the re-

search project, Genetic Risk Factors or Heart

Disease in Arican-Americans. Tis study is

the rst genome scan o an Arican-American

cohort and involves a close collaboration with

Dr. Charles Rotimi, Director o the NIH In-

tramural Center or Genomics and Health

Disparities (NICGHD).

Studies o genetic actors that are known to

increase the burden o disease in specic

populations are desperately needed. Arican-Americans are disproportionately predisposed

to heart disease, obesity, type 2 diabetes and

hypertension. Collectively, these diseases ex-

plain more than 80 percent o the health dis-

parity between Americans o Arican descent

and Americans o Western European descent.

Te high level o heritability or these diseases

indicates that genetic actors and/or gene en-

vironment interactions contribute to these

negative outcomes.

o identiy genetic risk actors, Coriell is work-

ing with Dr. Rotimi and his group to study

participants rom the Howard University Fam-

ily Study (HUFS) based in Washington, D.C.

and are determining the impact o copy-num-

ber variants on phenotypic variation.

A variety o analytical approaches are being

implemented in Helixtree, pLINK, Partek,

FBA and R. In parallel, data are being ana-

lyzed rom the Framingham Heart Study

(FHS) to identiy variants that show associa-

tion with disease in both populations (HUFS

and FHS). o replicate positive ndings, we

are also collaborating with the Scandinavian

FUSION study o type 2 diabetics and the de-

CODE group studying Icelanders.

Tis work is expected to continue throughout

the next year with a possibility that urther

targeted genotyping or deep resequencing o

particular individual DNAs will lead to iden-

tication o unctional variants. It is our hope

that data collected through this study will be

made accessible to other scientists through a

mechanism such as NIHs dbGAP.

Te research study was designed with the aim

o genotyping 2,000 Arican-Americans or

1 million single nucleotide polymorphisms

(SNPs) and preparing a phenotypic database

or analysis. A second aim o the study is to

analyze the resulting data in order to associ-

ate genetic variation with quantitative heart

disease-related traits, including hypertension

and type 2 diabetes.

o date, Dr. Rotimis group has reviewed

medical inormation and phenotypic measure-

ments rom each individual in the HUFS col-

lection; these data include measures o obesityand diabetic and hypertensive status collected

rom a single exam visit. Te inormation was

checked or accuracy and consistency. Further-

more, pedigree structure was conrmed using

genotyping results. Te participant dataset is

now rozen and ready or analysis. Research-

ers in Coriells Genotyping and Microarray

Center (GMAC) genotyped 763 males and

1,213 emales using the Aymetrix Human

6.0 GeneChip. Subsequent to the removal o

SNPs with low allele requency, low call rate

and those outside o Hardy Weinberg equilib-

rium, 841,156 genetic variations resulted and

are available or analysis or each individual.

We are now searching results or genetic vari-

ants that are associated with obesity and hy-

pertension using approaches based on 755

nuclear amilies within the HUFS population,

either separately or in combination with the

956 unrelated individuals. We are examining

association o SNPs with disease outcomes

Alan Herbert,MB.ChB., Ph.D., was recruited by Dr. Christman rom Boston University to join theCoriell Personalized Medicine Collaborative team and serves as a consultant to the Institute. Dr. Herbert is also

an associate proessor at Boston University. He received his Bachelor o Science degree in human biology rom

the University o Auckland in 1976 beore continuing on to earn his MB.ChB. in medicine and his Ph.D. in im-

munology. Dr. Herbert completed his post-doctorate or molecular immunology at the Massachusetts Institute o

echnology studying alternative DNA conormations and its eects on editing o genetic readout. Recently, Dr.

Herbert, along with Drs. Christman and Gerry, completed a whole genome scan o amilies rom a community-

based population in Framingham, MA, which involved the genotyping o 100,000 single nucleotide polymor-

phisms per individual. Tis scan was the rst o its kind and allowed the researchers to identiy a common variant

that increases risk or obesity.

Alan Herbert, MB.ChB., Ph.D.Consultant, Coriell Institute or Medical Research

Associate Proessor, Department o Genetics and Genomics, Boston University


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Mesenchymal stem cells (MSCs) are adult so-

matic stem cells that have enormous clinical

potential. MSCs can be ound in several tissue

sources including bone marrow and umbilical

cord blood (UCB) 1,2,3,4. Because MSCs are

stem cells, they are capable both o sel renew-

al as well as dierentiation into multiple cell

types, two hallmarks o stem cells. MSCs have

the capacity to dierentiate toward mesoder-

mal tissues, including cartilage, bone, at, and

muscle. Although MSCs can be ound in

multiple tissues o the body, they are present

at very low requency and thereore, extensiveex vivo expansion is used to generate sucient

numbers or their study or or clinical use.

Initially, MSCs grow quickly in culture, but ex-

hibit a limited lie span. Other stem cell types,

including embryonic stem cells, show

chromosomal instability under partic-

ular culture conditions 5. Additionally,

there is evidence that MSCs may lose

some o their stem cell characteristics

and enter into a state o cell senescence

during attempts to expand the cells in

culture 6,7. Cellular changes that occur

during ex vivo expansion may render

MSCs, generated in this manner, un-

suitable or use as a cellular therapeutic

due to loss o the pluripotency pheno-

type. In addition, the clinical utility

o MSCs will depend on the ability to

perorm ex vivo expansion while main-

taining genomic stability. Tereore, a

thorough understanding o how culture

conditions and time in culture aect the

ers o several genes known to be important to

maintenance o pluripotency.

First, we examined the cell prolieration kinet-

ics o the MSCs (Figure 1). As expected, the

MSCs grew quickly in culture ater the initial

isolation, with the cell rate slowing as the time

in passage progressed. Eventually, the cell cul-

ture became senescent, which was dened as

subcultivations that did not result in at least

one cell population doubling ater three weeks

in culture.

Next, we looked at the CFU-F ability o the

cells at various time points (P) in culture

(Figure 2). We chose to examine the cells at

P1 (passage 2, ~ 22 CPD), P2 (passage 5,

~28 CPD), P3 (passage 9, ~38 CPD) and

P4 (12 passages, ~40 CPD). At the rst two

pluripotency phenotype, genomic stability,

prolieration, and dierentiation capacities o

MSCs is critical to developing sae protocols

or their therapeutic use.

Te aim o our recent work has been to de-

termine the eects o ex vivo expansion on

the capacity o UCB-MSCs to sel renew, and

on the expression o various stem cell regula-

tors. MSCs rom UCB were cultured until

they reached senescence at approximately 40

population doublings. At our time points

during this continuous culture, we examined

sel-renewal, cell prolieration kinetics andcolony orming unit-broblast (CFU-F) abil-

ity, as well as expression o pluripotency genes.

Additionally, we were able to examine the oc-

cupancy o acetylated histones on the promot-

Eects o Ex Vivo Expansion on Mesenchymal Stem Cell Phenotype

Margaret A. Keller, Ph.D., is a molecular geneticist recruited to direct the New Jersey Stem Cell Resource (NJSCR) in all 2006. Dr. Keller

came to Coriell rom Tomas Jeerson University (JU), where she was a research assistant proessor in the Cardeza Foundation or Hemato-

logic Research in the Division o Hematology in the Department o Medicine. Her research, in collaboration with Dr. Saul Surrey at JU, has

ocused on lineage commitment o hematopoietic stem cells. Currently, in collaboration with Coriell and UMDNJ-SOM aculty, Dr. Keller is

studying the dynamics o the pluripotency phenotype o mesenchymal stem cells isolated rom umbilical cord blood (UCB) and expanded in the

laboratory. In her role as Director o the NJSCR, Dr. Keller oversees isolation, characterization, banking and distribution o biomaterials derived

rom UCB or use in basic research. She is also involved in the design and implementation o the Coriell Personalized Medicine Collaborative

(CPMC) research study. Dr. Keller is working with Coriell scientists, genetic counselors and inormation technologists, as well as with cliniciansand researchers outside o Coriell in many aspects o the study. In addition, Dr. Keller is an adjunct proessor at JU and was recently appointed

Assistant Proessor o Medicine at UMDNJ.

Margaret A. Keller, Ph.D.Associate Proessor, Coriell Institute or Medical ResearchAssociate Director, Cell Culture Laboratories, Coriell Cell Repositories

Shannon Morgan, Ph.D., is a Postdoctoral Fellow in the New Jersey Stem Cell Resource Laboratory at the Coriell Institute. Dr. Morgan holdsa Bachelor o Science degree in biology rom Te Catholic University o America, Washington, D.C. She earned her Doctorate in Microbiology

and Immunology rom emple University, Philadelphia, PA.

Shannon Morgan, Ph.D.Postdoctoral Fellow, New Jersey Stem Cell Resource Laboratory, Coriell Institute or Medical Research

Figure 1: MSC Liespan Analysis: UCB-MSC were seeded in 25 asks

at ~4x105 cells/ask in DMEM, 0.02M Hepes, and 15%FBS. Cells

were grown or 7 days, at which time cell yield was counted and cells

were used to seed another ask. Cumulative population doubling is cal-

culated as [log10(NH)-log10(NI)]/log10(2)=x, where NH=cell harvest

number, NI=inoculum number and x= population doublings. Cell cul-

tures were considered senescent when sub-cultivations did not results in

at least one cell population doubling ater 3 weeks in culture.

Figure 2: CFU-F Ability o MSC: UCB-MSC were seeded in

triplicate in 3 -25 asks at a density o 3 cells/cm 2 in DMEM

with high glucose and 10% FBS. Ater 14 days o incubatio

at 37C with 5% CO2, the colonies were stained with 0.5%

crystal violet in methanol or 10 minutes at room temperature

Purple colonies were counted to determine the percent CFU-F

ormation. Percent CFU was calculated as [(colonies counted

cells seeded)*100]. Average CFU was determined or each trip

licate set.


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9

time points, the MSCs showed 20 and 27 percent o the cells placed in

culture were capable o generating colonies. However, at the later two

time points, this ability or sel-renewal was lost.

We examined mRNA expression during this time course using the

aqMan Low Density Array (LDA) Pluripotency Panel (Applied

Biosystems, Foster City, CA) which simultaneously monitors changes

in expression o ~90 genes via real-time PCR. We assessed the expres-

sion state o genes involved in prolieration or stemness as well as those

involved in dierentiation at P1 (Figure 3). Several genes known tobe necessary or pluripotency were expressed in the UCB-MSCs at this

early time point, including NANOG, SOX2, and OC4.

Additionally, the LDA panels were used to examine the relative

change in gene expression during time in culture. We ocused on genes

that showed changes in expression when the two early time points were

compared to the two later time points, since this would correspond to

the loss o sel-renewal capacity demonstrated by the CFU-F assay. We

examined relative expression o ve genes: NANOG, LIN28, SOX2,

GAA6, and OC4, as these genes are known to play a role in the

pluripotency and dierentiation capabilities o pluripotent stem cells

!

8,9,10,11 (Figure 4). NANOG, LIN28, and SOX2 showed decreases in

expression comparing the P1 and P2 to P3 and P4. Conversely,

GAA6, which is known to be expressed during the dierentiation o

stem cells into other cell types 12,13,14, showed increased expression at

P4 when compared to P1.

Finally, we wanted to determine i the changes seen in gene expression

correlated with changes in epigenetic regulation o these genes. Chro-

matin immunoprecipitation assays using an anti-histone H3 antibody

were perormed ollowed by quantitative PCR o the promoter regionso the genes. We compared P2 to P4 and were able to show that

there was an increase in histone H3 occupancy on the promoters o

NANOG, LIN28, SOX2, and OC4 at P2 when compared to P4

(Figure 5). Te promoter o GAA6 showed a decrease in histone H3

occupancy at P2 when compared to P4. Because histone H3 occu-

pancy is oten associated with regions o transcriptionally-active chro-

matin, this suggests that these genes may be under epigenetic control.

ogether, these data demonstrate that as the cumulative population

doublings o the UCB-MSCs increase during repeated ex vivo cultur-

ing, the pluripotency phenotype is lost. Tis includes the decrease in

expression o regulators o stem cell prolieration as well as the loss o

the MSCs capacity or sel-renewal. Tis work highlights the need or

vigilant monitoring o MSCs expanded ex vivo and urther develop-

ment o culturing conditions to preserve their stem phenotype and di-

erentiation capacity or use in vivo.

Tis work was done in collaboration with Dr. Biagio Saitta (Coriell Institute) and Robert

Nagele (University o Medicine and Dentistry o New Jersey School o Osteopathic Medi-

cine and Institute or Successul Aging) and was supported by a grant rom the New Jersey

Commission on Science and echnology.

1. Digirolamo, C.M., et al., (1999) Br J Haematol107:275-281.2. Goodwin, H.S., et al., (2001) Biol Blood Marrow ransplant7:581-588.3. Lee, R.H., et al., (2004) Cell Physiol Biochem 14:311-324.4. Uccelli, A., et al., (2008) Nat Rev Immunol.5. Imreh, M.P., et al. (2006)J Cell Biochem 516.6. Bonab, M.M., et al., (2006) BMC cell biology7:14.7. Wagner, W., et al., (2008) PLoS ONE3:e2213.8. Lewitzky, M., Yamanaka, S. (2007) Current opinion in biotechnology18:467-473.9. Park, I.H., et al., (2008a) Nature protocols3:1180-1186.10. Park, I.H., et al., (2008b) Nature451:141-146.11. Yamanaka, S. (2008) Cell prolieration 41 Suppl 1:51-56.12. Cai, K.Q., et al., (2008) Dev Dyn 237:2820-2829.13. Fujikura, J., et al., (2002) Genes Dev16:784-789.14. Zhao, R., et al., (2008). Developmental biology317:614-619.

Figure 3: mRNA Expression Analysis o Early Passage MSC: mRNA expression analysis

was done using aqMan Low-Density Arrays (LDA, Applied Biosystems) contain-

ing pluripotency genes, dierentiation genes, and endogenous controls. UCB-MSC was

seeded in a 25 at 2x104 cells/cm2 in DMEM, 0.02M Hepes, and 15%FBS. Cells were

grown until conuent, at which time total RNA was obtained using RNeasy Mini Kit(Qiagen). otal RNA (100 ng) was used or cDNA synthesis using the High Capacity

RNA-to-cDNA Kit (ABI) and cDNA was combined with PCR master mix and loaded

onto the microuidics card. Te card was spun to distribute the reaction mix to each

well o the card and cycled in an ABI 7900 real-time instrument. Expression at the earli-

est passage (P2) is categorized as not expressed i the C was undetermined.

Figure 4: Gene Expression Changes During MSC Expansion: UCB-MSC was seeded in a

25 at 2x104 cells/cm2 in DMEM, 0.02M Hepes, and 15% FBS. Cells were grown until

conuent, at which time total RNA was obtained using RNeasy Mini Kit (Qiagen). otal

RNA (100 ng) was used or cDNA synthesis using the High Capacity RNA-to-cDNA Kit

(ABI) and cDNA was combined with PCR master mix and loaded onto a aqMan Low

Density Array. Te card was spun to distribute the reaction mix to each well o the card

and cycled in an ABI 7900 real-time instrument. Relative expression was determined by

using the C method and by comparing later passages (P4, P9, and P12) to the earliest

passage (P2). Five genes that show changes between early (P2, P4) and late (P9, P12) in

two replicate assays are shown.

Figure 5: Occupancy o acH3 on Stemness Gene Promoters Changes During MSC Ex-

pansion: UCB-MSC was seeded in a 25 at 2x104 cells/cm2 in DMEM, 0.02M Hepes,

and 15% FBS. Cells were grown until conuent, at which time proteins and DNA were

crosslinked with 1% ormaldehyde. Immunoprecipitation was carried out with anti-his-

tone H3. Cross-links were reversed and DNA was puried by QiaQuick purication kit

(Qiagen). qPCR was carried out on the recovered DNA in an ABI 7900 real-time instru-

ment using primers specic or the promoters o the indicated genes. Relative expression

was determined by using the C method and by comparing P4 to P12.


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10

Biagio Saitta, Ph.D., is Associate Proessor and Director o the Laboratory o Stem Cell and Matrix Biologyat the Coriell Institute or Medical Research. He also has a aculty appointment in the Department o Medicineat UMDNJ/RWJMS. Dr. Saitta received his Ph.D. in Biological Sciences rom the University o Messina in Italy.He then completed ellowships at the Pharmacology Institute Mario Negri o Milan and at the Developmental

Biology Institute o CNR o Palermo, Italy. He was subsequently a postdoctoral ellow and junior aculty mem-ber at Tomas Jeerson University in Philadelphia. Dr. Saitta joined the Coriell Institute in 2002, and the ocuso his laboratory research is the study o multipotent mesenchymal stem cell (MSC) populations isolated romhuman umbilical cord blood (UCB) and their dierentiation potential into specic cell lineages, including myo-cytes and chondrocytes. Dr. Saittas work has shown evidence that UCB-MSCs can be used as a complementarysource o stem cells as in vitro models or basic research. He is also interested in studying the role o extracellularmatrix proteins (ECM) involved in cell-specic lineage dierentiation and analysis o ECM modulation duringstem cell response to cardiac and skeletal muscle cell injury.

Biagio Saitta, Ph.D.Director, Stem Cell and Matrix Biology Laboratory

Associate Proessor, Coriell Institute or Medical Research

Regenerative Potential o Human Cord Blood Mesenchymal Stem Cellsstem cells, and cord blood-derived stem cell

transplants have been successully perormed

or a number o hematologic and oncologic

diseases 9.

Since MSCs can dierentiate into several cell

types and have less immune-related issues,

these cells have been used in several preclini-

cal and clinical trials (http://www.clinicaltri-

als.gov), as well as in trauma and myocardial

inarction (MI) conditions.

Institute where stem cell-rich cord blood is

collected and distributed or transplantation

and research. UCB is collected at birth rom

male and emale inants rom a variety o eth-

nic backgrounds, whose parents have provided

inormed consent prior to donation. UCB has

been increasingly explored as an alternative

source to bone marrow or multipotent MSCs

and or use in regenerative medicine 8. UCB

is a widely accepted source o hematopoietic

Stem cells can be isolated rom either adult or

embryonic tissue and dier in their capacity

to dierentiate into multiple cell lineages 1.

Te evolving eld o regenerative medicine

utilizes stem cells as a strategy to repair dam-

aged tissue and preserve or regain unction 2,3.

Adult stem cells or mesenchymal stem cells

(MSCs) are multipotent, broblast-like cells

that were rst described in the mid-1970s

and were ound in bone marrow 4. Cultured

bone marrow MSCs have been transplanted

in children with osteogenesis imperecta (OI),

a disease causing bone ractures and ragility.

When MSCs were engrated into the deective

bone, reduced bone ractures and increased

bone density resulted 5.

MSCs are attractive or a number o therapeu-

tic applications, as they are known to migrate

to some tissues, particularly when injured or

under pathological conditions 2. Our research

team has ocused on isolating and characteriz-

ing MSCs rom low volumes o umbilical cord

blood (UCB) samples (Figure 1).

We also demonstrated that MSCs can be e-

ciently propagated and dierentiated into

adipogenic, chondrogenic and osteogenic

cell lineages and can serve as in vitro models

or basic research 6,7. We obtained UCB sam-

ples rom the New Jersey Cord Blood Bank

(NJCBB), a acility housed within the Coriell

Figure 1. Distinct morphology o six dierent single isolated UCB-MSC colonies. By phase contrast microscopy, UCB1, UCB3

and UCB5 cells are spindle-shaped, small cells that grow to conuence, while UCB2, UCB4 and UCB6 cells are larger and grow

in ocal patches (passage 3 or all six MSCs). UCB1 and UCB2 were derived rom the same donor (Markov et al, 2007), and

UCB3 and UCB4 were also isolated rom a single donor. UCB5 and UCB6 were obtained rom two dierent donors. Tis mor-

phology was maintained through all population doublings or all six UCB-MSCs. Te magnications shown are 40x and 200x.


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11

Our laboratory o Stem Cell and Matrix Biology has ocused on under-

standing molecular mechanisms involved in tissue repair o these adult

stem cells. With support rom the New Jersey Commission on Science

and echnology (NJCS), we are investigating the ability o UCB-de-

rived MSCs to respond to myocardial damage in an in vitro model. o

simulate the eects o MI at the molecular level, we analyzed injured

rat cardiac cells (myocytes and broblasts) or apoptosis, necrosis and

viability. We also identied specic extracellular matrix (ECM) and an-

giogenic genes expressed by the UCB-MSCs that are modulated when

exposed to the hypoxic adult rat cardiac cells. O these specic genes,

the matrix metalloproteinases (MMP-1, MMP-2) and tissue inhibitors

o metalloproteinases (IMP-1, IMP-2) are involved in remodeling

o injured and brotic tissues. Other genes involved in vascular remod-

eling o injured and brotic tissues include: angiopoietins (ANGP1

and ANGP2), chemokines (CXCL1 and CXCL6), broblast growth

actors (FGF1 and FGF2), and interleukins (IL6 and IL8). Tese genes

encode critical ECM and angiogenic proteins that provide a substrate

or cells to migrate, grow and dierentiate. As such, the matrix is an in-

tegral regulator o cell and tissue unction. Our preliminary data show

increased expression o genes involved in the synthesis and remodeling

o cardiac tissue and supports our aim o nding potential mechanisms

involved in the process o cardiac repair. Trough a collaborative eort

with Drs. Steven Hollenberg and Joseph Parrillo, clinicians and car-

diovascular researchers at Cooper Heart Institute, these studies are be-

ing extended to understand unctional outcomes o our in vitro model.

Tis approach will determine i MSCs can improve the contractility othe injured cardiac cells.

In addition, our laboratory has initiated several collaborative projects

on both a national and international level aimed at strengthening the

stem cell biology program at Coriell. Collaborative projects include:

i) studying the engratment o our UCB-MSCs into the ventricles o

a rat model o MI. With Dr. Marisa Jaconi, University o Geneva,

Switzerland, we demonstrated that the cells invade and engrat into

the myocardium and ventricular wall in the region o inarct and orm

vessel-like structures at our weeks post-inarct. Markers indicated that

these structures originated rom the transplanted human UCB-MSCs

(Figure 2), suggesting that MSCs dierentiate toward endothelial lin-

eages and may contribute to new blood vessels in the damaged tissue;

ii) testing the potential o UCB-MSCs toward myogenic lineage di-

erentiation in an in vivo model o skeletal muscle regeneration using

a population o UCB-MSCs as a source or in vivo

muscle repair. Our initial work, in collaboration with

Drs. Pier Paolo Parnigotto and Maria eresa Con-

coni, University o Padova, Italy, ound muscular

engratment and myogenic dierentiation (Figure 3),

without immuno-rejection (Figure 4), o these stem

cells ater muscle injury; iii) investigations with Drs.

Margaret Keller and Jay Leonard, Coriell Institute,

and Dr. Robert Nagele, UMDNJ-SOM, to examine

the saety o cord blood MSCs or cellular therapeu-

tics by examining their stability ater being grown in

the laboratory.

It is essential to dene the biological mechanisms involved in the re-

sponses o stem cells and tissue-engineered products to injury at a mo-

lecular level. Our uture goal is to combine stem cell technology with

Figure 2. Engratment o UCB-MSCs transplanted into the heart o a rat model o cardiac inarct. (A) Let panels (top)

show UCB-MSC-containing biogel transplanted into the ventricle. Middle panels demonstrate engratment o UCB-MSCs

expressing green uorescent protein (GFP) into ventricular wall (black arrows). Red arrowheads show vessel-like structures

(100x magnication).

Figure 3. Hematoxylin-Eosin (H&E) stain o rat tibialis anterior (A) shows muscle damage with

Bupivicaine Hydrochloride solution. Let panel: 48 hours ater bupivacaine, muscles had degenera-

tive changes and an inammatory response. Many muscle bers showed necrosis (pale bers) and

internal nuclei reected macrophage invasion (50x magnication). Te right panels show H&E

stain o A skeletal muscle sections ater injection o MSC suspension (a-c, treated) or saline solu-

tion (d, control). Ater 7 days (a, 100x and b, 200x) with UCB-MSCs, there was no sign o severe

immunological response, with only occasional endomysial macrophage inltration (a) and perivas-

cular inammation (b). Te bers showed normal size variation and were not pale. Ater 14 days

(c, 50x) the muscle appeared intact with a native skeletal muscle appearance showing distinctive

longitudinal myobers and peripheral nuclei (dark points). Ater saline solution injection, necrotic

areas began to disappear, but bers remained pale (d, 100x).


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12

tissue engineering approaches to develop tissue-

specic extracellular matrix-based grats or test-

ing in injury repair, both or in vitro and in vivo

models. Tis approach will lead to optimization

o conditions and identication o actors that

can be modulated to improve outcomes.

Tese studies will provide important new inor-

mation on the mechanism o MSC action andnew targets or therapeutic discovery.

1. Scadden, D.., (2006) Nature441:1075-1079.

2. Caplan, A.I., (2007)J Cell Physiol213: 341-347.

3. Grete, S., et al., (2007) Stem Cells Dev16:857-868.

4. Friedenstein, A.J., et al., (1976) Exp Hematol4:267-274.

5. Horwitz, E.M., et al., (1999) Nat Med5:309-313.

6. Markov, V., et al., (2007) Stem Cells Dev16:53-73.

7. William, D.A., et al., (2007) Dev Biol305:172-186.

8. Harris, D.., et al., (2007) Expert Opin Biol Ter7:1311-1322.

9. Broxmeyer, H.E., (2005) Cytotherapy7:209-218.

Figure 4. Immunouorescence analysis o A muscle sections. Ater 7 days (a-i) the GFP-labeled MSCs had engrated into the A

muscle (Green Fluorescent Protein a, e, and i) and their human origin was conrmed by immunohistochemistry using HLA-A2 anti

body (b). Fibers showed immunostaining or both the early myogenic markers My-5 () and MyoD (j), which are nuclear regulatory

actors controlling prolieration and dierentiation during embryogenesis and muscle growth and repair. At 14 days (m-p), GFP

labeled MSCs were detected in A muscle that also stained or sarcomeric tropomyosin (p). (Green: GFP, Red: specic antibody

Blue: DAPI, Merged: usion o three color channels. All images at 400x magnication).

Recent Innovations in Stem Cell echnology

In November 2007, research groups headed by James Tompson at the

University o Wisconsin-Madison and Shinya Yamanaka at Kyoto Univer-

sity in Japan described methods to reprogram adult humansomatic cells

into a pluripotent state 1,2. Using viral vectors containing our transcrip-

tion actors (Oct4, Sox2, and either Nanog and Lin28 or Kl4 and cMyc,

respectively), human broblasts were genetically modied to behave like

embryonic stem (ES) cells. Tese induced pluripotent stem (iPS) cell lines

are similar to ES cells in morphology, prolieration, surace antigens, gene

expression, telomerase activity and developmental potential. Tese stud-

ies utilized viral vectors that rely on integration o DNA sequences into

the human genome, thus carrying the potential o tumor ormation. In

order to develop iPS cells amenable or in vivo use, researchers have devel-

oped non-viral means o generating iPS cells 3,4 and have explored meth-

ods that do not utilize the cMyc oncogene 3. Tese advances would allow

or the development o patient-specic iPS lines that would constitute an

enormous step towards personalized medicine.

In June 2008, George Daley o Harvard Medical School and colleagues

demonstrated that iPS cells could be established rom a wide variety o

disease-specic broblasts, which he obtained rom the NIGMS Reposi-

tory at Coriell 5. Disease-specic iPS cells will be invaluable tools with

which researchers can examine the development o disease tissues in cul-

ture. Tey have the potential to urther our current understanding o

the underlying mechanisms o disease development and to allow de-

velopment o new treatments to slow or even stop the progression o a

number o devastating diseases.

Following the publication o these reports describing the creation o

iPS cells rom broblasts rom both normal individuals and individuals

with inherited diseases, Coriell has distributed 146 NIGMS broblast

cell lines, representing more than 50 dierent diagnoses, to research-

ers who have stated their intention o creating disease-specic iPS cell

lines.

Coriell recently restructured its stem cell repository capabilities in order

to oer researchers the opportunity to bank their iPS lines at the Repos-

itory. Further, Coriell will bring in-house the capabilities to establish,

maintain, characterize, bank and distribute this important stem cell re-

source. Tus, Coriell is doing its part in providing the highest quality

genetic resources to the biomedical research community.

1. akahashi, K., et al., (2007) Cell131:861-872.2. Yu, J., et al., (2007) Science318:1917-1920.3. Huangu, D., et al,. (2008) Nat Biotech.4. Stadteld, M., et al., (2008) Science.5. Park, I., et al., (2008) Cell134:877-886.


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13

Karen Fecenko-acka, Ph.D., is a sta scientist or the Coriell Cell Repositories Cell Biology ResearchLaboratory. Prior to her promotion and appointment earlier this year, Dr. Fecenko-acka served as a post-doctor-ate ellow and later a research associate at the Coriell Institute. She also served as a member o the science aculty

responsible or organizing and teaching the Stem Cell raining Course at Coriell in January 2006. Dr. Fecenko-

acka holds a Bachelor o Science degree with a major in biology and minor in neuroscience rom Kings College

in Wilkes-Barre, PA. She later earned her doctorate in neuroscience rom State University o New York Upstate

Medical Center, Syracuse, NY.

Karen Fecenko-acka, Ph.D.Sta Scientist, Coriell Cell Repositories, Cell Biology Research Laboratory

Coriell Institute or Medical Research

Characterization oHerpesvirus saimiriransormed -lymphocytesormed cell lines were incubated with EBV at

an MOI o 10. -lymphocyte cultures were

stimulated with 20 U/ml IL-2 and immedi-

ately incubated with H. saimirior stimulated

with IL-2 or 24 hours prior to incubation

with H. saimiri. A control ask was stimulat-

ed with growth actors but not inected with

virus. Samples were maintained in RPMI, 2

mM glutamine, 20% FBS and observed daily

or signs o transormation (clumps o small

bireringent cells).

EBV-transormed cells were cryopreserved

once cultures reached 1 x 108 cells. Since the

growth characteristics o H. saimiri-trans-

ormed cultures are quite dierent than EBV-

transormed cultures, the same criteria or

evaluating transormation o the cells could

not be used. During the rst six to eight weeks

o cell culture, normal growth o -lympho-

periodic restimulation with antigens or mito-

gens 5. Like EBV-transormed B-lymphocytes,

HVS-transormed human -lymphocytes

express a limited number o viral genes and

inectious virus is not detectable 5,6, making

this virus an ideal method to obtain a con-

tinuous cell line or donors who are not easily

transormed by EBV. Tereore, a study was

undertaken by CCR to compare the standard

transormation protocol using EBV to an al-

ternative transormation protocol using H.

saimiri.

Samples were collected rom healthy individu-

als and lymphocytes were isolated by centriu-

gation on Ficol-Histopaque gradients. Cells

were plated at a density o 1.5x106 2.5x106

in a standard 25 ask containing RPMI 1640

medium, 2 mM glutamine, 20% FBS and 10

ng/ul phytohemaglutinin (PHA). EBV-trans-

ransormation o B-cells by Epstein-Barr Vi-

rus (EBV) is used by the Coriell Cell Reposi-

tories (CCR) to produce renewable cell lines

and DNA to urther genomic and proteomic

studies o inherited diseases. With the ocus o

science shiting to encompass whole-genome

studies, specically, genetic analysis or suscep-

tibility to complex diseases, publicly available

cell lines and DNA are becoming more widely

sought. However, as much as two percent

o clinical peripheral blood samples do not

transorm with EBV ater repeated attempts

due to either genetic disease, such as Brutons

agammaglobulinemia, or to transormation

resistance 1,2. Tereore, it is o interest to op-

timize alternative methods o transormation

o human lymphocytes.

Alternative methods o lymphocyte transor-

mation, such as usion hybridomas and the

use o the transorming virus, HLV-1, have

proven inecient or unsuitable or large-scale

laboratory usage or the study o genetics 3,4.

However, a method similar to EBV transor-

mation has been developed using a -lym-

photrophic virus ound in squirrel monkeys,

Herpesvirus saimiri(H. saimiri). H. saimiri is

a gamma-herpesvirus capable o producing

stable transormation o -cells in humans 5,6.

Although the transormed lymphocytes oten

remain IL-2 dependent, they do not require

able 1: Chart depicts the transormation eciency o EBV and H. saimiriusing whole blood. Adult lymphocytes were isolated

by centriugation on Ficol-Histopaque gradients. Cells were plated at a density o 1.5x10 6 2.5x106 in a standard 25 ask.

Lymphocyte cultures were grown in RPMI, 2 mM glutamine, 20% FBS and 10 ng/ul PHA (lectin). Flasks were either incu-

bated with EBV to produce EBV transormed cell lines or was stimulated 20 U/ml IL-2 or 24 hours prior to incubation with

H. saimirito produce -lymphocyte cultures. A control ask was stimulated with growth actors but let uninected. For EBV

inected cultures, transormation was assessed as continued prolieration to 1 x 108 cells and recovery rom cryopreservation. For

H. saimiriinected cultures, transormation was determined by continued prolieration as compared to uninected control and

recovery rom cryopreservation.


14/28

14

chromosomal abnormalities (able 2).

Once it was determined that H. saimiricould pro

duce continuously growing cell lines and chromo

somal stability o the cells lines were assessed, i

was important to determine whether the cell line

retained characteristics o their respective lineages

Lineage delity was assessed by cell surace anti

gen characterization and gene expression analy

sis. Characterization o cell surace antigens wa

perormed to determine the subtypes within th

EBV- and HVS-transormed populations. EBV

transormed cell lines were classied on the basis o

CD45, CD19 and CD3. Surace antigen CD45 i

present on all lymphocytes, while CD19 and CD3

are mutually exclusive to B-cells and -cells, respec

tively. HVS-transormed -cell lines were subtyped

on the basis o CD8 or CD4 expression. Surac

antigen CD8 is indicative o cytotoxic -cells, whil

CD4 is expressed by -helper cells; however, thes

antigens are not exclusive and may be simultane

ously expressed by the same cell (double positive).

As expected, the EBV-transormed cell line most

ly comprised CD19+ B-lymphocytes with a very

small percentage o CD3+ -lymphocytes (Figur

2B). All imaged cells were CD45+; however th

intensity o the expression o CD19+ varied rom

cell to cell (Figure 2A). HVS-transormed cell line

contained both single positive CD8 (68.2%) and

CD4 (3.07%), along with double positive popula

tions (22.8%; Figure 2C-D).

Finally, Aymetrix expression arrays were analyzed

in the Coriell Genotyping and Microarray Cente

to identiy which genes were up- or down-regulate

upon exposure to the transorming viruses. Based

on our cell surace antigen classication, we ex

pected EBV-transormed cell lines to express high

levels o genes associated with B-cell dierentiatio

ing to manuacturers protocol

(Roche). As seen in Figure 1A, it

was determined that cellular pro-

lieration was not signicantly

diminished in concentrations o

IL-2 as low as 2 U/ml when the

prolieration o transormed cells

( ) was compared to uninected

control

cells ( ), indicating that the con-

centration o IL-2 can be de-

creased ollowing the establishment o the cell

lines. Figure 1B depicts the average cell proli-

eration o ve transormed cell lines during a

period o three weeks in response to dierent

IL-2 concentrations, urther indicating that

IL-2 concentration can be reduced to maintain

the transormed cell lines without a signicant

decline in cell number.

EBV-transormed cell lines are used extensively

in the study o genetics and the genetic basis o

disease and thus, it is important to determine

that genome stability is retained ollowing

transormation and culturing. It has been es-

tablished that karyotypes o

EBV-transormed cell lines

show chromosomal stability

up to high passages 7. In or-

der to assess the stability o

lines produced byH. saimiri

transormation, matched

EBV and H. saimiricell lines

(same individual) were ana-

lyzed or chromosomal

abnormalities by kary-

otyping (g-banding).

In two o the three H.

saimiri cultures, mul-

tiple chromosomal

abnormalities were

detected, suggesting

that the lines may be

somewhat chromoso-

mally unstable (able

2). Only one o the

three EBV cell lines

contained signicant

cytes was observed and the cells were not

considered transormed. Following this initial

rapid prolieration, the cells went through a

crisis period until a small outgrowth o virally-

inected cells was observed. ransormed cells

were cryopreserved once cultures reached 1 x

108 cells, 56 days or H. saimiritransormed

lines and 32 days or EBV. Upon recovery

rom cryopreservation, cells were assessed or

viability and continued cell growth. It was

determined that EBV-transormed cell lines

were obtained with an eciency o 100%,

while the eciency or H. saimiritransorma-

tion was approximately 66% when inected 24

hours ater stimulation with IL-2 (able 1).

Following transormation using H. saimiri

in 20 U/ml o IL-2, we wanted to determine

whether we could maintain the cell lines in

decreased concentrations o IL-2. Tereore,

H. saimirilymphocyte cultures were plated at

a density o 5 x 105cells/well on 6-well dishes

in ve dierent IL-2 concentrations (0-20 U/

ml). Prolieration was assessed at week 1 and

week 2 by X cell prolieration assay accord-

Figure 1: A) Histogram depicts cell prolieration o uninected cells ( ) as compared to a

transormed cell line( ) in response to IL-2 concentration. Lymphocyte cultures were either

inected with H. saimirior let uninected and grown in RPMI supplemented with glutamine

(350ug/0l), 10% etal cal serum, and IL-2 (20U/ml). Ater 44 days in culture, 5 x 10 5cells/

well were plated on 6-well dishes in 5 dierent IL-2 concentrations. Prolieration was assessed

by X cell prolieration assay according to manuacturers protocol (Roche). (B) Histogram

depicts average cell prolieration o transormed cells over three weeks in response to dierent

IL-2 concentrations. Cell counts were also perormed at various time points to conrm the

X results using Vi-Cell XR 2.03.

able 2: Chart outlines the karyotype and percentage o abnormal cells within

the cell population. Both EBV and HVS transormed cell lines were g-banded in

accordance with our standard operating procedures.


15/28

15

heat map indicating that genes were dieren-

tially expressed in EBV-transormed cell lines

as compared with HVS-transormed lines.

Further elucidating genes that showed the

greatest changes in gene expression between

transormants, the volcano graph (Figure 3C)

shows genes that were highly expressed (right

upper quadrant) and signicantly down-reg-

and linage. On the other hand, H. saimiri-

transormed cell lines should express higher

levels o genes associated with -cell dier-

entiation and lineage. Figure 3A is a popula-

tion comparative graph showing that EBV-

transormed cell lines grouped together and

were considered a separate population rom

HVS-transormed cell lines. Figure 3B is a

ulated (let upper quadrant) between popula-

tions. As expected, genes showing the greatest

up-regulation in HVS-transormed cell lines

were genes associated with -cell signaling and

dierentiation, such as CD2, indicating the

transormed cell lines retain characteristics o

cell lineage.

In this study, we have evaluated the transorma-tion process using H. saimiriin comparison to

the traditional transormation protocol using

EBV. We show data that -lymphocytes trans-

orm ollowing H. saimiri inection and that,

although the -cells remain IL-2 dependent,

the concentration o IL-2 can be reduced 10-

old without a signicant reduction in proli-

eration. Furthermore, we show that H. saimiri-

transormed lymphocytes retain characteristics

o -lymphocytes, such as cell surace antigens

and gene expression as assessed by microarray

analysis. A larger study is currently underway

to urther investigate the use oH. saimirias

an alternative transormant to the traditional

EBV-transormation protocol.

1. Bass, H., et al., (2004) Eur J Immuno 31:87.2. Beck, J., et al., (2001) Cancer Epi Biomark & Prev10:551-554.

3. Dzhambazov, B., et al., (2003) Folia Biologica 49:87-94.4. Anderson, M., et al., (2004)J Virolog Met116:195-202.5. Kaschka-Dierich, C., et al., (1982)J Virol44:295-310.6. Grassmann, R., et al., (1994)Adv Cancer Res63:211-44.

7. Neitzel, H., (1986) Hum Genet73:320-326.

Figure 2: Surace Antigen Analysis o EBV transormed cells. (A) Dot plot showing the CD19+ population (red) o cells

within the larger population o CD45+ (pink) lymphocytes (B) Dot plot showing the CD3+ population (blue) o cells

within the larger population o CD45+ (pink) lymphocytes. Surace Antigen Expression oH. saimiritransormed cells. (C)

Dot plot showing the CD3+ population (blue) o cells within the larger population o CD45+ (pink) lymphocytes (D) Dot

plot showing the dierent subtypes o cells that comprise the CD3+ cells, CD4+ population (green), CD8+ population

(yellow) and CD4+,CD8+ population (orange).

Figure 3: Gene Expression Analysis o RNA isolated rom cell lines using Ametrix Platorm. (A) Principal Component Analysis (PCA) where triangles are EBV transormed cell lines and

squares are H. saimiritransormed cell lines (B) Heat Map o dierences in gene expression between EBV and H. saimiritransormed cell lines where down-regulation o gene expression is in-

dicated by blue and up-regulation o gene expression is indicated by red (C) Volcano Graph o analyzed genes, points in the upper two quadrants show the greatest changes in gene expression

between transormants (let is down regulation and right is upregulation)


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Using Copy Number Analysis as a ool or Cytogenetics

Jay C. Leonard, Ph.D., joined the scientic sta o the Coriell Cell Repositories (CCR) in 1992 as directoro its Cytogenetics Laboratory. He is also an associate proessor or the Coriell Institute. Dr. Leonard and his sta

conduct applied research to ensure that cytogenetic analyses or the CCR meet the best and most current stan-dards o practice. Tey are also involved in mining the CCRs extensive set o cytogenetics data. Prior to joining

the Institute, Dr. Leonard served as the Cytogenetics Laboratory Director o the Wilson Genetics Center, where

his laboratory specialized in prenatal diagnosis. At that time, he was also an adjunct assistant proessor in the

Program or Genetics. In 1990, Dr. Leonard became a Diplomate o the American Board o Medical Genetics in

Clinical Cytogenetics, and rom 1991-1992, he directed the Cytogenetics Laboratory in the Pediatrics Depart-

ment o the Albert Einstein Medical Center in Philadelphia, PA.

Jay C. Leonard, Ph.D.Associate Proessor, Coriell Institute or Medical Research

Director, Cytogenetics Laboratory

cytogenetic methods.

Our study includes more than ve hundred

cultures rom the National Institute o Gener-

al Medical Sciences (NIGMS) Chromosomal

Aberrations Collection. Cultures selected or

the study have gains and/or losses o chromo-

somal segments that were previously dened

by G-band karyotype analysis or FISH analy-

sis and, in some cases, by molecular analyses

provided rom the investigator who submit-

ted the culture(s). Tese analyses have been

reviewed by the NIGMS Repository sta and

by two independent reviewers prior to inclu-

sion in the NIGMS Repository catalog.

Sample preparation, hybridization and scan-

ning were perormed using GeneChip In-

strument System hardware according to man-

uacturers specications (Aymetrix, Santa

Clara, CA). Analysis was perormed using

the Aymetrix Genotyping Console sotware.

Te samples met Aymetrix-recommended

values or contrast QC (SNP) and MAPD

QC (CNV). Te intensities o both SNP and

CNV probes were used to determine segments

that varied in copy number. Te segment re-

port was restricted to regions o 100kb or

greater with ten or more consecutive probes

that diered signicantly rom the expected

normalized diploid values. SNP intensities

were normalized against the values or the

is detection o both balanced and unbalanced

changes, even when only a small number o

cells in a sample are aected.

Copy number analysis by microarray is an

indirect method that measures imbalances

between genomes through the relative hybrid-

ization intensities o uorescent labeled DNAs

to a series o oligonucleotide probes. Its inher-

ent limitations are that it cannot detect bal-

anced changes in chromosomal organization

and that it is limited in the detection o low-

requency mosaic changes. However, since

the majority o chromosomal abnormalities

that underlie genetic disease involve gains and

losses o chromosomal segments in all cells,

copy number analysis promises to transorm

cytogenetics because o the greater sensitivity

and resolution that it provides.

Te Genome-Wide SNP Array 6.0 contains

nearly one million single nucleotide polymor-

phism (SNP) probes and nearly one million

copy number variant (CNV) probes. Tis

results in marker spacing in the range o 7

Kb. Te large number and regular spacing o

both types o probes permit a cytogeneticist

to dene gains and losses o chromosomal

segments with greater resolution and preci-

sion than standard cytogenetic techniques.

Furthermore, loss o heterozygosity can be

assessed, which is not possible with standard

Jay Leonard, Ph.D., Director o the Cytoge-

netics Laboratory, and Norman Gerry, Ph.D.,

Director o the Genotyping and Microarray

Center, together with James Collins o A-

ymetrix, are evaluating the Aymetrix Hu-

man Genome-Wide SNP Array 6.0 or cyto-

genetic analysis.

Cytogeneticists advance the practice o clini-

cal genetics by dening specic changes in the

genome that are the basis o specic genetic

diseases. Karyotype analysis, based on direct

microscopic analysis o metaphase chromo-

somes, detects changes in chromosome num-

ber and structure in a single cell. Unbalanced

structural changes involving gains and losses

o chromosome segments o 5 to 10Mb or

greater can be inerred rom changes in chro-

mosome banding patterns. Changes that do

not involve gains or losses, but rather a re-

shufing o chromosome segments, are also

detected; however, breakpoint placement is

more dicult.

Fluorescence in situ hybridization to meta-

phases detects the presence or absence o

specic segments (commonly 10 to 100 Kb)

and chromosome location. While this is a sig-

nicant gain in sensitivity, data are only ob-

tained or the segments that are probed and

most laboratories hybridize only two or three

probes at a time. Te strength o these assays


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Accurate Characterization o Contiguous Gene Syndromes:

microscopic and submicroscopic

Te Coriell/Aymetrix collaborative study included microscopic (>5

Mb) and submicroscopic (1 2 Mb) deletions and duplications. Mi-

croarray analysis consistently detected loss o critical regions to mi-

crodeletion syndromes, which had previously been FISH-conrmed by

the Coriell Cytogenetics

group. Successul appli-cation o copy number

analysis to the character-

ization o FISH-veried

microdeletion cell lines

led us to use this mi-

croarray or other micro-

duplication or microde-

letions with molecular

characterization by the

submitter, but or which

commercial FISH probes

are not available. Our

results to date show the

Aymetrix GeneChip

6.0 is a reliable method or characterizing these disorders.

Copy number analysis is particularly useul or reliable detection o

small duplications which are not resolvable by metaphase FISH and

it does so without the technical challenges inherent in ber FISH or

interphase FISH. Pelizaeus-Merzbacher disease is an X-linked recessive

disorder resulting rom dysmyelization o the central nervous systemdue to disruption o proteolipid protein-1 (PLP1). Tis has been related

to mutation or duplication o PLP1. Te PLP1 gene is included in the

HapMap 270 samples. Copy number intensities or the group were

normalized against themselves.

Complete Ascertainment o Large Unbalanced Rearrangements

Ascertained by G-band Karyotype Analysis

Each sample previously judged to be unbalanced by traditional G-band

analysis was conrmed to be unbalanced by SNP array 6.0 analysis.

G-bands are alternating regions o light and dark staining along meta-phase chromosomes o approximately 5 Mb in length. Tese are most

commonly produced by controlled exposure to a proteolytic enzyme

ollowed by Giemsa or Wrights stains. Tere was a high degree o con-

sistency between the breakpoints involved in the abnormalities as called

by the CNV data and by the G-band data, according to the National

Center or Biotechnology Inormation (NCBI) and University o Cali-

ornia Santa Clara Genome Browsers. In most cases, the calls coincided.

For the other cases, the copy number data indicated the breakpoints

occur in adjacent bands or sub-bands. Tis was not unexpected since

small ractions o chromosome bands oten are not visualized by mi-

croscopy.

Improved Resolution o Breakpoints

GM50122 was established rom a patient with chromosome 18q de-

letion syndrome. Standard G-band analysis indicated 46,XY,del(18)

(pter>q21.3:). FISH analysis with probes to the 18p and 18q subtelo-

meric region showed the deletion to be terminal. Te karyotype in our

catalog is thereore specied 46,XY,del(18)(pter>q21.3:).ish del(18)

(VIJ-YRM2102+,18qtel11-). Copy number analysis renes the break-

point to band 18q21.33 as seen in Figure 1.

Further analysis by Katz et al.1

placed the breakpoint between SER-PINB4 (SCCA2) and SERPINB3 (SCCA1). As seen in Figure 2, we

ound the breakpoint to be within SERPINB4. Te dierence between

these calls was approximately 10 Kb.

Figure 2. High Resolution View o the 18q breakpoint in GM50122. rack 1,

copy number state calculated by HMM in dark green. rack 2, deleted segment in

red. rack 3, oronto Database o Genomic Variants in gray. rack 4, ReSeq genes in

light green. Te breakpoint or the deletion 18q is shown to be within SERPINB4 in

18q21.33. SERPINs are serine protease inhibitors. Clade B o the SERPIN multigene

amily is located in this area o chromosome 18; B4 and B3 are within the Copy Number

Variant 5059.

Figure 1.View o deletion 18q chromosome in GM50122. rack 1, Allele dierence

rack 2, LOH. rack 3, log2 ratio. rack 4, Hidden Markov Model calculation o Copy

Number State. Copy number segment: deletion shown in red. rack 5, oronto Database

o Genomic Variants. rack 6, ReSeq genes. rack 7, chromosome bands. rack 8, Bp

rom pter.


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18

Figure 4. Consistent detection o Dystrophin deletions and in cultures rom dierent tissues rom the

same person and in relatives. Figure 5 plots copy number changes in the Dystrophin genein the orm

o a heat map: 0 copies = red; 1 copy = yellow; 2 copies = green. Order rom top to bottom: GM04099,

GM04100, GM05107, GM05115, GM05169, GM02339, GM03064, GM03781, GM03780,

GM03782, GM05126.

Detection o Copy Number Changes

Within Genes

Figure 4 displays copy number changes within

the Dystrophin gene or cultures established

rom one carrier mother and ten males a-

ected with Duchenne Muscular Dystrophy.

Consistent results were ound among rela-

tives and between cultures established romdierent tissues rom the same donor. Te

same deletion was seen in GM04100 and in

GM04099, his unaected carrier mother. An-

other deletion was seen consistently in two

brothers, GM03181 and GM03182, and in

GM3780, a lymphoblastoid cell line rom the

brother who was the donor o the broblast

culture, GM03781. Te submitter ound no

Dystrophin deletion o GM05126 by mul-

tiplex PCR. We have detected a duplication

o DMD o approximately 750 Kb located

within chrX:31224486-31977208.

Te smallest deletion included in the study

removes exon alpha o the SNRPN gene in

cultures rom an unaected ather and his son

and daughter who are aected with Prader-

Willi Syndrome 3. Tis deletion was about 38

Kb in size (Figure 5).

Based on these results, copy number analysis

is now a standard component o cytogeneticanalysis perormed by the NIGMS Human

Genetic Cell Repository. We are currently

in the process o posting descriptions o the

disorder-specic copy number changes on our

website to aid users in the selection o lines or

use as reerence materials.

1. Katz, S.G., et al., (1999) Hum Mol Genet8:87-92.

2. Warshawsky, I., et al., (2006) Clin Chem 52:1267-75.

3. Sutclie, J.S., et al., (1994) Nat Genet8:52-8.

Figure 3. GM11005 a case o Pelizaeus-Merzbacher disease due to duplication o the proteolipid pro-

tein-1. Figure 3 is exported rom Aymetrix Genotyping Console Browser. It shows a duplication o

Xq22.1>Xq22.2 detected in two separate experiments (copy number state = red and green lines, calculated

duplication = blue lines). Reseq genes, chromosome bands and base pairs rom pter are given in the lower

three tracks.

Figure 5. Consistent detection o a deletion o part o the SNRPN Gene. Tree

paired tracks show log2 ratio above and HMM calculated copy number state below.

Color code: red GM13354 unaected ather, blue GM13355 proband PWS aected

son, green GM13556 aected daughter. Te Re Seq gene track in light green shows

the SNRPN gene mapped against chromosome band 15q11.2 and bp rom pter

band: 22.6 Mb to 22.78 Mb.

PLP1


19/28


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20

with one another and with environmental components (e.g. diet or

airborne exposure to toxins). Te complex path to neurodegenerative

disease makes it challenging to discover genetic susceptibility, and thus

slows the search or successul therapeutic intervention. At present,

the best strategy available to solve genetically complex disease involves

high-throughput genetic studies o large numbers o subjects.

Accomplishments and Goals o the NINDS Repository

Te NINDS Repository was ounded in 2002 to serve as a centralized

national and international resource to support investigators in over-

coming the challenges o managing thousands o biospecimens and as-

sociated clinical data. With samples rom more than 23,000 individuals

added since its inception, the NINDS collection is the astest growing

repository in the history o Coriell. During the rst six years o the

project, 16,737 DNA samples, 164 lymphoblastoid cell lines, and 200

DNA panels were distributed to researchers worldwide.

Te NINDS Repository was among the rst publicly available sources

or biomaterials associated with phenotypic and genotypic data. Eachsample in the NINDS collection is linked to clinical data including

gender, race, age, diagnosis, and medical history or hypertension, heart

disease, cancer, and neurological disorders. Te NINDS Repository,

playing a pioneering role in housing and distributing genotype/pheno-

type data, was a predecessor to dbGaP, the international resource that

now serves this unction through the National Center or Biotechnol-

ogy Inormation (NCBI).

A series o samples are available rom Coriell that have been character-

ized or mutations known to be associated with Parkinsons disease (e.g.

a-synuclein triplication) and ALS (e.g. superoxide dismutase). Addi-tionally, genome-wide single nucleotide polymorphism (SNP) data are

available or many samples. In an eort to increase the rate o discovery,

all o these data are reely and publicly available via the NINDS Reposi-

tory catalog: (http://ccr.coriell.org).

Samples and data in the NINDS Repository have been used or more

than ninety studies published in peer-reviewed scientic journals in-

cluding Science, American Journal o Human Genetics, and PLOS

One. Te pace o discovery is rapid, with reported results ranging rom

the discovery o disease-associated mutations (e.g. Simon-Sanchez et

al, 2007) to genomics tool development (e.g. Purcell, Sham and col-leagues, 2007). Tis state-o-the-art combination o biology and bioin-

ormatics sets the stage or breakthroughs that will translate the public

investment made by NINDS into improved diagnostic and prognostic

tools, new targeted therapeutics and, ultimately, relie or millions that

suer consequences o neurodegenerative disease.

Selected publications utilizing resources rom the

NINDS Repository:

Paisn-Ruz, C., Singleton, A.B., et al., (2008) Comprehensive analysis

o LRRK2 in publicly available Parkinsons disease cases and neurologi-

cally normal controls. Human Mutation 29:485-490.

Lesnick, .G., Maraganore, D.M., et al., (2008) Beyond Parkinson

disease: Amyotrophic lateral sclerosis and the axon guidance pathway.

PLOS One2(12): e1254.

Purcell, S., Sham, P.C., et al., (2007) PLINK: a toolset or whole ge-

nome association and population-based linkage analyses. American

Journal o Human Genetics81(3):559-75.

Simon-Sanchez, J., Singleton, A., et al., (2007) Genomewide SNP as-

say reveals mutations underlying Parkinson disease. Human Mutation

29:315-22.

Gwinn K., Corriveau, R.A., Keller, M.A., et al., (2007) Amyotrophic

lateral sclerosis: An emerging era o collaborative gene discovery. PLOS

One2(12): e1254.

For a ull listing o publications that use NINDS Repository samples

and data, please visit: http://ccr.coriell.org/Sections/Collections/NINDS/

Publns.aspx?PgId=490&coll=ND


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Collaborative For Discovery o Huntington Disease Markers:Call or Participants

Cooperative Huntingtons Observational

Research rial (COHOR) is a large

worldwide collaborative research eort

by Huntington Study Group (HSG) re-

search centers. Te goal o this study is to

discover phenotypic and biological mark-

ers o Huntingtons disease (HD) that can

be used to improve the treatment and prognosis o HD.

Advances in understanding the pathogenesis o HD have largely been

limited by the lack o availability o suitable phenotypic data that are

linked to biological markers or disease progression in individual pa-

tients. COHOR addresses this need by collecting biological samples

and phenotypic data rom consenting individuals who are aected by

HD or who are part o an HD amily. Te large descriptive COHOR

phenotypic database is used to study the natural history and progression

o HD, while biological specimens are used to investigate relationships

among HD genotypes and phenotypes. Biological specimens are pro-

vided to scientists or identication o useul biomarkers or HD.

Renewable biological resources or HD in the COHOR Repository,

together with corresponding clinical data, are a rich resource designed

to stimulate worldwide eorts or biomarker discovery in HD.

Who is eligible to participate in COHOR?

For individuals 18 years o age and older, the ollowing may partici-

pate:

Individuals who have HD or have tested positive for an HD gene

expansion.

Parents, grandparents, children, grandchildren, siblings, and spouses

o individuals who have HD or have tested positive or an HD gene

expansion. Family members who have tested negative or an HD gene

expansion are still included.

For individuals under the age o 18, only those who have HD are eli-

gible to participate.

How does one register to participate andprovide a sample or inclusion in COHOR?

Te Huntington Study Group (HSG) is an international association

o more than 200 clinical investigators, coordinators, scientists, and

sta rom 55 participating hospitals and universities in North America,

Europe, and Australia. Formed in 1993, the HSG strives to advance

knowledge about the cause, process, and clinical impact o HD in order

to develop and test promising therapeutic interventions.

I you are interested in learning more about this study contact the Hun-

tington Study Group at (800) 487-7671 or www.Huntington-Study-

Group.org, or visit the Huntington Project website at www.hunting-

tonproject.org. Te HSG and the Huntington Project are supported

by the Huntingtons Disease Society o America, the Hereditary Disease

Foundation, the Huntington Society o Canada, and the CHDI, Inc.

!


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Donald L. Coppock, Ph.D., is Associate Proessor and the Assistant Director o the Coriell Cell Reposi-tories (CCR). In this role, Dr. Coppock is the Principal Investigator o three NIH cell and DNA Repositories:the National Institute o General Medical Sciences Human Genetic Cell Repository, the National Institute on

Agings Aged Cell Repository, and the National Human Genetic Research Institutes Human Genetic Sample

Repository. Te goal or all three is to provide the scientic community with high-quality cell lines and DNAsamples rom individuals with a wide range o inherited conditions, samples or investigation o aging at the cel-lular level and or the study o human genetic variation. Among several other projects, Dr. Coppock is develop-ing a bioinormatic integration o the data rom the CCR with the inormation rom the other genetic databases.Previously, as the director o the Oncology Research Lab at Winthrop University Hospital in Mineola, NY, andas Associate Proessor at State University o NY, Stony Brook, Dr. Coppock developed two programs in cancerresearch: one investigating how the malignant melanoma cell diers rom the normal melanocyte in the regula-tion o cell growth and cell death, and the other investigating the mechanism by which normal cells arrest growthand how this diers rom tumorigenic cells. In the uture, his goal is to use the genetic and genomic inormationat Coriell to make an integrated resource or the study o human disease.

Donald L. Coppock, Ph.D.Associate Proessor, Coriell Institute or Medical Research

Assistant Director, Coriell Cell Repositories

Finding a Needle in the HaystacksBAPD was concentrated in only a ew am-

ily lines. Not only does bipolar behavior

contrast sharply with the communitys quiet

ways, making it easier to diagnose, but several

possible conounding behaviors are absent or

extremely rare: alcoholism, drug abuse, unem-

ployment, divorce and violence. At the time

Dr. Egeland initiated her studies in the 1960s

and 1970s, there was very limited acceptance

that one could nd a genetic basis or a psy-

chiatric disorder. Te study was unded by the

NIMH in 1976 and in 1987, Dr. Egeland

published a major paper on a potential link-

age to a marker or the cause o BAPD on

chromosome 11 1. Later, as the study was ex-

tended to include additional amily members

and controls, the probability o this linkage

coming rom Switzerland in the 1700s, pro-

vide a natural laboratory or genetic research.

A close-knit group that strongly discourages

marriage outside the community, they have re-

mained largely separate rom the surrounding

populations. Because o the genetic similarity

and the insularity, they are a special popula-

tion that provides a unique opportunity or

the study o inherited disorders. In addition,

the Amish have large amilies (seven children

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