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Human stem cell-derived cardiomyocytes for regenerative medicine and disease modeling

Leiden University Medical CenterAnatomy and Embryology Robert Passier

robert.passier@utwente.nl

Disclosure

• No conflict of interest

• For regenerative medicine:– Cardiac repair and regeneration - replacement of cells or tissue– The heart does not regenerate (myocardial infarction)

• To understand the underlying mechanisms of human cardiovascular disease– Drug discovery and development

• To understand cardiac development and differentiation

Why do we need human stem cell-CMs? Why do we need human stem cell-CMs?

• To develop human-based models for predicting drug-induced cardiotoxicity– High attrition rate of drugs in during process of drug development

• Because of low efficacy and unexpected toxicity• cardiotoxicity is a major problem

– Current (disease) models are not predictive enough• Animals, cell lines

• There is urgent need for human predictive models

Highattritionrateduringdrugdevelopment

Cardiovasculair,33%,

Cardiotoxicityisamajorproblem

Human pluripotent stem cells

“1998:Humanembryonicstemcells”hESC

“2007:Humaninducedpluripotentstemcells”hiPSC

How to make heart cells from human pluripotent stem cells?

Embryonic stem cellsInduced pluripotent stem cells

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Heart development at a glance Differentiation of hPSC to the cardiac lineage

NKX2.5

Lessons from cardiac development

Genetic manipulation of hPSC

Differentiation of targeted hPSC reporter line to cardiomyocytes

Elliot et al. Nat. Meth. 2011, 8:1037

Purification of human cardiomyocytesHomologous recombination following electroporation in hPSC

GFP+ beating cardiomyocytes

> 50% Cardiomyocytes

Defined, HT differentiation

Differentiation in 96-well plates (scalable technology)

Scaling up

hPSC

Using hPSC-CM for cardiac repair

Survival of hESC-CM following transplantation

Whole hearts

Heart sections

Selected survival

Experimental setup• 2×105 - 1×106 cells from beating areas GFP-HES3

(~20% cardiomyocytes)• Intubation, ventilation, thoracotomy SCID

(immunodeficient) mouse• Intramyocardial injection• Follow-up 2d-13w (n=6 to 10 per timepoint)• Cryo sections • Immunostaining• CLSM: emission spectrum

van Laake et al. SCR 2007

Effect on cardiac function?

Model of acute myocardial infarctionMale SCID mice (n=13-15 per group)• MI (LAD ligation) +

– 1 million GFP-HES3 from beating areas END-2 co-culture (20% CM)– 1 million non-CM differentiated from GFP-HES3

• MRI (9.4 T) after 2 days, 4 weeks, 12 weeks

4 Chamber view

Cardiac function improvement at 4 weeks not sustained!

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Remuscularization of the infarcted macaque heart with human cardiomyocytes.

Chong et al. Nature 2014

Blood vessels extend from the host coronary network into the graft.

Human cardiomyocytes are electrically coupled 1:1 to the infarcted host macaque heart after transplantation

Ventricular arrhythmias after hESC-CM transplantation

Heart repair: transplantation of cardiovascular cell layers

Cell sheet technology using rat cardiomyocytes and GFP+ endothelial cells (Sekine & Okano Japan)

Engineered Heart Tissue

Zimmermann (Göttingen) & Eschenhagen (Hamburg)

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Using hPSC-CM for cardiotoxicityscreening and disease modeling

MEA Chip

Clump of approximately 500 cardiomyocytes

Safety pharmacology assay development• Unanticipated drug-induced cardiac arrhythmias may lead to sudden cardiac death (long QT

syndrome)• Predominantly by interference with the HERG potassium channel (lacking in mice) à

Ikr currents

• MEA: field potential measurement

The effects of various compounds on field potentials of hPSC-CM

9 drugs were correctly predicted, 2 Iks blockers did not show a response

Braam et al. SCR 2013

Patiënt (KCNH2 mutatin, Ikr)

Precision medicine

IKr IKsK+ K+

IKs blocker

No effect on action potential

IKs blocker

Prolongation of action potential duration

+control

IKr IKsK+ K+

iPS cells-CM derived from patients with mutation in KCNH2 (encodes Ikr channel) leads to prolonged field potential duration (Matsa et al. Eur Heart Journal, 2011)

+

Precision medicine

CTRL

iPSC

CTRL

iPSC

Braam et al. SCR 2013

Iks blockers induce prolonged FPD in iPSC-CM with mutation for Ikr

Treatment with Iks blockers

Ikr mutation

“Patient at risk”

Precision Medicine

How can we mimic the human heart?

The heart consists of multiple cardiac subtypes with specific functions:can we generate these cells from hPSCs?

development of functional human stem-cell based cardiomyocyte subpopulations

12d

hPSCs mesoderm cardiac progenitors

CM

UH 1d 3d 6d 9ddifferentiation

atrial

ventricular

nodal (pacemaker)

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HowtomaturehPSC –cardiomyocytes?

Contraction force of single cardiomyocytes

Microcontact printingof1%gelatinlines(usingPDMSstamp)on5%polyacrylamidegels(5.8kPa)embeddedwithfluorescentbeads

Humanstemcell-basedcardiomyocytemultiplextechnology

Ribeiro etalBiomaterials2015

Cardiac disease modeling in “mature” hPSC-cardiomyocytes

• Hypertrophic cardiomyopathy (HCM) – Affecting 1:500 of the population (decreased

contractility)– Mutation in MYBPC3

• Improved maturation of hPSC-CMs– Adding: Thyroid hormone (T3), Dexamethasone

(Dex) and IGF-1

Birket et al Cell Reports 2015

Summary and future outlook

• hPSC-derived cardiomyocytes can be used for:– Cardiac repair

• Integration and survival?– Disease modeling and toxicity screening

• Maturation?

• Adjusting differentiation culture conditions allow cardiac subtype specification(atrial cardiomyocytes, pacemaker cells)

• Cardiomyocytes can be more matured in vitro (molecularly and functionally)– Facilitating disease modeling (hypertrophic cardiomyopathy)

Next steps:• Mimic the human heart as close as we can get for disease modeling and drug

discovery• Tissue engineering using multiple defined cardiac (subtype) cell populations• Multidisciplinary approach: “heart-on-a-chip” (microfluidics, nanotechnology, biomaterials,

mechanics, electronics)

Acknowledgements

Harsha Deepti DevallaMarcelo RibeiroSabine Den HartoghVerena RönzJantine MonshouwerLu Cao

Christine MummeryRichard DavisMatthew BirketDorien WardLeon Tertoolen

David ElliotAndrew ElefantyEd Stanley

Stefan Braam

Janssen Pharmaceutica

University of Nottingham

Chris Denning

Dept. of Applied Stem Cell Technologies

Robert PassierMarcelo Ribeiro

Rolf SlaatsKim VermeulLu Cao

Andries van der MeerHeleen MiddelkampYusuf Arik

Annette van der BergStanislav Paul Xention

AMCArie VerkerkArthur WildeAbdelaziz Beqqali