Charles E. Murry, MD, PhD

ES/iPS Cells for Cardiac Repair
Chuck Murry, MD, PhD
Pathology, Bioengineering and Cardiology
Center for Cardiovascular Biology
Institute for Stem Cell and Regenerative
Medicine
University of Washington, Seattle
Current Projects
Therapeutics
-cell transplantation
-tissue engineering
Pluripotent
Stem Cell
Cardiomyocyte
“Omics” via NextGen Seq
-Histone Modifications
-DNAse Hypersensitivity
-RNAseq
Maturation
iPSCs and
Cardiomyopathy
-cell phenotyping
-tissue phenotyping
Myocardial Infarction
Coronary Thrombosis
Putting Stem Cells in the Heart:
What Could Go Wrong?
Me
Keratin
Embryonic Stem Cells:
The Need to Control
Differentiation
Ectoderm
Cartilage
Mesoderm
Goblet cells
Endoderm
5x105 Undifferentiated mESCs Grafted
Nussbaum et al., FASEB J 2007
Two Cardiac Differentiation Protocols
Laflamme et al, Nature Biotech 07—High Density Monolayers, 20% O2
Day -4
Day 0
Plate in
MEF-CM
Day 1
Activin A
Day 5
BMP4
Day 14
RPMI + B27 + KO-SR
Yang et al, Nature 08—Embryoid Bodies, 5% O2
Yield: 10^8 cells/run Purity: >60% without sorting
Human Cardiac
Directed Differentiation
(Sharon Paige, UW MSTP;
Nina Tan, Undergraduate Researcher)
IMR90 Lung Fibroblast-Derived hIPSCs
Monolayer Activin A + BMP4 Differentiation
a-actinin / Nkx2.5 / DAPI
hESC-Cardios Prevent Heart Failure
Post-Infarct Hearts Studied by MRI at 4 wks
Wall Thickening (%)
80
Uninfarcted
Cardiac cells / PSC
PSC-only
SFM-only
Non cardiac cells / PSC
NS
60
40
**
**
**
20
0
Infarcted Wall
Uninfarcted Wall
Laflamme et al, Nature Biotech 2007
A Village Production



Murry lab: Sarah Fernandes, Nathan Palpant,
Hans Reinecke
Laflamme lab: Jay Gantz, Yuji Shiba
Kotlikoff lab (Cornell): Noreen Tallini
GCaMP3
Calcium Indicator Protein
from Kotlikoff, 2007
Knock-In of GCaMP
AAV-S1 Locus via Zn Finger Nucleases
GCaMP3 Cardios in Guinea Pig Heart
108 cells, 20d post-transplant
Synchrony between Human Graft
and Guinea Pig Heart
Human
Ca2+
Transient
Guinea Pig
EKG
0
1
2
3
4

The developing heart grows in response
to preload and afterload.
◦ Fiber architecture
◦ Hyperplasia
◦ Hypertrophy

In situ growth and maturation is
desirable for implants
◦ Infarct repopulation
◦ Pediatric applications, e.g. outflow tracts
•hESC- or hiPSC-derived
cardiomyocytes in collagen gel +
basement membrane prep
•Trabecula-shaped, 1 cm x ~1
mm
•Unstressed, static tension,
cyclic tension (1 Hz, 5% strain)
•Cardio-only or “tri-cell” with
HUVECs and hMSCs
N. Tulloch,
In revision
No Stress
Static Stress
Cyclic Stress
Static Cyclic
No
Stress Stress Stress
DNA Synthesis
(Normalized)
BrdU β-MHC
2 Week Compound Interest
Basal: (1.15)^14 = 7x
Cyclic: (1.41)^14 = 122x
Non-normalized BrdU incorporation rates were 1530% in unstrained cultures w/ overnight pulse.
Cardiomyocyte Area
(Normalized)
No
Stress
Static
Stress
Cyclic
Stress
Cardio-Only Constructs
Spont. Ca2+
Transients
Concurrent
Length
Shortening
Ca2+ Transient Parameters
No Strain (SE)
Static Strain (SE)
Departing velocity 0.42 ± .04
4.2 ± .3
Peak Height
0.02 ± .002
0.13 ± .004
Return velocity
-0.10 ± 0.01
-0.31 ± 0.01
Active Force (mN/mm^2)
C
0.09
0.08
0.07
0.06
0.05
0.04
y = 0.0012x - 0.1043
R² = 0.9897
0.03
0.02
0.01
0
100
120
140
160
length (%)
180
200
Engineered vs. Fetal Human Myocardium:
Which is Which?
α-actinin/DAPI
A
Engineered—2 weeks
B
Fetal—~100 days

Broad Goal: use iPSC-derived cardiomyocytes
to understand genotype-phenotype relations

Focus on MYH7 in HCM and DCM

Pediatric vs. adult cases
◦ Early onset disease is most likely to be manifest in
stem cell-derived cardiomyocytes

Down the road: drug screens
B
Fluorescence intensity
A α-actinin/Hoechst
Fluo4-AM Quantification
5
10
Time (secs)
15
20



Make relevant cells and tissues
They are human, so correct myosins, ion channels etc.
Gene function—good for reverse genetics
 Transgenesis is easy
 Knock-outs and knock-ins possible, getting easier


Can use techniques of somatic cell genetics.
Gene/variant discovery?
◦ Missing the ability to recombine prospectively by sexual
reproduction (ethics)
◦ Can take advantage of lots of natural sexual reproduction

No cage charges or per diem!
Thanks also to...
UW CV Biology
Mike Laflamme
Dan Bowen-Pope
Beverly Torok-Storb
Bill Mahoney
Steve Schwartz
Elina Minami
Gordon Keller
UW Stem Cell Institute
Randy Moon
Tony Blau
Carol Ware
Tom Reh
Hannele Ruohola-Baker
Bioengineering
Mike Regnier
Steve Korte
Buddy Ratner
Kip Hauch
Marta Scatena
Genome Sciences
John Stamatoyanopolous
Sean Thomas
Funding
•NIH
•UW ISCRM