Stem Cell Therapy for Vascular Disease

Qingbo Xu, MD, PhDCardiovascular DivisionKing’s College London

China-UK Forum in Biomedical Innovation 17-21 March 2008

*Background

*Stem cells

*Cardiovascular Disease

*Stem cell repairing/therapy

ProteomicsManuel Mayr

Stem Cells and Vascular Diseases

Cell biologyLingfang Zeng

Animal ModelYanhua Hu

Clinic/humanStefan Kiechl

Proteomics workflowSample Prep

Image analysis

Sample labelling

2-D Separation

Automated spot picking

Automated spot digestion

Automated MALDI spotting

Mass spectrometry

Image acquisition

Endothelial cells

Stem cells

SMCs

Adult and embryonic stem cell culture and differentiation into vascular cells

Mouse models established in our laboratory

• Spontaneous atherosclerosis

• Endothelial injury of artery

• Vein graft atherosclerosis

• Heart infarction

• Heart transplantation

With MoviesFor SurgicalProcedures

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

Derivation of the ES cell lines. The ES cell lines were generated from the blastocysts. These cells were isolated by immunosurgeryand 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.

Egg Sperm

Day 7

Progenitor cell mobilisation and homing

Stem cellpool

in bloodAorta

Adipose

BM Spleen

Liver

Intestine

SMP EPC

AS

Xu Q. Circ. Res. 2008

*Background

*Stem cells

*Cardiovascular Disease

*Stem cell therapy

Endothelial cells

Smooth muscle cells

Stem cells

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

CoronaryArtery

Treated with a balloon catheter, i.e. angioplasty

Endothelial cells

Repairing with stem cells

Endothelial cell lost

*Background

*Stem cells

*Cardiovascular Disease

*Stem cell therapy: Mechanism of cell differentiation

Attached “progenitors”A B C

D EWild-type 2 months ApoE-/- 2 months ApoE-/- 18 months

ApoE-/- 18 months

Endothelial cells

Stem cells

SMCs

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

?

?

Flow Shear Induces Stem Cell Differentiation:Model & Hyppothesis

Xu Q. Curr. Opin. Lipidol. 2007.

Shear stress

VEGF receptor

Endothelial markers

Akt

eNOSHDAC3

p53

p21

NO

HoxA

Smooth muscle markers

HDAC7

PI3K FAK

HDAC7 splicing

MEF2C

PDGF-BB Collagen IV

Receptor-ββ

Flow Shear Induces Stem Cell Differentiation toward Endothelial Cells

Static/H.E

Shear/H.E

Shear/X-gal

Zeng et al. J. Cell Biol. 2006

In vitro In vivo matrigel

Flow Shear Induces Stem Cell Differentiation toward Endothelial Cells

Flk-1

eNOS

α−tubulin

ES ST SH ST SH

12h 24h

CD31

CD133

CD144

Flk-1

Flt-1

β-actin

ESC Sca1+

C ST SH C ST SH

Zeng et al. J. Cell Biol. 2006

Flow Shear Induces Stem Cell Differentiation:Impact of HDAC3

Flt-1

p53K317

α-tubulin

p53

HDAC1

p21

st sh st sh st sh st sh

CTL CTLsi HDAC1si

st sh st sh st sh st sh

CTL CTLsi HDAC3si

Flt-1

p53K370

α-tubulin

p53

HDAC3

p21

Flow Shear Induces Stem Cell Differentiation:Model & Hyppothesis

Xu Q. Curr. Opin. Lipidol. 2007.

Shear stress

VEGF receptor

Endothelial markers

Akt

eNOSHDAC3

p53

p21

NO

HoxA

Smooth muscle markers

HDAC7

PI3K FAK

HDAC7 splicing

MEF2C

PDGF-BB Collagen IV

Receptor-ββ

HDAC7 and SMC differentiation

HDAC7

SMA

Calponin

α-tubulin

105KD

42KD

34KD

50KD

0 1 3 5 7 9 mSMC

Days of ES/Col IV

HDAC7 SMA Merge

HDAC7 Calponin Merge

HDAC7 and SMC differentiation

1.0

2.0

3.0

4.0

SMA-Luc SM22-Luc

R L

U

2.0

4.0

6.0

8.0

SMA-Luc SM22-Luc

R L

U

Mock

HDAC7

HDAC7-1

HDAC7-2

Mock

HDAC7

HDAC7-1

HDAC7-2

**

** *

*

HDAC7 and SMC differentiation

Adenovirus-HDAC7-1

0 2 5 10 20 MOI

HDAC7

SMA

α-tubulin

Calponin2.0

4.0

LucSM22

PBSPDGF

ctlsi HDAC7siR

L U

siRNA-HDAC7-1

Endothelial cells

Stem cells

SMCs

ConclusionsHDAC3 is crucial for stem cell differentiation into endothelial cells, while HDAC7 is for SMCs

HDAC3

HDAC7

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

β-gal CD31X-gal

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

Endothelial cells

Stem cells

SMCs

Stem cell differentiation

Mouse model of vein bypass graft atherosclerosis

Carotid artery

Vessel segment

QuickTime™ and aYUV420 codec decompressor

are needed to see this picture.

stem cell-derived vessel

Ongoing work: replacing diseased artery with 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 M Mayr Mr Z Zhang Dr G Pula

Dr A Zampetaki Dr X YinMs X Bai Ms F Zhang Dr Q Xiao

Dr L Zeng

Dr A Margriti

Mr D MartinDr U Mayr Dr Y Hu

Mrs S King Ms A E Pepe

Ms A Sidibe

Ms Z Luo

Dr E De Falco

Dr J P KirtonDr H LiMs M Prokopi Mr S Alam

Dr T N Tsai