INNOVATIVE METHODS OF APPLICATION OF STEM CELLS IN MEDICINE

Michał Tendera MDWojciech Wojakowski MD

THIRD DIVISION OF CARDIOLOGYMEDICAL UNIVERSITY OF SILESIA, KATOWICE, POLAND

POLISH PROJECT ON VERY SMALL EMBRYONIC-LIKE STEM CELLS(VSELs) IN CARDIOLOGY

VSELs

Cytometry A. 2009 January; 75(1): 4–13.

IMAGE STREAM

Cytometry A. 2009 January; 75(1): 4–13.

Sca-1+lin-CD45+

Sca-1+lin-CD45-

2 m 2 m 2.5 m

1 m 2 m

4 m

A

B

1 m 1 m

Kucia et al. Leukemia 2006,20:857-869

TEM of murine Sca-1+lin- CD45- (VSEL) and Sca-1+lin- CD45+ (HSC) cells

VSELs ARE PROGENY OF EPIBLAST-DERIVED CELLS

Shin et al. Leukemia, 2009: 23(11): 2042

REGULATION OF QUIESCENCE AND PLURIPOTNCY

RECOVERY OF GENOMIC IMPRINTING DURING FORATION OF VSEL-DS

Shin et al. Leukemia, 2009: 23(11): 2042

REGULATION OF QUIESCENCE AND PLURIPOTNCY

MURINE VSEL-DERIVED CM

C2C12 myoblasts7 days

Re-sorting

GFP+ VSEL

Tripsinization

GFP+ VSEL

C2C12

C2C12

DMEM + 2% FBS37oC, 5% CO2.

Wojakowski et al. Int J Onc, 2010, in press

VSEL-DERIVED CM

5x104 CELLS

CA

RD

IAC

DIF

FE

RE

NT

IAT

ION

0

20

40

60

80

100

120

Nkx 2.5/Csx GATA-4 Troponin I Mybpc3 actinin 2 actinin 3

MNC

VSEL

DAY 4

DAY 6

DAY 9

DAY 12

DAY 16

m

RN

A(f

old-

diff

eren

ce)

Wojakowski et al. Int J Onc, 2010, in press

VSEL-DERIVED CM

SORT II

Cardiac pre-differen.

VSEL Expansion culture

C2C12 feeder layer 9 days

EGFP transgenic mice [C57BL/6]

BM isolation SORT I

VSELs

HCSs

WT mice [C57BL/6]

afterinfarction

Injection 5 days6 monthsof follow-up

Injection

MURINE MODEL OF ACUTE MI

0

10

20

30

40

50

60

70

80

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0

1

2

3

4

5

6

7

* † * † * †

%

BSL 48 h 1 mo 3 mo 6 mo

LV Ejection Fraction

mm

† * †

BSL 48 h 1 mo 3 mo 6 mo

LV End-systolic Diameter

Infarct Wall Thickness(Diastole)

* †

BSL 48 h 1 mo 3 mo 6 mo

mm

* P < 0.05 vs. Vehicle-treated † P < 0.05 vs. HSCs

Gr I (Vehicle)

Gr II (HSCs)

Gr III (VSELs expanded and pre-differentiated)

IMPROVEMENT OF LV FUNCTION

Zuba-Surma EK et al. Circulation (AHA 2008)

Zuba-Surma EK et al. Circulation (AHA 2008)

EGFP+ MYOCYTES AFTER 6 MONTHS

VSEL-DERIVED CM

STUDY LIMITATIONS

limited gene-array and no proteomic data no EP data

murine transplantation model

RESEARCH PLAN

comparision with ESC-derived CM throughout differentiation electrophysiology study differentiation of human BM-VSELs implantation of EGFP+ VSELs in murine model of reperfused MI

HUMAN VSELS

Cytometry A. 2009 January; 75(1): 4–13.

CIRCULATION OF VSELs

MOBILIZATION OF VSELS

Wojakowski, W. et al. J Am Coll Cardiol 2009;53:1-9

Peripheral BloodBone MarrowSkeletal MuscleOther Tissues

MYOCARDIAL INFARCTION

MOBILIZATION OF CXCR4+ CELLSIncreased Expression ofCardiac/Endothelial Markers

Mobilization Homing/Engraftment

Kucia et al.. Circulation Res. 2004, 95:1191-9Ratajczak et al.. Biol. Cell 2005, 97:113-146. Wojakowski et al. Heart 2008 94(1):27-33

HUMAN VSELs

Wojakowski, W. et al. J Am Coll Cardiol 2009;53:1-9

13.5µm

12.4µm

Monocytes

Granulocytes

Lymphocytes

8.4µm

Erythrocytes

7.5µm

VSEL

7.04 µm

MOBILIZATION OF VSELs

MOBILIZATION OF VSELs INDUCED BY ISCHEMIC STROKE

Paczkowska, E. et al. Stroke 2009;40:1237-1244

MOBILIZATION BY PHYSICAL EXERCISE

HEALTHY SUBJECTS AND PATIENTS WITH FIRST ACUTE MI <45y

VSEL analysis

VSEL 0h (p

rzed

wys

iłkie

m)

VSEL 2h p

o wys

iłku

VSEL 6h p

o wys

iłku

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

p=0,0344

% o

f ev

ents

in V

SE

L g

ate

HYPOTHESIS : MOBILIZATION OF VSELs AND THEIR FUNCTION IS REDUCED IN PATIENTS WITH HEART FAILURE

MOBILIZATION

MOBILIZATION OF VSELS DURING HEART SURGERY

Mieno, S. et al. Circulation 2006;114:186-192

SDF-1

MOBILIZATION OF VSELs DURING CARDIAC SURGERY

CHILDREN VS. ADULTS

GENE EXPRESSION PROFILE

ON-PUMP VS. OFF-PUMP

PRESENCE OF VSELs IN HUMAN HEART

AIMS

Bone marrow

Thymus10m

Spleen

Brain

Heart

Pancreas

Kidneys

Liver Skeletal muscles

Testes

Lungs

Oct-4 and 7-AAD

Oct-4 and 7-AAD

Images of Oct-4+ VSELs from organs – by ImageStream system

CLINICAL APPLICATION

REGENT

LABELLING

ANTI-CD34

II STEP

LABELLINGANTI-CXCR4

I STEP

CD34+CXCR4+

PT SELECTION

RANDOMIZATION

UNSELECTED SELECTED CONTROL

CELL INFUSION

MRI + VENTRICULO + CLINICAL F-U

CELL INFUSION

MRI + CLIN F-U

MRI

VENTRICULO VENTRICULO

6 MONTHS

Tendera et al..Eur Heart J. 2009 Jun;30(11):1313-21.

CLINICAL APPLICATION

REGENT

Tendera et al..Eur Heart J. 2009 Jun;30(11):1313-21.

LVEF (M RI)

Med ian 25%-75% MIn .-Max.

C ontrols U nselected Selected

-20

-10

0

10

20

30

40

50

LV

EF

[%]

p=0.17 p=0.75

p=0.19

-4 3 5

Wilcoxon test

Changes of LVEF for patients w ith in itia l LVEF <m edian (37% )

G roup 'selected'

0 6 months10

20

30

40

50

60

G roup 'unselected'

0 6 months10

20

30

40

50

60

p=0.01 p=0.00736

3129.5

34.5

INTRACORONARY RADIOLABELLED SELECTED 34+ CELLS

Musiałek et al., TCT 200999mTc-extametazime-labeled

CLINICAL APPLICATION

REGENT-VSELs

„Randomized, prospective, double-blinded, placebo controlled trial to assess the effects of BM-derived immunoselected CD133+ cells on LV perfusion and function in patients with inducible ischemia and refractory angina”

1. Efficiency of intracoronary infusion of BM-derived CD133+ cells after succesful recanalization of the CTO of the artery in patients with reractory angina and incucible ischemia.

2. Efficiency of NOGA-guided direct intramyocardial injection of BM-derived CD133+ cells in patients with reractory angina and incucible ischemia ineligible for recanalization of CTO.

AIMS

PODZADANIE 7.5

INCLUSION CRITERIA

60 patients with CTO in qualifying angiogram scheduled for PCI

Inclusion criteria: angina CCS class ≥2, symptoms despite optimal medical therapy, chronic total occlusion of the artery supplying viable myocardium, LVEF ≤ 50%, ineligible for surgical revascularization

Exclusion criteria: acute coronary syndrome, myocardial infarction < 3 months, LVEF <30%, renal failure (GFR <30 mL/min/1.73m2), malignancy, bleeding diathesis

PROTOCOL

CELLS ISOLATION (CliniMACS)

RANDOMIZATION

CTO PCI

SCREENING

INFORMED CONSENT

SUCCESSFUL

1. Clinical evaluation: CCS, NYHA, quality of life2. Exercise test3. SPECT imaging4. MRI

UNSUCCESSFUL

INTRACORONARY CD133+ CELLS

INTRAMYOCARDIAL CD133+ CELLS

PLACEBOPLACEBO

1. Clinical evaluation: CCS, NYHA, quality of life2. Exercise test3. SPECT imaging4. MRI

BASE

LIN

E3

MO

NTH

S

Warszawa, 09.01.2010Innowacyjne metody wykorzystania komórek macierzystych w medycynie30

PODZADANIE 7.5

PROGRESSION OF ATHEROSCLEROSIS

1. INRAVASCULAR ULTRASOUND WITH VH2. OPTICAL COHERENCE TOMPGRAPHY3. MICROCIRCULATORY FUNCTION4. ENDOTHELIAL REACTIVITY

1. CARDIAC DIFFERENTIATION OF BM-DERIVED VSELS

2. MOBILIZATION AND FUNCTION OF CIRCULATING VSELS IN PATIENTS WITH HF, MI <45y AND UNDERGOING CARDIAC SURGERY

3. PHYSICAL EXERCISE AND MOBILIZATION

4. PRESENCE OF VSELs IN HUMAN HEARTS AND ARTERIES

5. CLINICAL TRIAL

SUMMARY

TEAM

III DIVISION OF CARDIOLOGY MEDICAL UNIVERSITY OF SILESIA

BONE MARROW TRANSPLANT UNITDEPARTMENT OF HEMATOLOGY

MEDICAL UNIVERSITY OF SILESIA

STEM CELL INSTITUTEUNIVERSITY OF LOUISVILLE

DEPARTMENT OF CHILDREN CARDIAC SURGERY

JAGIELLONIAN UNIVERSITY

DEPARTMENT OF CARDIAC SURGERYMEDICAL UNIVERSITY OF SILESIA

DEPARTMENT OF BIOTECHNIOLOGY

JAGIELLONIAN UNIVERSITY