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Stem cell therapy for cardiovascular disease –

where does it stand?

Yerem Yeghiazarians, MDProfessor of Medicine

Leone-Perkins Family Endowed Chair

December 16, 2016

Disclosures

I have nothing to disclose

Today’s Topics

• Background on Stem Cells • Update on Cardiac Stem Cell Therapies• Update on Direct Differentiation• Future of Stem Cell Therapy

* Based on data from the Atherosclerotic Risk in Communities study (ARIC) of the National Heart, Lung, and Blood Institiute, 1987-94. Includes Americans hospitalized with definite or probable MI or fatal CHD, not including silent MIs. American Heart Association, 1998 Heart and Stroke Statistical Update

Epidemiology of Myocardial Infarction and Angina in the U.S.

n Single Largest Cause of Death– >500,000 deaths in the U.S./year – 1 in every 4.8 deaths

n Incidence– 1,500,000 Americans will have a new or

recurrent MI each yearn Prevalence

– ~14,000,000 with a history of MI, angina, or both

– ~5,000,000 Americans with Heart Failure

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Myocardial Infarction

Death of cardiomyocytes

Scar tissue formation

Decreased pumping capacity

Left ventricular remodeling

Aneurysmal thinning

Myocardial Infarction and Heart Failure

Courtesy of Randall Lee and Karen Christman

Current Therapeutic Options For Patients with Large MI and Severe Heart Dysfunction

• Revascularization therapy• Medications --- anti-platelet therapies, statin,

ace-inhibitor, beta-blocker, aldactone• Prophylactic ICD to decrease risk of sudden

death• Cardiac resynchronization therapy• Biventricular assist devices• Heart Transplantation None of these therapies lead to myocyte generation

Stem Cell -- “Fountain of Youth?” Potential Beneficial Mechanisms of Stem Cell Therapy after MI

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Classification of Stem CellsPrimary distinction

1) Embryonic stem cells2) Non-embryonic stem cells

- adult stem cells (bone marrow cells;circulating endothelial progenitor cells; mesenchymal stem cell; native cardiac stem cells; adipose derived stem cells, skeletal myoblast cells …..)- cord blood

3) Induced Pluripotent Stem cells4) Somatic cell nuclear transfer cells

Stem Cell Embryology

In the 3-5 day old embryo,called a blastocyst, a groupof about 30 cells called the inner cell mass gives rise to specialized cells that make up an adult organism

Adapted from UCSF Program in Development and Stem Cell Biology

Körbling and Estrov NEJM 349;6, 2003

Embryonic Stem Cell Differentiation Stem Cell Embryology

Adapted from UCSF Program in Development and Stem Cell Biology

Induced Pluripotent Stem Cell Lines (iPS)

Four factors (Oct 4, NANOG, Sox 2, LIN28) sufficient to reprogram a human somatic stem cell (fibroblast) to

a pluripotent cell with all the characteristics of hES(Yu J, et al., Thomson J, Science 2007)

Generation of iPS from human dermal fibroblasts by transduction of four transcription factors (Oct3/4, Sox2,

Klf4, and c-Myc). iPS with all characteristics of hES(Takayashi K, et al., Yamanaka S, Cell 2007)

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The Nobel Prize in Physiology or Medicine

2012

✦Shinya Yamanaka (UCSF)✦Sir John B. Gurdon

Patient-SpecificSomatic Cell

Nuclear Transfer

Perry ACF, NEJM 2005

Which Stem Cells to use?

Embryonic Stem Cells Adult Stem Cells

? Cell Therapy? Regenerative Medicine? Tissue Engineering

iPS Cells

How many cells to use?Which patients will benefit?How to deliver the cells?What is the fate of the cells?What are the risks/benefits?How are the benefits achieved?Can we make the cells work better?Many other questions …..

Human ESC vs. iPS cellsAdvantages

• Highly Expandable• Pluripotent

Disadvantages

• Ethical objections (not with iPS)• Difficult to isolate• Risk of rejection (not with iPS) • Immune-suppressive Rx

required (not with iPS)• Arrhythmogenic potential• Risk of teratocarcinomas• Lack of specific markers

Strauer BE and Kornowski R 2003;107: 929-934

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Adult Stem Cells

Advantages

• Likely more easily obtainable• No ethical objections• Highly compatible• Autologous transplantation• No need for

immunosuppressive Rx• Clinical application already

realized

Disadvantages

• Lack of specific markers• ? arrhythmogenic

Strauer BE and Kornowski R 2003;107: 929-934

Today’s Topics

• Background on Stem Cells • Update on Cardiac Stem Cell Therapies• Update on Direct Differentiation• Future of Stem Cell Therapy

StemCellsusedforCardiacRepairinClinicalTrials

SkeletalMuscleSatellitecells(myoblast)

BoneMarrowMononuclearcells(BMMNCs)MesenchymalstemcellsHematopoieticstemcells(CD34+)

HeartResidentCardiacStemCells

BloodVesselEndothelialProgenitorcells(Hemangioblasts)

Strauer BE et al Circulation 2002;106: 1913-1918

Procedure of Intracoronary Cell Transplantation into Infarcted Myocardium in Humans

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Transendocardial Injection Technique

Perin EC et al Circulation 2003;107: 2294-2302

NOGA Myostarinjection catheter

Injection catheterAdvanced into LV

Use of various types of stem cell therapies in patients with cardiovascular disease

Sanganalmath S, Bolli R. Circulation Research 2013;113:810-834

Copyright © American Heart Association

Strauer BEetalCirculation 2002;106:1913-1918

FirstBoneMarrowCellTherapyReport

• 10patientsfollowingMIvs10historicalcontrols

• Stop-flowballoontechnique

• BMMNCs downtheinfarctartery

REPAIR-AMI Trial (Schachinger V. et al)

ST-elevation MI (Rx’ed <24 hrs)

Bone Marrow Aspiration (days 3-5)

Placebo#103

B.M. treated#101

Intracoronary Infusion (236 million cells; 98% viable)

Baseline and F/U LV-angiogram at 4 monthsAHA 2005

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Schachinger V. American Heart Association Scientific Sessions 2005; Nov 13-16, 2005; Dallas, TX.

Improvement in ejection fraction (%) from baseline (3-6 days post MI) to four months Group Stem

cells Placebo p

All patients 5.5 3 0.014Patients with baseline EF<49% 7.5 2.5 0.002Patients with baseline EF>49% 4.0 3.7 0.81Patients treated within 5 days of MI

4.5 3.9 0.62

Patients treated after 5 days 7.0 1.9 0.004

Clinical events at four months

Schachinger V. American Heart Association Scientific Sessions 2005; Nov 13-16, 2005; Dallas, TX.

Event Events in stem-cell group, n

Events in placebo group, n

Death 2 2Recurrent MI 0 5Hospitalization for CHF

0 2

Revascularization 19 28

Courtesy of Buddhadeb Dawn, MD Circulation 2012; 126:551-568Fisher SA et al Cochrane Database Sys. Rev 2015

Most recent Cochrane Database Systematic Review 2015:- 41 clinical trials; 2732 participants- Cell therapy was safe but found no improvement in LVEFor quality of life in the short or long-term

TIME,Late-TIME– U.S.Trials

• NHLBIsponsored• PatientswithMI,treatedwithprimaryPCI• Intra-coronaryBMMNCs at3days,7daysor2weekspost-MI.

• Controlsreceivedcell-freemediumusingstop-flowtechnique.

• NoeffectonregionalorglobalLVfunction.

TraverseJ,etalJAMA 2012TraverseJ,etal.JAMA2011

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BAMITrial– BoneMarrowDerivedMononuclearCelladministrationinacuteMI

• 1:1Randomized,controlled(3,000patients)• noplacebogroup• ICBMCvs.standardofcare• Primaryendpoint: All-causemortality• Inclusion:LVEF<45%,3-6daysaftersuccessfulreperfusion

• Aim:toreduce2-yearmortalityby25%• 11Europeancountries

POSEIDON-DCM:Allogenic vs.AutologousStemCellsinNon-IschemicDilatedCardiomyopathy–

• Randomized,parallelstudy• Patientswithnonischemic dilatedcardiomyopathyrandomizedto

transendocardial injectionin10leftventricularsiteswiththeNOGAcatheterofallogenic (n=18)versusautologousstemcells(n=16)

• LVEF<40%;f/u12months• Theprimarysafetyoutcome,treatment-emergentserious

adverseeventswithin30days,occurredinnoneoftheallogenicstemcellgroupversusnoneoftheautologousstemcellgroup

• ChangeinLVEF:8.0%intheallogenic groupversus5.4%intheautologousgroup(p=0.49)

• Changein6-minutewalktest:37mintheallogenic groupversus7.3mintheautologousgroup(p=0.012)

AHA2016;JACC2016

Cardiac Repair in Humans

• Traditional ViewThe adult heart is a terminally differentiated organ without regenerative capacity after injury.

• Current ViewThe adult heart has some regenerative capacity after injury and several stem/progenitor cells have been identified.

Endogenous cardiac stem cells

Advantage of cardiac stem/progenitor cells:

Ø differentiate into cardiac cells; unlikely to form teratomas

Ø can be isolated and expanded from myocardial biopsy

Ø can be used for autologous transplantation

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Method of Isolating CSsCut heart to small piece (2 mm3) and digest by enzymes

Culture the small piece (explants) for 1 to 7 weeks

Fibroblast-like cells grow out from adherent explants first

Small, phase-bright cells (putative CS forming cells) appeared on top of the fibroblast-like cells

Harvest putative CS forming cells and transfer them to Poly-D-lysine coated plate

Cardiospheres

CSs40x

An Explant cultured 1st day, 40x

CSs forming cells around an explant30th day, 40x

Cardiospheres Beating

Neonatal Mouse

Lancet 2012E. Marban and colleagues

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Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction

(CADUCEUS): a prospective, randomisedphase 1 trial

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Caduceus Trial

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How about combined cell therapy?

• Ixmyelocel-T – combination of two bone marrow cells: mesenchymal stem cells and activated macrophages

• Treated patients with ischemic heart failure• Prospective, randomized double-blind trial

(51 Placebo vs. 58 Ixmyelocel-T)

Lancet 2016;387:2382

Ixmyelocel-T Trial (continued)

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• Patients treated with ixmyelocel-T had a significant reduction in the primary endpoint

• 37% to 48% reduction in cardiac events compared to placebo

• Driven by a reduction in mortality and cardiac hospitalizations

• Fewer patients with SAEs observed in the ixmyelocel-T group compared to the placebo group

• No significant changes in LVEF or LV volumes, NYHA or 6-minute-walk

Potential Mechanisms for the Observed Benefits with Stem Cell Therapy after Acute MI?

Progenitor Cells

? Improved cardiac function? New muscle? Less apoptosis? New blood vessels

Cell transdifferentationCell fusion

Soluble factorsCell-to-cell contact

? other

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UCSF Translational Cardiac Stem Cell Program

• Tale of Three Cells: à Bone Marrow Derived Cellsà Native Cardiac Stem Cellsà Human Embryonic Stem Cells

10

20

30

40

50

60

70

Baseline Day 2 post-MI Day 28 post-MI

LVEF

%

P<0.0005

Infarction Day 0

n=8/group

P=0.045

HBSS

P=0.03

GFP+ mBMC

Injection Day 3

Bone marrow cells injection 3 days after MI

00.010.020.030.040.050.060.070.080.09

0.1

C57BL-mBM C57BL-Vehicle

SCID-Vehicle

SCID-hEPC SCID-mBM SCID-hBM

Baseline Pre-injectionDay28

Less left ventricular dilatation with cell therapy

P=0.016

Volu

me

(cc)

00.010.020.030.040.050.060.070.080.09

0.1

C57BL-mBM C57BL-Vehicle

SCID-Vehicle

SCID-hEPC SCID-mBM SCID-hBM

Baseline Pre-injectionDay28

0

10

20

30

40

50

60

C57BL-mBM C57BL-Vehicle

SCID-Vehicle SCID-hEPC SCID-mBM SCID-hBM

Infa

rct s

ize

(%)

P=0.005

Infarct size smaller with cell therapy Change of LVEF 25 days post-injection of different cells

-12-10-8

-6-4-202

468

LVEF

(%)

Control

BM

Cs

Extr

act

CSs

Sca-

1

hCM

s*

* * **

NS

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Change of infarct size after injection of different cells

Infa

crt S

ize

of L

V (%

)

Extr

act

CSs

Sca-

1

hCM

s

01020304050607080

Control*

*

*

*

*

NS

BM

Cs

25 days post-injection60 days

post-injection

Mechanism?

In detailed histologic analysis, we saw no new cardiomyocytes forming

and in fact, very rare GFP+ cells were even identified in the hearts.

Additional controls are enlightening…

10

20

30

40

50

60

70

Baseline Day 2 post-MI Day 28 post-MI

LVEF

%

saline

live mBMCsP=NS

P=0.048

mBMC extract

Yeghiazarians, Y, at al. Mol Ther. 2009 Jul;17(7):1250-6. Epub 2009 Apr 21.

Limitation of infarct size in BMC and extract groups

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Yeghiazarians, Y, at al. Mol Ther. 2009 Jul;17(7):1250-6. Epub 2009 Apr 21.

Enhanced vascularity at the infarct border zone

Yeghiazarians, Y, at al. Mol Ther. 2009 Jul;17(7):1250-6. Epub 2009 Apr 21.

Reduction in cardiomyocyte apoptosis in the extract group

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What is in the extract?

Protein(s)?Extracellular vesicles (exosomes)?MicroRNA?

Copyright ©2008 American Heart Association

Gnecchi, M. et al. Circ Res 2008;103:1204-1219

Paracrine/autocrine mechanisms in adult stem cell therapy

Today’s Topics

• Background on Stem Cells • Update on Cardiac Stem Cell Therapies• Update on Direct Differentiation• Future of Stem Cell Therapy

Can non-cardiomyocytes be directed to differentiate into

cardiomyocytes (Direct Reprogramming)?

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iPS Cell Generation

Courtesy of Dr. B Conklin – Gladstone

Direct Reprogramming

Courtesy of Dr. B Conklin – Gladstone

Howaboutabioartificial heart?

• Decellularizedheartsbycoronaryperfusionwithdetergents

• Preservedtheunderlyingextracellularmatrix• Producedanacellularperfusablevasculararchitecture• Reseededtheconstructwithcardiacandendothelialcells• Toestablishfunction,maintainedtheheartsforupto28daysbycoronaryperfusioninabioreactorthatsimulatedcardiacphysiology

Ott HC et al (Doris Taylor) Nature Medicine 2008

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Today’s Topics

• Background on Stem Cells • Update on Cardiac Stem Cell Therapies• Update on Direct Differentiation• Future of Stem Cell Therapy

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Issuesforcardiacstemcelltherapy

• Whichcell(viability/potency)• BonemarrowcellsfromolderpatientswithCV

riskfactorsandpost-MImightbelessfunctionalthancellsfromhealthyyoungerdonors

• Howmanycells• Whatdisease(acuteMIvs.chronicHFvs.

ischemic/non-ischemicCMP)• ModeofDelivery• TimingofDelivery• Singleversusmultiple• Combinationsofcells• Cells+scaffoldsorcytokines• Whopays?

Conclusion • Therapy with some of the cells appears safe but

more research is certainly required• Understanding the components of the extract derived

from bone marrow cells is a must • Improving cell retention using biodegradable

scaffolds after delivery is under investigation and appears promising

• Direct Reprogramming of non-CM into CM is of great interest

• Bioartificial organs potentially have a bright future

THANK YOU