Klinikum derJohann Wolfgang Goethe Universität

Frankfurt am Main

What´s new: On cell processing and What´s new: On cell processing and mobilisation?mobilisation?

Stefanie DimmelerStefanie Dimmeler

Supported by:DFG (SFB 553, FOR 501), the European Network of Excellence(EVGN) and the Transatlantic Network of Excellence (Leducq Foundation)

Vas

cula

r zon

eV

ascu

lar z

one

Stromal cells

HematopoieticProgenitor Cells

Ost

eobl

astic

zon

eO

steo

blas

tic z

one

mKitL

PhysiologicalPhysiological

VEGFVEGF SDFSDF ExerciseExercise

IschemiaIschemia

Mobilisation and Homing of Endothelial Progenitor Cells

(Signal)(Signal)

sKitL (SCF)

PharmacologicalPharmacological

G-CSFG-CSF GM-CSFGM-CSF EPOEPO StatinsStatins Dickkopf-1Dickkopf-1

G-CSFVEGF (gene therapy)ErythropoietinCXCR4-inhibitorsIntegrin 4 inhibitorsDickkopf-1

Mobilisation of progenitor cells

StatinsExersizeEstrogenPPAR ag.ACE inh?

Pro-inflammatory?Gene therapy? Permeability?Thrombotic? In clinical trialsMay block homing of EPCEffect on homing??

Positive effects on progenitor cell functionsModerate „mobilisation“

Mobilisation Pros and Cons

G-CSF:

Mobilisation by G-CSF

-Benefit in experimental studies (early, combination therapy)-Clinical benefit lacking in most trialsMetaanalysis: Zohlnhoefer et al, AHA 2007

-Useful to enrich progenitor cell populationsErbs et al, Circ Res 2006 (chronic coronary artery occlusion);Losordo et al, Circ 2007 (intractable angina)

Why does G-CSF fail?

-Early treatment necessary (?)-Cells are scavanged in other organs (animal studies have been done with splenectomy)-Cleavage of the CXCR4 receptor inhibits homing

SDF-1

Before after G-CSF

6H8+ EPC% of cultivated EPC absolute number/µl blood

Before after G-CSF

0

5

10

15

20

mig

rate

d ce

lls (x

1000

)

* *

0

10

20

30

40

50

60

%

6H

8+ cul

tivat

ed E

PC

0

10

20

30

6H8+

EPC

/ µl b

lood

Before after G-CSF

Mobilisation

Bone marrow

StemCells

CXCR4

SDF-1(stroma-derived

factor-1)

6H8+ epitop

G-CSF

Proteases

SDF-1 Hypoxia(Yamaguchi Circulation 2003;

Ceradini Nat Med 2004; Askari, Lancet 2004; Walter Circ Res 2005)

CXCR4

Honold et al, ATVB 2006

(Levesque JCI 2003)

5-6aa

Homing of progenitor cells

Mobilisation by G-CSF

Ost

eobl

astic

O

steo

blas

tic

zone

zone

Vasc

ular

Va

scul

ar

zone

zone

Osteoblasts

Stem cell Stem cell nicheniche

Integrins (VCAM)SDF-1/CXCR4 Heart

Beneficial effects in animal models

(mobilisation, direct effect on cardiac myocytes)

Increase inflammation -> atherosclerosis, plaque

stability?

G-CSF:

Mobilisation by G-CSF: pro-inflammatory effects?

Mobilisation without inducing inflammation?Mobilisation of tissue-resident stem cells?

Osteoblast

Osteoclast

Mobilisation of progenitor cells by RANKL and Wnt antagonists

Proteases(CathK, MMP9)

Wnt

-RANKL

Spencer, J Cell Sciences 2005

RANKL

+

RANKL

Dickkopf

• RANKL induces the mobilisation of immature SKL cells but not inflammatory cells (Kollet et al,Nat Med 2006)

Mobilisation

Bone marrow stem cell niche

TOP-gal transgenic mice identify sites of Wnt signaling in the bone marrow

Tracecular bone

X-gal staining

Long bone

Wnt

AxinAPC

GSK3catenin

AxinAPC

GSK3

catenin

Dishevelled

LEF/TCFcatenin lacZ

FrizzledLRP5/6

?Dkk-1

Dkk-1 treatment blocks Wnt signaling in the bone marrow stem cell niche

control

DKK-1

Control

Axin 2

GAPDH

PBS Dkk

Inhibition of Wnt-targetgene expression:

Dickkopf stimulates mobilisation of EPC without inducing a pro-inflammatory effect

PBS Dkk PBS Dkk

Day 4 Day 7

0

5

10

15

20

25

30

35

P<0.05 P<0.01

Num

ber o

f EC

-CFU

per

wel

l

Day 4 Day 7

PBS DkkG-CSF PBS DkkG-CSF

P<0.01

Num

ber o

f Gr-

1+ CD

45+ g

ranu

locy

tes

(% o

f con

trol

)

0

50

100

150

200

Progenitor cells Inflammatory cells

•Dkk-1 significantly increases Sca-1/Flk-1 and Sca-1/c-kit cells

Dkk and RANKL interaction, mobilisation and neovascularization

EC-C

FU

050100150200250300350400450

PBS RANKL

*

0100200300400500600

PBS RANKL

*nu

mbe

r of S

M+

vess

els

(% P

BS)

GAPDH

PBS24h 48h 48h 48h 72h

mDKK-1

RANKL

00,51

1,52

2,53

3,5

PBS 24 h 48 h 72 h

RA

NK

L m

RN

A e

xpre

ssio

n (r

atio

Dkk

/GA

PDH

)

RANKL Expression EPC-Mobilisation

Neovascularization

Summary

•Active Wnt signaling in the stem cell niche(Stabilization of -catenin results in expression of -galactosidase (blue) mainly in endosteal cells )

Without Dkk-1 With Dkk-1

Endosteal layer

LEF/TCFcatenin lacZ

LEF/TCFcatenin lacZ

Mobilization of progenitor

cells

RANKL

•Inhibition of Wnt signaling in the stem cell niche•Expression of RANKL in osteoclasts•Mobilisation of progenitor cells•No induction of inflammation

What new:Cell processing and

isolation?

-Number and viability are not the only parameter that count: function is important-Check the quality of the cells in vivo

-Details matter!

rela

tive

perf

usio

n (%

left

limb)

0102030405060

day 7 day 14

*

Hind Limb Ischemia

Recovery of perfusion after injection of 1x106

o/n stored BMC

* p<0.05 vs. Lymphoprep

n=8

Lymphoprep

Ficoll *

(Seeger et al, Eur Heart Journal 2007)

SDF-1

Inflammatorycytokines

HMGB-1

VEGF Hypoxia

Inflammation

Necrosis

?

(Palumbo JCB 2004Zacheo et al, 2005)

(Yamaguchi Circulation 2003; Ceradini Nat Med 2004; Askari,

Lancet 2004; Walter Circ Res 2005)

CXCR4

VEGF-R1VEGF-R2

Receptor Stimulus

RAGE(TLR)

Integrin activation

Regulation of progenitor cell homing

(Asahara et al, 1999;Iwaguro et al Circ 2000)

EPC

In vitro Migration capacity predicts functional In vitro Migration capacity predicts functional improvement in vivoimprovement in vivo

Migratory capacity

60

40

20

0

-20

-40

-60

Infa

rct s

ize

[ml]

(late

enh

ance

men

t by

MR

I)

p = 0.006

low highLaser Doppler-derived blood flow (ischemic/non-ischemic)

SDF-

1-in

duce

d m

igra

tion

0

50

100

150

200

250

0 0.2 0.4 0.6 0.8 1.0

r = 0.78p < 0.001

Hind limb ischemia model Hind limb ischemia model TOPCARE-AMI trialTOPCARE-AMI trial

Heeschen et al, Circ 2004 Britten et al, Circ 2003

matrigel 24 hours± SDF-1

Storage experiments I

Inva

sion

(x10

3 ) /1

06 B

MC

x-vivo 10serum

0

20

40

60

80

100

120

basal SDF-1 basal SDF-1 basal SDF-1

0.9% NaClserum

x-vivo 10heparin-plasma

+

#*

§

§

§

Isolation of BMC according to the Ficoll-protocol

over-night storage

Invasionassay

§ p<0.05 vs. basal

+ p=0.05 vs. SDF-1 x-vivo 10 + serum # p<0.05 vs. basal x-vivo 10 + serum

* p<0.05 vs. SDF-1 x-vivo 10 + serumn=4

Storage experiments II

Isolation of BMC according to the Ficoll-protocol

over-night storage

Invasionassay

Inva

sion

(x10

3 ) /1

06 B

MC

basal SDF-1

RT RT4°C 4°C

* p< 0.05 vs. RT

0

20

40

60

80

100

120

140

*

CXCR4-FACS-Analysis after Storage

mean CXCR4 expression(area under curve)

CXCR4 positive cells per 10 ml BM

CXC

R4

posi

tive

cells

(106 p

er 1

0 m

l BM

)

Ficoll Lymphoprep

400

800

1200

1600

*

* p<0.05 vs. Ficolln≥7

mea

n C

XCR

4-ex

pres

sion

(are

a un

der c

urve

)10

20

30

40

*

Ficoll Lymphoprep

Can we identify markers of cell quality and cell function that

determine functionalrecovery in AMI patients of the

REPAIR-AMI trial?Number, surface markers (CD34, CD133, KDR), MSC, colony assays, Migration, contamination with inflammatory cells or red blood cells

1.21.00.80.60.40.20

30

20

10

0

-10

-20

Abs

olut

e ch

ange

in

LVEF

(bas

elin

e to

4 m

onth

s (%

)

Contamination with erythrocytes (RBC, * 109)

n = 91, r = 0.14, p = 0.23

Placebo

1.21.00.80.60.40.20

30

20

10

0

-10

-20

Contamination with erythrocytes (RBC, *109)

n = 94, r = -0.25, p = 0.02

BMC

Contamination of the BMC preparation with RBC is associatedContamination of the BMC preparation with RBC is associatedwith reduced contractility improvement after 4 monthswith reduced contractility improvement after 4 months

Tertiles of SDF-1 induced BMC migration normalized for RBC contaminationTertiles of SDF-1 induced BMC migration normalized for RBC contamination

1. tertile 2. tertile 3. tertile

10

8

6

4

2

0

1. tertile 2. tertile 3. tertile

P for trend 0.39 P for trend 0.03

Placebo BMC

SDF-1 induced migration normalized for RBCcontamination predicts functional recovery

Abs

olut

e ch

ange

in g

loba

l LVE

F (

%)

RBC addition abolishes SDF-1 induced BMC migration in vitro

Experimental confirmation

1 x 106 BMC ± X* 106 RBC

0

10

20

30

40

50

60

70

80

90

100

BMC only BMC+5*106 RBC BMC+1*106 RBCBMC+0.1*106 RBC

SDF-

1 in

ducc

ed in

vasi

on(a

bs. m

igra

ted

cells

)p = n.s.

p < 0.01

p < 0.01p < 0.01

P for trend (control) = 0.05P for trend (SDF-1) = 0.003

controlSDF-1 N > 4

24 hours

Matrigel

± SDF-1

Overall (primary endpoint)Overall (primary endpoint)

Baseline LV-EFBaseline LV-EF 48.9 % 48.9 % (n= 93)(n= 93)

> 48.9 % > 48.9 % (n=94)(n=94)

Timing Timing of infusionof infusion(days after AMI)(days after AMI)

4 days 4 days (n=107)(n=107)

5 days5 days (n=80) (n=80)

ProcessingProcessing& delivering time& delivering time(aspiration to infusion)(aspiration to infusion)

same daysame day (n=101) (n=101)

next daynext day (n=86) (n=86)

-2-2 00 22 44 66 88favoursfavours

BMCBMCfavoursfavoursplaceboplacebo

Treatment effect on LVEF Treatment effect on LVEF ((% with 95% CI)% with 95% CI)

p forp for interactioninteraction

p = 0.020p = 0.020

p = 0.029p = 0.029

p = 0.81p = 0.81

Timing ofTiming ofinfusioninfusion(days after AMI):(days after AMI):

4.25 4.25 1.61.64.36 4.36 1.11.1(p = 0.32)(p = 0.32)

Predictors for improvement in LVEF after 4 months

Multivariate analysis for improvement in LVEFafter 4 months within the BMC group

Time to treatment

Baseline LVEF

SDF-1 induced migration(normalized for contaminating RBC)

0.70

0.03

0.02

p-value Standardized Coefficient

0.04

-0.24

0.23

Significance (ANOVA)=0.01

Baseline LVEF and SDF-1 induced migrationindependently predict contractility recovery

Conclusion

Quality of cell isolation (Viability and RBC contamination) and Functionality of the infused cells (SDF-1 induced migration) after

the isolation procedure

determine the extent of contractile recovery after intracoronary

BMC infusion in acute MI.

These results demonstrate an association between functionality of the infused BMC and functional recovery suggesting a bioactivity response relationship very much like a dose-response relationship in drug trials.

For future clinical trials RBC contamination may be used as ad hoc quality control in addition to functional testing of the infused cells.

Steering CommitteeSteering Committee

Hamburg

LeipzigBad Berka

Suhl

Bad Oeynhausen

Lippe

Homburg/Saar

ZürichZürich

Mannheim

Ludwigshafen Frankfurt(2 centers)

BadNauheimGiessen

Mainz

Kassel

Study Centers and Core Facilities

S. Erbs / R. HambrechtS. Erbs / R. Hambrecht

V. Schächinger /V. Schächinger /B. Assmus / S. DimmelerB. Assmus / S. Dimmeler

A. M. Zeiher (PI)A. M. Zeiher (PI)

A. Elsässer / M. Stanisch /A. Elsässer / M. Stanisch /T. Dill / Ch. HammT. Dill / Ch. Hamm

W. HaberboschW. Haberbosch

H. Hölschermann /H. Hölschermann /H. TillmannsH. Tillmanns

J. Yu / B. LauerJ. Yu / B. Lauer

R. Corti / T. LüscherR. Corti / T. Lüscher

D. Mathey / T. TüblerD. Mathey / T. Tübler

T. Süselbeck / M. Brückmann /T. Süselbeck / M. Brückmann /K. HaaseK. Haase

G. Nickenig / N. Werner /G. Nickenig / N. Werner /M. BöhmM. Böhm

J. HaaseJ. Haase

C. Hansen / J. NeuznerC. Hansen / J. Neuzner

A. Germing / A. MüggeA. Germing / A. Mügge

B. Mark / J. SengesB. Mark / J. Senges

C. Hoffmann / M. Farr /C. Hoffmann / M. Farr /D. HorstkotteD. Horstkotte

A. Cuneo / U. TebbeA. Cuneo / U. Tebbe

S. Genth-Zotz /S. Genth-Zotz /T. MünzelT. Münzel

Bochum

Cell Processing CenterCell Processing Center

T. Tonn / N. Krzossok/T. Tonn / N. Krzossok/E. SeifriedE. Seifried

Safety CommitteeSafety CommitteeT. Bonzel / W. KasperT. Bonzel / W. Kasper

Coordinating CenterCoordinating CenterH. BraunH. Braun

MRI Core LabMRI Core Lab

Doppler Core LabDoppler Core Lab

Echo Core LabEcho Core Lab

Angio Core LabAngio Core Lab

www.REPAIR-AMI.orgwww.REPAIR-AMI.org400 km

Klinikum derKlinikum derJohann Wolfgang Goethe UniversitätJohann Wolfgang Goethe Universität

Frankfurt am MainFrankfurt am Main

Collaborators:Collaborators:

Pediatric Cardiology, GiessenPediatric Cardiology, GiessenS. Rupp, D. SchranzS. Rupp, D. SchranzWeizman InstituteWeizman InstituteO. Kollet, T. LapidotO. Kollet, T. Lapidot

Andreas ZeiherAndreas Zeiher

V. SchächingerV. SchächingerB. AssmusB. AssmusR. LehmannR. LehmannJ. HonoldJ. HonoldU. Fischer-RasokatU. Fischer-RasokatM. Britten/C. TeupeM. Britten/C. Teupe

Clinical Studies:Clinical Studies:

Experimental Experimental Studies:Studies:

C. UrbichC. UrbichC. HeeschenC. HeeschenA. AicherA. Aicher, K. Sasaki, K. SasakiL. Rössig,L. Rössig,I. SpyridopoulosI. SpyridopoulosF. SeegerF. SeegerE. ChavakisE. ChavakisC. BadorffC. BadorffM. KoyanagiM. KoyanagiM. IwasakiM. Iwasaki

* p<0.05 vs. Lymphoprep

Invasion capacity after storage

20

40

60

80

100

120

140

*

*

Inva

sion

(x10

3 ) /1

06 BM

C

+

basal SDF-1 basal SDF-1

Ficoll Lymphoprep

Healthy controls

basal SDF-1 basal SDF-1

*

Inva

sion

(x10

3 ) /1

06 BM

C

+

Ficoll Lymphoprep

CAD-patients

20

40

60

80

100

120

140

Mobilizable circulating mesoangioblasts (MAB)

Vessel- associated mesoangioblast

differentiate todifferentiate toblood, cartilage, blood, cartilage, bone, and musclebone, and muscle

Embryonic dorsal aorta

Endothelial markers (KDR) / mesenchymal markers (CD73)Lack of hematopoietic markers

• Do circulating MAB resemble embryonic stem cells ?• Can specific factors mobilize MAB?

Circulating children-derived cells resemble embryonic mesoangioblasts

Questions

Mobilization of mesoangioblasts in children and adults during open heart surgery

child cells

CD45

KDR

CD73

GAPDH

adult cellsH 2O

Cardiopulmonary bypass mobilized cellsMobilisation in adults?

200

100

0

300

400pre post

*

EGF

OSM Ct1

IL16

bNGF

IL12

VEGF-

DBD

NF IL2

VEGF

BMP-

7IL

18al

pha

TGFb

eta

IL10

BMP-

4TG

Falp

haan

gios

tatin IL6

SCF

angi

opoe

tin2

IL1b

eta

SDF-

1IL

1alp

haPD

GFRbe

taIG

F1PD

GFRalp

haHG

FPD

GF-AA

Groalp

haPD

GF-BB

% (post / pre-operation)

pre

post HGF

Cytokine profile during extracorporal circulation?

HGF induces the migration of cardiac stem cells-> Mobilisation?

Infusion of HGF mobilizes mesoangioblast-like cells in vivo

c-Met

different donors

GAPDH

c-Met

cMABisotype control

Human MAB express the HGF-receptor

PBS

HG

F

different rats

H 2O

-RT

CD45KDR

CD73GAPDH

colo

nies

/

10 m

l blo

od

PBS

HGF

*Mobilisation of MAB in rats (colonies)

Mobilisation of MAB in mice (FACS)

PBS

HGF 1µg/kg

HGF 5µg/kg

num

ber /

106 c

ells

num

ber /

µl

PBS

HGF 1µg/kg

HGF 5µg/kg

HGF mobilizes mesoangioblast-like cells in experimental models

Wnts

is expressed in MSCpromotes proliferation of MSCincreases osteoblasts

(Gregory et al. JBC 2003)

Dkk

Role of Wnt and Dickkopf (Dkk) in the bone marrow stem cell niche

are expressed by HSC and stromal cellspromote self renewal, clonal expanison of HSC

(Reya et al. Nature 2003; Willert et al. Nature 2003, Trowbridge et al. Nat Med 2006)

Wnt

Dsh

GSK-3ß

ß-cateninstabilisation

Dkk

Mao et al. Nature 2002

Rattis et al., Curr OpinHematol 2004

Self-renewal cues

Self-renewal cues

Trabecular bone

Long bone

Dkk-1 increases the number of EC-CFU

Num

ber o

f EC

-CFU

per

wel

l0

5

10

15

20

25

30

35

40

PBS

0.1 0.5

Dkk (mg/kg); Day 7

P<0.05

PBS Dkk PBS Dkk

Day 4 Day 7

0

5

10

15

20

25

30

35

P<0.05

P<0.01

Num

ber o

f EC

-CFU

per

wel

l

0

10

20

30

40

50

60

PBS Dkk sFRP G-CSF

Num

ber o

f EC

-CFU

per

wel

l

Day 7

P<0.01

P<0.01

P<0.01

Comparison of EC-CFU mobilization by Dkk-1, sFRP-1, and G-CSF

0

10

20

30

40

50

60

70

80

PBS Dkk G-CSF PBS Dkk G-CSF

Num

ber o

f GM

-CFU

per

wel

l

G-CSF, but not Dkk-1 increases the number of GM-CFU

Day 4 Day 7

P<0.001

X-gal staining in the BM of TOP-gal mice is preferentially found at the endosteal layer

Osteoblast

Hematopoietic cell

Hematopoietic cell

Osteoclasts

Osteoblast

Hematopoietic cell

Osteoblast

Osteoblasts

Summary II

Stabilization of -catenin results in expression of -galactosidase (blue)

mainly in endosteal cells

Without Dkk-1

With Dkk-1

Mechanisms?

1. Release of factors by endosteal cells to

selectively target EPCs

2. Blockade of HSC formation and facilitation of EPC differentiation

Endosteal layer

LEF/TCFcatenin lacZ

LEF/TCFcatenin lacZ

Mobilization of EPCs

Regulation of progenitor-cell homing

Walter et al. 2005SDF-1 Hypoxia

AMIYamaguchi, Circulation 2003

Ceradini, Nat Med 2004Askari, Lancet 2004

Walter, Circ Res 2005

CXCR4

recruitment / homing of EPC

EPC/BMC

Placebo BMC

Impr

ovem

ent i

n LV

EF

Impr

ovem

ent i

n LV

EFSDF-1 induced migration

(normalized for RBC contamination)SDF-1 induced migration

(normalized for RBC contamination)

n = 91, r=-0.16, p=0.14 n = 94, r=0.33, p=0.001

Functional capacity (SDF-1 induced migration) and purity of isolation (RBC contamination) predict BMC-mediated

contractile recovery in patients with AMI

Influence of ex vivo cell migration on functional recovery