Check Against Delivery.Embargoed until 3:45 PM, 6 November 2010

Targeting endogenous stem and somatic cell

by Karl Lenhard RudolphUlm University, Germany

Session 10, Workshop 10.2 „Emerging Therapies“Our Common Future, Essen, November 6th, 2010

Our Common Future, Hannover/Essen, 2-6 November 2010 (www.ourcommonfuture.de))

Targeting endogenous stem and somatic cells

Human life expectancy

Africa50.000 b.c.

Europe15.000 b.c.

19001980

Trauma

10 20 30 40 50 60 70 80 90 100

Age (years)

Sur

viva

l (%

)

100

50

44

Decrease inorgan function

T Rando, Nature 2006

Mechanisms of stem cell aging

Miller, Richard A.'Accelerated aging': a primrose path to insight?.Aging Cell 3 (2), 47-51.

Rothmund-Thomson Syndrome:near normal ifespan, cataracts, osteoporosis, hairgreying, alopeciaMutation in a DNA-helicase of unknown functionWerner Syndrom:- average lifespan: 50 years, alopecia, hair greying,osteoporosis, arteriosclerosis, diabetes, cataract, skinatrophy, teleangiektaisas mutation of WRN gene: DNA-helicase involved in DNA-repair by NHEJAtaxia teleangiectasia:skin atrophy, immunodeficiency, hair greying, neuronalcell lossATM-gene is involved in DNA damage signallingHutchinson-Gilford-Progeria:average lifespan 13 years, alopecia, wrinkling of skin,cachexia, arteriosklerosislamin A mutation: nuclear structure protein

Reviewed in Lieber et al., Nature Reviews 2004

DNA damage: a molecular mechanism of aging

Eliizabeth Blackburn

End replication problem of DNA-Polymerase:Telomeres shorten with each round of cell division

- simple repeat elements (TTAGGG)- no coding sequence- main function: chromosomal capping

cell nucleitelomere

chromosome

Telomeres

8

Telomere shortening limits proliferation ofhuman cells

Telo

mer

e Le

ngth

Cell Division Senescence Crisis

somatic cells

Hayflick 1968, Allsopp et al. 1990, Wright and Shay, 1992, Vaziri et al. 1994, Bodnar et al. 1998Hayflick 1968, Allsopp et al. 1990, Wright and Shay, 1992, Vaziri et al. 1994, Bodnar et al. 1998

p53p53stem cells

?

9

Telomere shortening in human tissues and stem cells

Affected organs:

Accelerated in chronic diseases:Anemia, arteriosklerosis, hepatitis, colitis ulcerosa, HIV, etc.

Blood cells, arteries, epithelia of the GI tract, kidney, liver, spleen, among others

Stem cells:CD34+ hematopoietic cells (Vaziri et al. PNAS 1994)

Telomerase mutations are linked to stem cell and organ failure:Dyskertaosis congenita, aplastic anemia, idiopathic lung fibrosis(Dokal, Besser, Collins, Amanios, Garcia and others)

Peptidomics (CE-TOF-MS) : biomarker oftelomere dysfunction and DNA damage

1

1

2

2

mTE

RC

-m

Terc

-/-m

Terc

+/+

jung alt

0 20 40 60 80 10020

40

60

80

100

Age

Co

mb

inati

on

sco

re

rho=0.5328 P<0.0001

0 20 40 60 80 1002

4

6

8

10

Age

Telo

mere

len

gth

(kb

)

rho=-0.318 P=0.0001

11

0.51-Specificity

1Young30±3.8

Old85±8.1

Old patients73±8.5

1

2

3

Mar

ker-

Com

bina

tion

P<0.001

P<0.001P<0.001

1

0.5 0.99720.98940.8863

Proteins from telomere dysfunctional cells arebiomarkers of human aging & disease

P<0.001

P<0.001P<0.001

12

G3 mTERC-/- (18 months)

mTERC+/+(22 months)

Impaired Wound Healing

Crypt Atrophy

Premature Aging

M1

mTERC+M1

G4 mTERC-

Impaired Stem CellMaintenance & Function

G4TERC-/-TERC+/+

mTERC+

G4mTERC-

mTERC+/+ G3 mTERC-/-

Impaired Liver Regeneration

Telomere dysfunctional mice

Rudolph et al. Cell 1999

surv

ival

(%)

20

40

60

80

100

3 9 15 21 27Age (months)

mTERC+/+ G1 G2-3 G4-5 G6

Reduced lifespan of telomere dysfunctional miceReduced lifespan of telomere dysfunctional mice

14

Correlation betwen telomere shortening andCorrelation betwen telomere shortening andhuman lifespanhuman lifespan

Cawthon et al. Lancet 2003

Cirrhosis- high regenerative reserve of hepatocytes- cirrhosis evolves after long latencies: 20-40 years- cirrhosis is characterized by an impaired regenerative capacity

Telomere shortening & organ regeneration

RNRN

FS

FSRN

100

200

300

controlsHC SC

p<0.0001 p<0.0001

TFI

HCcirrhosis

20%

60%

100%

cirrhosiscontrols

27/31

1/11

p=0.002S

enes

cent

HC

(%)

Wiemann et al. FASEB J. 2002

Hepatocellular telomere shortening and senescencecharacterize human cirrhosis

Telomere dysfunction induces DNA-damage checkpoints

Brown & Sedivy 1997

Chk2P

P

P

p53

p21

ATMGamma-H2AX

Apoptosis Cell CycleArrest

ATR

Chk1P

P

P

Exo1 RPARPA

PUMA

5 10 15 20

25

50

75

100

Age (months)

Surv

ival

(%)

G4 G4, p21-/-

Deletion of p21 elongates lifespan oftelomere dysfunctional mice

p21 limits stem cell functionin response to telomere dysfunction

19

Impaired self renewal and function of HSCs

and intestinal stem cells

CD34

G4

p21-

/-

M1M1

M1M1

10 0

E

vent p2

1+/+

10 110 2

10 3 10 410 0 10 1 10

2 103

104

mTR+FSC

0 200 400 600 800 1000

Sca1

Line

age

cKit

101

102

10

210

3

104

Choudhury et al., Nature Genetics 2007

Telomere dysfunc/on induces environmentaldefects limi/ng stem cell func/on

100

101

102

103

104

FL4-H: APC-CD11b

100

101

102

103

104

FL

1-H

: F

IT

C-B

22

0

31.7

59.5

100

101

102

103

104

FL4-H: APC-CD11b

100

101

102

103

104

FL

1-H

: F

IT

C-B

22

0

24.3

63.8

100

101

102

103

104

FL4-H: APC-CD11b

100

101

102

103

104

FL

2-H

: P

E-B

22

04.3

80.9

100

101

102

103

104

FL4-H: APC-CD11b

100

101

102

103

104

FL

2-H

: P

E-B

22

0

18.1

57.2

G4 mTR-

mTR+

Transplantation into mTR+ recipient

Transplantation of mTR+ HSC

G4 recipient

100

101

102

103

104

FL4-H: APC-CD11b

100

101

102

103

104

FL

1-H

: F

IT

C-B

22

0

3.98

87.2

100

101

102

103

104

FL4-H: APC-CD11b

100

101

102

103

104

FL

1-H

: F

IT

C-B

22

0

46.7

43.5

mTR+ recipient

Ju et al., Nature Medicine 2007

B22

0

Telomere dysfunction affects the stem cell niche and the macroenvironment

10 10 10 10 10

10

10

10

10

10

0.054

10 10 10 10 10

10

10

10

10

10

0.026

10 10 10 10 10

10

10

10

10

10

0.055

10 10 10 10 10

10

10

10

10

10

0.052

c-KitSca

12

mon

ths

12 m

onth

s

iF1 iG4

% o

f don

or d

eriv

edm

onon

ucle

ur b

lood

cel

ls

12 month old recipients

iF1iG4

0.260.55

iF1

11.560.48

iG4

p=0.0112

50

100

2 month old recipients

Unconditioned RecipientsUnconditioned Recipients Lethally irradiated RecipientsLethally irradiated Recipients

Telomere dysfunction induces environmental defectslimiting stem cell engraftment

Is it possible to increase lifespan

1000 year old sponges in the arctic ocean

Calorie reduced diet

- Increases lifespan of yeast- increases lifespan (30%) and reduces tumor risk in mice- Delays aging in primates

Aging telomere dysfunctional mice exhibit weight loss andCR-induced metabolic changes

High glucose diet increases lifespan of telomeredysfunctional mice

Glucose re-feeding reverts CR-profile of telomeredysfunctional mice

Group I HealthyGroup II MalnourishedGroup III Malnourished fed with high protein/calorie diet

Beaumont et al.

Glucose re-feeding ameloriates DNA damage signalling andimproves cell proliferation and organ maintenance

Calorie restriction may delay tissue aging by improving the clearance ofdamaged cells but this response could turn out to be deleterious at advancedage when tissues accumulate high rates of damaged cells.

- cell intrinsic checkpoints limit the function of adult stem cells in response toDNA damage and telomere dysfunction

- the abrogation of checkpoints can improve stem cell function and organmaintenance

- cell extrinsic checkpoints can limit the function of adult stem cells and theengraftment of transplanted cells possibly limiting cell transplantation therapies

- deficiencies in energy supply limit lifespan in the context of telomeredysfunction by amplifying checkpoint responses

Conclusions

Max-Planck-Partner Group atILAS, CAMS, Beijing, China:

Zhenyu Ju

Karin KleinhansAndré Lechel Hye-Min LeePallavi Mahaddalkar Pavlos MissiosKodandaramireddy NalapareddySatyavani RavipatiSundaram ReddyTao Si Zhangfa SongTobias SperkaHelen Qian SunStefan TümpelJianwei WangGuido von FiguraYuan Zhou

Cooperation:

Wolf-Georg Forssmann, Medical School Hannover – Serum FractionsDr. Thomas Illig, HZM - MetabolomicsChristoph Klein, Regensburg – Stem cell gene expression analysisMichael Manns, MHH, Hannover – human cirrhosis & HCCHarald Mischak, Mosaiques Diagnostics - PeptidomicsMichael Speicher, Austria – ArrayCGHRudi Westendorp, Leyden – human agingLars Zender, HZI – Deep Sequencing

Lab member:

Ali Hyder BaigKerstin Bauer

Yvonne Begus-NahrmannMartin BurghalterTang Duozhuang

Parisa EshragiClaire Fallandry

Juan FengLuis Miguel Guachalla

Cagatay GuenesDaniel Hartmann

Elena HoffmanAnett Illing

Sarah-Fee KatzAlexander Kleger

Funding:

Deutsche Forschungsgemeinschaft

Deutsche Krebshilfe e.V.

European Union (GENINCA,TELOMARKER)

Else-Kröner-Fresenius FoundationII E

lse-K

röner-

Fresen

ius S

ympo

sium

on S

tem C

ell A

ging,

May, 1

9-22 2

011

at the

Reis

ensb

urg in

Germ

any