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Teaser: Promising results of experimental cell therapy did not translate into equivalent clinical benefit yet. Feasibility and safety of endothelial and mesenchymal progenitor cell transplantation have been verified in multiple clinical trials over the past decade. For conventional 'bone marrow transplantation' it took 20 years from early steps until the first proof of cure. Lessons learned from one million patients who received lifesaving blood stem cell transplantations since will be discussed and compared to our still limited mechanistic insight into cardiovascular regenerative cell therapy.

dirk.strunk@pmu.ac.at

www.pmu.ac.at/zelltherapie

Anna & Anna

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LECTURE OUTLOOK

1.Strunk – Lab.: Cell Tx & Reg. Med.

( Why ECFC & MSPC ?)

2.Cell – Tx. in Cardiology / CV Surgery

(hematopoietic > CM; endoth. & mesench. cells)

3.Learning from HSCT (new strategies / next steps )

Adult stem cells can improve organ regeneration

after ischemic / metabolic / toxic injury.

OPERATIVE MECHANISMS

vascular

REPAIR

somatic

REPAIR BYSTANDER

EFFECT

cellular humoral

Re generative

S tem Cell T herapy

BM-DERIVED CANDIDATE

REGENERATIVE CELLS

• HSPC (< 1%)

• MSPC (< 0.01%) (incl.MAPC; USSC; RS1; BMSC; MIAMI < 0.00001)

• EPC (< 0.0001%)

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LATE OUTGROWTH COLONIES = ECFC

Define “EPC”

Hirschi et al, ATVB 2008 Lin, Hebbel et al. 2000

Ingram, Yoder et al. 2004, 2005 Rohde, Strunk et al. 2006

Rohde, Strunk et al. 2006

Reinisch, Strunk et al. 2009

>1x108

somatic ECFCs

ISOLATION

EXPANSION

Blood 113(26):6716-6725 (2009)

MAKE ECFC A CLINICAL PRODUCT

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SLICE THICKNESS

78µm

Bi Bii

Biii Biv Bv

Bvi Bvii Bviii

78µm slice thickness, 2h10min aquisition

C

MAXIMUM INTENSITY PROJECTION

2.5mm

Darkfield Microscopy

with:

Clemens Diwoky,

Rudolf Stollberger

TU Graz

Bruker WB 300 7 Tesla

Bruker WB 500 11,7 Tesla

Karlsruhe, Germany

ECFC FUNCTION IN VIVO

hu. CD31

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MSPC

ECFC

1 – 2 mio. 1 : 4 Matrix

300µL

s.c. implantation

immune-

compromised

mice

Reinisch et al., Blood 113(26):6716-6725 (2009)

A

Mouse tissue

Matrigel®

plug

+ Human cells

D CD31

F mTer119 mTer119 G mIgG H vWF E

C Vimentin Vimentin B

see also: Koike, Nature 428:138-9 (2004)

Au, Blood 111:1302-5 (2008)

Melero-Martin, Circ Res 103:194-202 (2008)

MNC

wash (2-3x) primary culture

2-3 colonies / flask (15-25 days)

remove non adherent cells (day 3)

bio

logy

an

d f

un

ctio

n

cord

blo

od

density gradient centrifugation

1st passage 4 x CF-4; 30/cm2; 14 days

MSC HPL vs. MSC FBS

clinical scale expansion

a-MEM / 10% pHPL

a-MEM / 10% FBS

(14 days)

MSC HPL vs. MSC FBS

MSC HPL vs. MSC FBS

ECFC

s

MSPC

s Regen Med. 2007 2:371-82. Blood 2009 113: 6716-6725.

>1x108

somatic ECFCs

ISOLATION

EXPANSION

Isolation & Large Scale Expansion Protocols

PMU | ExCT | Experimental & Clinical Cell Therapy Institute | Prof. Dr. D. STRUNK

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Patient Center Sex Age Diagnosis Indication Donor Infusions MSC/kg

D.F. Hematology

Graz m 33 AML aGvHD Third party 2 0.2-1x107

U.K. Orthop.

Graz m 35 Critical size bone defect Tissue repair Autologous 2 local 2+6 x 108

H.E. Hematology

Graz f 65 AML aGvHD Third party 1 2x106

N.S. St. Anna

Vienna f 8 JMML Graft failure Third party 1 1.5x107

T.T. PHO

Graz m 11 Cycl. Neutropenia aGvHD Sibling 3 1-2x107

K.J. St. Anna

Vienna f 15 SAA Graft failure Third party 1 5x106

W.TJ. PHO

Graz f 8 AML aGvHD Parent 3 1-2x106

R.K. PHO

Graz f 15 AML aGvHD Third party 5 1x107

L.S. PHO

Graz f 15 Neuroblastoma aGvHD Third party 3 1.8x106

S.EM. Ped Hem

Geneva f 10 SAA aGvHD Third party 2 2x106

E.A. Hematology

Graz m 31 AML GvHD Third party 3 2-4x106

F.J. Hematology

Innsbruck m 33 AML GvHD Third party 2 1.8x106

D.A. PMU, Salzburg m 27 Critical size bone defect Tissue repair Autologous 2 local 2 + 6x108

E.W. PMU, Salzburg f 48 Non-union fracture Tissue repair Autologous 1 local 4x108

O.R. Hematology

Vienna m 57 CLL GvHD Third party 3 1.7 – 3.5x106

2006 - 2012 SALZBURG

Cantanhede

Zürich, Genf

Tucson

Wien

London

Houston (MDACC +TX Heart)

Vancouver

Los Angeles

Göttingen, Mannheim, Ulm, Hannover Tübingen, Bergisch Gladbach, Berlin

Langenfeld Innsbruck, Graz / Salzburg

Oslo

Washington Bethesda (NIH)

Our Contribution:

Standard pooled Human Platelet Lysate (pHPL; 3-5L): Production / Testing / Cryopreservation in Salzburg.

Distribution on dry ice to partner institutions and reference laboratories.

Indianapolis Padua

12

Maskat (Oman)

see also: Human Alternatives to FBS… Stem Cells 27:2331-41 (2009)

Ottawa

Commercially available since: IV / 2012

pHPL Contact: k.schallmoser@salk.at

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2 x 106 BM-MSPCs +

300 µl Matrigel™

HPL

culture

medium

FBS

culture

medium

NSG mouse

24 hrs OsteoSense™ 750

i.v., 150 µl

4 weeks

fixation in newly formed

Hydroxyapatite, emission 750 nm

Donor

A

Donor

B

matrigel

implant

HPL-cultured FBS-cultured

knee

Near-Infrared fluorenscence,

Maestro™ in vivo imaging

N. Etchart-Liechtenstein (manuscript in preparation)

Donor N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 All

HPL-derived MSPC

Bone + + + + + + + + + + + + + + + + + 17/17

Marrow + + - + - - + - + + - + + - - + + 10/17

FBS-derived MSPC

Bone - n.t. - - - - + + n.t. - n.t. - - n.t. n.t. - n.t. 2/11

Marrow - n.t. - - - - + - n.t. - n.t. - - n.t. n.t. - n.t. 1/11

BM Fib. UC WAT

H/E

M

ova

ts„

5C

Score = 0 Score : a = 2; b = 2

a b

Score : a = 3; b = 5

a b

Score = 1

a

b

Score : a = 4; b = 5

4

5

3

2

1

0

Bo

ne

Sc

ore

BM WAT UC Fib. no cells

Matrigel, „non-inductive“

TCP/HA, „osteo-inductive“

A

B

D

C *

* *

OsteoSense™ Fluorescence

Bone Scoring System

0 = None

1 = Weak signal

2 = Clear signal, weaker than knee

3 = Clear signal, as intense as knee

4 = Signal more intense than knee

5 = Signal more than twice as intense

as knee

Rein

isch / S

trunk B

lood 2

015

HUMAN BONE + MOUSE MARROW

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PMU | ExCT | Experimental & Clinical Cell Therapy Institute | Prof. Dr. D. STRUNK

© PMU | Experimental & Clinical Cell Therapy Institute | Dr. D. STRUNK

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September 12, 1957 Vol. 257 No. 11

1st clinical MSC Tx Lazarus HM et al,

Bone Marrow Transplantation 1995 ~ 30 years

1st intra-coronary BM Tx Hamano K et al, (BMNC ~ CABG)

Jpn Circ J 2001

1st intra-myocardial HSPC Tx Stamm…Steinhoff, (CD133+ BMNC ~ CABG)

Lancet 2003

1st intra-myocardial MYOBLAST Tx Menasché et al, (5-Aza+skel. Myobl. ~ CABG)

Lancet 2001

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NUMBER OF CLINICAL TRIALS

EFFECT

BENEFIT FOR PATIENTS

IMBALANCE

IMMUNE SYSTEM &ORGAN REGENERATION

… …

…

Circ Res. 2015; 116:1346-1360

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Circ Res. 2015;116:1361-1377

hematopoietic

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Circ Res. 2015;116:1413-1430

PMU | ExCT | Experimental & Clinical Cell Therapy Institute | Prof. Dr. D. STRUNK

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There are 3 possible strategies to utilize stem cells (SC) for regeneration: 1.) Local recruitment of resident SC towards an injury. 2.) Mobilization of SC from bone marrow or other distant sites via BLOOD circulation. 3.) Transplantation of SC to the injury site vs. systemic delivery.

invasive SC harvest

BM WAT Heart…

non - invasive SC harvest

UC UCB Placenta Blood Apheresis

… Urine

Mictionation

iPSCs 3rd party SC

(living or cadaveric donor)

PMU | Exp. & Clin. Cell Therapy Institute | D. STRUNK, MD

PMU | Exp. & Clin. Cell Therapy Institute | D. STRUNK, MD

CD90 CD73 CD105 CD29

97.39% 99.99% 98.64% 99.56% 40.53%

NG-2

CD45 CD34 HLA-DR CD14 CD19

2.76% 1.86% 2.56% 2.09% 1.42%

USCs 007 p1

p2

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Osteocyte

Osteoblasts

Pericyte

VASCULAR

NICHE

ENDOSTEAL

NICHE

CAR/MSPC

Osteoclast

MARROW

HSCs Adipocyte

Endothelial cell

Endothelial colony forming

progenitor cell (ECFC)

Pericyte

Osteoblast

Osteocyte

Osteoclast

Multipotent mesenchymal

stromal cell (MSC)

Hematopoietic stem cell (HSC)

Hematopoietic progenitor cell (HPC)

Adipocyte

Hematopoietic cluster

RBC

BONE

HSPC

Osteoclasts

Hematopoietic

Cluster

from:

REINISCH, STRUNK.

Concepts to facilitate UCB-Tx.

in: Regenerative Therapy using Blood-

Derived Stem Cells

Springer, NY (2012)

1.2%

CD41

2.4%

CD19

0.7%

GpA

3.4%

CD56

6.7%

CD14 21.6%

CD3(2)

63.0%

CD15

Fluorescence 2

SS

C

NON-MOBILIZED PB

TRANSFUSION 54:315 (2005)

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<1% SC Human LT-HSC: … CD34+/LinNEG/CD38-/CD90+/CD45RA- Human MSC: … CD34-/LinNEG/CD38-/CD90+/CD45RA-

Human EPC: … CD34+/LinNEG/CD38-/CD90-/CD45RA-

CD34+ Hematopoietic Stem / Progenitor Cells

BONE MARROW TRANSPLANTATION

(HSCT = Hematopoietic SC Tx)

>99% CD45+ WBC (BM; MPB)

Highlights

• The number of cardiomyocytes remains

constant during the human lifespan

• Endothelial and mesenchymal cells

increase into adulthood and show high

turnover

• Cardiomyocyte turnover decreases

exponentially with age and is <1% per

year in adults

• The cardiomyocyte turnover rate is

equal in the main subdivisions of the

human heart

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Hematopoietic stem

/ progenitor cells

What is the mechanism ?

PMU | ExCT | Experimental & Clinical Cell Therapy Institute | Prof. Dr. D. STRUNK

2 = FIX FOR BROKEN HEARTS C Templin / A Reinisch / D Strunk / U Martin et al.; Circulation 2012

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What is the best model ?

in vitro – Rodent – large Mammal – non-human Primate – clinical Trial …

humanized

syngeneic

mechanistic

side studies

multicenter

prospective

randomized

dog; minipig

horse

100% predict/reflect in vivo pathology / mechanism

AVATAR

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What is the mechanism ?