Stem Cell Therapy for Vascular Disease Qingbo Xu, MD, PhD Cardiovascular Division King’s College London China-UK Forum in Biomedical Innovation 17-21 March 2008

*Background *Stem cells *Cardiovascular Disease *Stem cell repairing/therapy

Stem Cells and Vascular Diseases Proteomics Manuel Mayr

Cell biology Lingfang Zeng

Animal Model Yanhua Hu

Clinic/human Stefan Kiechl

Proteomics workflow Sample Prep

2-D Separation

Image acquisition

Sample labelling

Mass spectrometry

Image analysis Automated spot digestion Automated spot picking

Automated MALDI spotting

Adult and embryonic stem cell culture and differentiation into vascular cells

SMCs

Stem cells

Endothelial cells

Mouse models established in our laboratory • Spontaneous atherosclerosis • Endothelial injury of artery • Vein graft atherosclerosis • Heart infarction • Heart transplantation

With Movies For Surgical Procedures

Human/clinic studies, e.g. Bruneck Study

-Brief introduction about our work -Background about stem/progenitor cells -Human studies -Mechanistic studies on stem cell differentiation

Egg

Day 7

Sperm

Derivation of the ES cell lines. The ES cell lines were generated from the blastocysts. These cells were isolated by immunosurgery and plated on the MEF feeder layer. ES cells can be propagated in the undifferentiated state while being cultured on top of the MEF feeder layer. When they are cultivated in suspension as 3D cell aggregates (EBs), the ES cells differentiate into specialized cells.

Progenitor cell mobilisation and homing

Spleen

BM

Adipose

Liver

Stem cell pool in blood

Aorta

Intestine

EPC

SMP

AS Xu Q. Circ. Res. 2008

*Background *Stem cells *Cardiovascular Disease *Stem cell therapy

Endothelial cells

Stem cells

Smooth muscle cells

Atherosclerosis - a vascular blocking disease Treated with a balloon catheter, i.e. angioplasty

Treated with a balloon catheter, i.e. angioplasty

Coronary Artery

Endothelial cells

Endothelial cell lost

Repairing with stem cells

*Background *Stem cells *Cardiovascular Disease *Stem cell therapy: Mechanism of cell differentiation

Attached “progenitors” B

A

Wild-type 2 months

D

C

ApoE-/- 2 months

ApoE-/- 18 months

E

ApoE-/- 18 months

What is the mechanism for stem cell differentiation into vascular cells?

? Stem cells

SMCs

? Endothelial cells

Flow Shear Induces Stem Cell Differentiation: Model & Hyppothesis Shear stress

PDGF-BB

VEGF receptor

Receptor-ββ

PI3K

Akt HDAC3

eNOS

p53

NO

p21

HoxA

Endothelial markers

Collagen IV

FAK

HDAC7 HDAC7 splicing

MEF2C Smooth muscle markers Xu Q. Curr. Opin. Lipidol. 2007.

Flow Shear Induces Stem Cell Differentiation toward Endothelial Cells In vitro

In vivo matrigel Static/H.E

Shear/H.E

Shear/X-gal

Zeng et al. J. Cell Biol. 2006

Flow Shear Induces Stem Cell Differentiation toward Endothelial Cells

ESC

Sca1+

12h

C ST SH C ST SH

24h

ES ST SH ST SH CD31

Flk-1 CD133 CD144

eNOS Flk-1 Flt-1

α−tubulin

β-actin

Zeng et al. J. Cell Biol. 2006

Flow Shear Induces Stem Cell Differentiation: Impact of HDAC3 CTL CTLsi

HDAC1si

CTL CTLsi

HDAC3si

st sh st sh st sh st sh

st sh st sh st sh st sh Flt-1

Flt-1

p53K317

p53K370

p53

p53

p21

p21

HDAC1

HDAC3

α-tubulin

α-tubulin

Flow Shear Induces Stem Cell Differentiation: Model & Hyppothesis Shear stress

PDGF-BB

VEGF receptor

Receptor-ββ

PI3K

Akt HDAC3

eNOS

p53

NO

p21

HoxA

Endothelial markers

Collagen IV

FAK

HDAC7 HDAC7 splicing

MEF2C Smooth muscle markers Xu Q. Curr. Opin. Lipidol. 2007.

HDAC7 and SMC differentiation

Days of ES/Col IV 0

105KD

42KD

1

3

5

7

9

mSMC HDAC7

SMA

Merge

HDAC7

Calponin

Merge

HDAC7

SMA

34KD

Calponin

50KD

α-tubulin

HDAC7 and SMC differentiation

**

Mock

Mock

HDAC7

HDAC7

HDAC7-1

*

HDAC7-2 8.0

HDAC7-2 4.0

**

*

3.0 RLU

6.0 RLU

HDAC7-1

4.0

2.0

2.0

1.0

SMA-Luc

SM22-Luc

SMA-Luc

SM22-Luc

HDAC7 and SMC differentiation siRNA-HDAC7-1

Adenovirus-HDAC7-1 2

5

10

SM22

20 MOI

Luc HDAC7

PBS

4.0

PDGF SMA

RLU

0

2.0 Calponin

α-tubulin ctlsi

HDAC7si

Conclusions HDAC3 is crucial for stem cell differentiation into endothelial cells, while HDAC7 is for SMCs

HDAC7

Stem cells

SMCs

HDAC3

Endothelial cells

Can progenitor cells differentiate to endothelial cells in vivo in response to flow shear? Shearing stem cells in vivo

Isolation of sca-1+ progenitors

Apply the cells to denuded artery

Endothelial phenotype

Flow shear Induces Stem Cell Differentiation: Endothelial cell therapy

X-gal

β-gal

CD31

Zeng et al. J. Cell Biol. 2006

Flow Shear Induces Stem Cell Differentiation: Sca-1+ cell therapy

Xiao et al. ATVB 2006

Replace blocked vessels with a normal vessel, e.g. saphenous vein

blocked vessels

Normal vessel

Vein graft atheroma

Stem cell differentiation

SMCs Stem cells

Endothelial cells

Mouse model of vein bypass graft atherosclerosis Carotid artery

Vessel segment

QuickTime™ and a YUV420 codec decompressor are needed to see this picture.

Ongoing work: replacing diseased artery with stem cell-derived vessel

stem cell-derived vessel

Summary -Two types of stem cells: embryonic and adult cells -VEGF plays a crucial role of stem/progenitor cells homing to the damaged vessels -Signal pathways for endothelial differentiation involving in HDAC3-p53/p21 -Stem cell-derived endothelial cells can repairing damaged vessels in vivo

Innovation & Collaboration New drug discovery to direct stem cell differentiation into endothelial cells, e.g. HDAC7 inhibitor or HDAC3 enhancer Biomaterials for vascular production Stem cell therapy in patients

Prof Q Xu

Dr T N Tsai

Ms M Prokopi

Dr M Mayr

Mr Z Zhang

Dr A Zampetaki

Ms X Bai

Mr S Alam

Dr L Zeng

Dr G Pula

Dr A Margriti

Mrs S King

Ms A E Pepe

Ms Z Luo

Dr X Yin

Dr E De Falco

Ms F Zhang

Dr Q Xiao

Ms A Sidibe

Dr U Mayr

Mr D Martin

Dr H Li

Dr J P Kirton

Dr Y Hu