iPS cell-derived cardiomyocytes:

overcoming barriers to therapeutic use

Chris GeorgeSwansea University Medical School

(i) A retrospective look at approaches for heart repair

: autologous bone marrow-derived SC

: cardiac-derived (ckit+) SC

(ii) Contemporary approaches

: ES-derived CM

: iPS-CM

(Direct fibroblast-to-CM conversion)

(v) Systematizing iPS-CM maturation

(iii) Problems with cell type and maturation

(vi) Cellular determinism: towards predicting and

controlling phenotype

(iv) Excitation-contraction coupling and the network

organization of cell signalling

(i) Repairing the failing heart with bone marrow-derived stem cells (BMSC): the end of the line?

• Just 5/49 trials using BMSC were free from „discrepancies‟ (~10%)

• In those 5, mean EF effect size was zero

Repairing the failing heart with cardiac-derived SC:

the fall-from-grace of c-kit+ lineage

2001 2017‘03 ‘04 ‘09 ‘10 ‘11 ‘12 ‘14 ‘15 ‘16

Lin-/c-kit+

haematopoietic cells

repair mouse heart

Nature (2001) 410:701-5

Cardiac-resident

c-kit+ cells repair

post-MI rat heart

Cell (2003) 114:763-76

BMSC do not become

cardiomyocytes

Nature (2004) 428:664-8 BMSC contribute to

revascularization of

the damaged heart

Nature (2004) 428:668-73BMSC do not become

cardiomyocytes

Nat. Med. (2004) 10:494-501No evidence of c-kit+

SC myogenesis

PNAS (2009) 106:1808-13

Neonatal, but not adult,

c-kit+ SC can become

cardiomyocytes

Circulation (2010) 121:1992-2000

Lancet (2011) 378:1847-57

Clinical benefit of c-kit+ cell transplantation

Adult c-kit+ SC adopt

vascular fate; neonatal c-kit+

SC can become

cardiomyocytes

PNAS (2012) 109:13380-5

Lancet Expression of concern 2014

Between 2 and 78 y/o human heart is regenerated

8-times from c-kit+-mediated myogenesis

Circulation (2012) 126:1869-81

Retraction of the „8-new-

hearts‟ story

Circulation (2014) 129:e466

Lancet (2014) 60608

Circulation (2013) 128:122-31

c-kit+ SC improve cardiac

function in HF

‘13

Nature (2014) 506:337-41

c-kit+ SC are irrelevant as a source of

new cardiomyocytes in vivo

Nat. Commun (2015) 6:8701

Lineage tracing confirms c-kit+ SC do

not become cardiomyocytes

Circ. Res. (2015) 116:1216-30

Paracrine effects of c-kit+ SCCirc. Res. (2016) 118: 17-19

Drawing a line under things?

Boston Globe

(ii) Beyond c-kit+ SC - ES-derived cardiomyocytes

Nature (2014) 510:273-7

1x109 cells (suspension, post-freeze

73±12% CM)

Direct myocardial injection

2 weeks 3 month Evidence of CM maturation in situ

2 weeks

x4

Diameter = 43mm

2 mm

Circ. Res. (2014) 115:335-8

Accentuate the negatives

1. Teratomer risk

2. Long term immunosuppression

3. 100% incidence of arrhythmias

(especially early on)

4. Arrhythmogenic risk increases at

slower heart rates (humans)

5. Is cardiac function improved?

Development of alternans

6. Long term toxicity of pro-survival

cocktail (sarcoma proteins)

1. Risk overplayed; 98% CM in graft;

zero teratomers in > 1000 rats

2. Universal donors / HLA engineering

3. Potentially due to poor coupling from

injecting 1x109 cells (< 1% cells remain);

Human heart may need ~ 8x109

4. Unlikely to see increased risk in humans

5. Further work needed

6. As point 1

The response

Circ. Res. (2014) 115:e28-9

Beyond ES-derived CM: grafting EHT built from iPS-CM

Sci. Trans. Med. (2016) 8:363ra148

EHT ‘seeded’ at 65% CM (atrial CM)

Day21 : 95% vCM @ 50bpm

2 weeks post-graft

4 weeks

post-graft

IPS-CM in EHT• ‘Immature’• In situ maturation

: sarcomeric extension by ~ 10% but still smaller than GP CM: >95% conversion of MLCA to MLCV isoform

• No arrhythmias reported• Human DNA in spleen (2/7) and lungs (4/7); none in liver or kidney

(iii) The futility of selecting nodal-, atrial- and ventricular-like cells…....?

Hallmarks of cardiomyocyte immaturity

Yang and Murry. Circ. Res. (2014) 114:511-523

Nature (2011) 471:68-73

IPS and epigenetic imprinting: they remember where they came from….....

iPS-CM ready for the clinic??

A network approach to cellular signalling

Out

In

Na+ K+

(iv) Excitation-contraction coupling and the entrainment of

surface membrane and intracellular SR „clocks‟

Na/K-

ATPase

NCXNa/K-

ATPasePMCACa2+

Plateau

“Trigger Ca2+”

RyR

SR

SERCA Myofilaments

Mito

Vinogradova et al. (2004) Circ. Res. 94:802-9

Inhibition of SR clock unmasks an

unusual Ca2+ entrainment in IPS-CM

Out

In

Na+ K+

Na/K-

ATPase

NCX Na/K-

ATPase PMCA Ca2+

“Trigger Ca2+”

RyR

SR

Sarcolemma

SERCA Myofilaments

Mito

X

X

Control

CPA

Edwards (unpub.)

Building a network….........

β-AR AC

Adr

ATP cAMP

Multiple downstream effectors

(“Fight or flight” response)

PKA

+

βγ

α

out

in

Activation via the the beta-adrenoceptor pathway

http://expasy.org/cgi-bin/show_thumbnails.pl

The cell signalling „blueprint‟ – everything linked to

everything else

///SR

Excitation-

contraction coupling

Ca2+

release

Mitochondria

Surface ion fluxes

Metabolism

Apoptosis

Protein synthesis & degradation

Intra-cellular synchronization

Inter-cellular synchronization

Gene expression

Horizontal and vertical network organization

out

in

cyto

lumen

Basal

Restore

Restore

Disrupt

Perturbing the homeostatic state : network adaptation

out

in

cyto

lumen

Unstable

Disrupt

Activated

out

in

cyto

lumen

Disrupt

Adapt out

in

cyto

lumen

Pseudo-stable

George et al. (2012) Am. J. Physiol. 303:897-910.

RyR2SERCA

LTCCNCX

Day

2 3 4 5 6 70

20

40

60

80

100

Tro

po

nin

po

sitiv

e

(%

)

2 3 4 5 6 70

20

40

60

80

100

Day

Ce

llula

r a

lig

nm

en

t (%

)

***

*

Day

Day 2 Day 4 Day 7

DAPI

Troponin-T

Alignment of ES-CM in culture

Lewis et al. J. Biomol. Scr. (2015) 20:30-40

Maturation-linked refinement of Ca2+ handling

5 sec

ΔC

a2

+

(20 f.u

.)

C

a2

+

(20

f.u

.)

Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Δ

2 sec

Temporal heterogeneity index (THI) = σ(int1….intn)

Amplitude heterogeneity index (AHI) = σ(ampa….ampn)

Inter-transient noise (ITN) = Σ(SV1….SVn)

SV1 SV2 SVn

int1 int2 intn

ampa ampb

ampn

Sa Sb Sb Sn

Time (t) Flu

ore

scen

ce

(units)

ampc

Frequency = S s-1

F0

Amplitude* = (ampn-F0) / F0

ka kb kc kn

da db dc dn

Duration = (da + db + dc + dn) / n

Area* = Area under curve / F0

Rate of decay = (ka + kb + kc + kn) / n

a b c n

a b c n

a b c n

a b c n

1

2

3

4

a b c n a b c n a b c n

a 1 1 0

b 0 1 0c 0 0 1

n 0 0 1

a 1 0b 0 1

c 0 0n 1 0

a 0

b 0c 0

n 0

S

Cell no.

1

2

3

2 3 4

= 8/24 (33.3%)

Co-incidence of S Coincidence of S = 33.3% (8/24)

Synchronization = Coincidence (Sa…..Sn)

Cell

S

SALVO-based profiling of cellular Ca2+ : discriminating

signals from noise

George and Edwards (2017) Stem Cell-Derived Methods in Toxicology pp173-90 Humana Press

Intracellular Intercellular

synchronization

D4 D5 D6 D70.0

0.2

0.4

0.6

0.8

Ra

te (H

z)

****

****

******

**

D4 D5 D6 D70.00

0.05

0.10

0.15

0.20

0.25

0.30

Am

plitu

de

he

tero

ge

ne

ity

in

de

x (A

HI)

****

****

****

D4 D5 D6 D70.0

0.1

0.2

0.3

0.4

0.5

Te

mp

ora

l h

ete

rog

en

eit

y in

de

x (T

HI)

****

****

********

D4 D5 D6 D70.000

0.005

0.010

0.015

0.020

Inte

r-tr

an

sie

nt n

ois

e (

ITN

)

*

****

**

D4 D5 D6 D70

10

20

30

40

50

Sy

nc

hro

niz

atio

n (%

)

Maturation of Ca2+ handling is linked to reduced Ca2+

signalling variability

Ra

te

Basal C

a2+

ITN

-A

ITN

-B

ITN

-C

Am

pli

tud

e

Du

rati

on

Are

a

Ra

te U

p

Tim

e-t

o-p

ea

k

Ra

te D

ec

ay

Tim

e-t

o-d

ec

ay

No

ise

in

dec

ay

TH

I

AH

I

0

50

100

SALVO parameter

% o

f m

ax

0.6 0.8 1.3 1.2 - 0.5 0.4 0.7 0.6 0.4 0.6 0.4 0.8 1.7 0.8CoV

n=209

Massively variable homeostatic Ca2+ handling

CoV = SD/mean

Jones et al (2016) Front. Cell Dev. Biol. 3:89

(v) Systematizing iPS-CM maturation : EB vs. monolayer

culture

EB maturation is not associated

with morphological change

Increased post-disaggregation clustering from older EBs

Defining conditions for maximum Ca2+ synchronization

Unmasking Ca2+ signalling phenotype

Switch [Ca2+]ext from

400μM to 1.8mM [Ca2+]ext

(vi) Cellular determinism

1

2

3

4

4

5

6

7

8

9

10

11

12

12

13

14

15

16

17

18

19

20

20

21

22

23

24

25

26

27

28

28

29

30

31

32

33

34

35

36

36

37

38

39

40

41

42

43

44

44

45

46

47

48

49

50

51

52

52

53

54

55

56

57

58

59

60

Cause Effect

Cell #

Towards predicting cellular response and heterogeneity

Homeostasis

State 1

State 2

State 3

Predictably influencing cell-to-

cell coupling in computational

arrays

Variability of

cell-to-cell coupling

Boileau et al. (2015) Ann. Biomed. Engin. 43:1614-25

59

103

Rotors in iPS-CM (Edwards, unpub)

Progress made

• Maturity of cells

• Maturity of field

• More realistic endpoints

Barriers that still exist

• Transparency / dataset availability /

understanding of „nuisance variables‟

• Reliance on crude end-points of phenotype and function

• High levels of phenotypic variability

• Unpredictability

• Unknown impact of reprogramming / source

What‟s needed?

• New „network‟ approach to cellular variability

and determinism

SUMMARY

Cardiff

Catrin Williams

David Lloyd

Adrian Porch

Dimitris Parthimos

Nottingham

Chris Denning

Gary Duncan

Divya Mirrington

David Edwards

Aled Jones

Sarah Marsh

Kimberley Lewis

Catherine Hather

Nicole Silvester

Steven Barberini-Jammaers

Phil Ashton

Archana Jayanthi

Alice Mitchell

Jessica Wells

Swansea (Engineering)

Perumal Nithiarasu

Etienne Boileau

Sanjay Pant

Ankush Aggarwal

SIRPA+/ VCAM+ SIRPA+/ VCAM-0

10

20

30

Pro

po

rtio

n o

f c

ells

(%

) *

102

103

104

105

102 103 104 105

VC

AM

-54

6 (

un

its

)

SIRPA-488 (units)

SIRPA+ / VCAM+

SIRPA+ / VCAM-

Non-e

nrich

ed

SIR

PA+ / V

CAM+

SIR

PA+ / V

CAM-

0

20

40

60

80

100

Tro

po

nin

po

sitiv

e (%

)

********

****

SIRPA+/VCAM-

SIRPA+/VCAM+

Non-enriched

TnT

DAPI

Does CM enrichment improve phenotype?

5 sec

ΔC

a2

+

(20

f.u

.)

Non-enriched SIRPA+/VCAM+ SIRPA+/VCAM-

Non-enriched SIRPA+/ VCAM+0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Ra

te

(no

rma

lize

d)

Non-enriched SIRPA+/ VCAM+0.0

0.2

0.4

0.6

0.8

1.0

1.2

AH

I

(no

rma

lize

d)

*

Non-enriched SIRPA+/ VCAM+0.0

0.2

0.4

0.6

0.8

1.0

1.2

TH

I

(no

rma

lize

d)

*

Non-enriched SIRPA+/ VCAM+0.0

0.2

0.4

0.6

0.8

1.0

1.2

ITN

(n

orm

alize

d)

****

Non-enriched SIRPA+/ VCAM+0.0

0.5

1.0

1.5

2.0

Sy

nc

hro

niz

ati

on

(no

rma

lize

d)

*

Non-FACS (Day 7)