Human Embryonic Stem Cells for Cardiac Regeneration

Human Embryonic Stem
Cells for Cardiac
Regeneration
Robert A. Kloner, MD, PhD
Professor of Medicine
Keck School of Medicine at University of
Southern California
Director of Research
Heart Institute, Good Samaritan Hospital
Los Angeles, CA
TCT; Sep 2010
Washington, DC
Conflict of Interest
Dr. Kloner’s lab receives a grant form Celavie for Stem
Cell Research
Several recent exciting findings
suggest that:
Cardiomyocytes derived from human embryonic stem
cells may form stable grafts in myocardial
ischemia/infarction models and contribute to improved
LV function
Cardiac progenitor cells can be isolated and transplanted
into myocardial infarction models improving LV function
Humoral factors (hepatocyte growth factor, insulin-like
growth factor) may stimulate local cardiac progenitor
cells to improve LV function
Pluripotent stem cells induced from skin fibroblasts could
generate functional cardiac myocytes (would negate the
need for immunosupression)
BUT … there are hurdles to
overcome:
─ small graft size in some studies
─ cell death-necrosis vs. apoptosis or both
─ poor retention of cells
─ rejection
─ true contribution to contraction
─ only transient improvement in LV function
Methods
 In recipient athymic nude rats, the proximal left anterior
descending coronary artery was occluded for 15 minutes;
then the coronary artery was reperfused (ischemic with
minimal infarction).
 A cell suspension of two million cells containing GFP positive
beating immature cardiomyocytes derived from human
embryonic stem cells was injected directly into the
myocardium within the ischemic risk area at 5 minutes after
left coronary artery occlusion.
 The hearts were harvested at various time points (hours, 1, 2
or 4 weeks) later and processed for histology,
immunohistochemical staining, and fluorescence and
immunofluorescence microscopy.
Results
 In the nude rats, transplanted hESC-derived
cardiomyocytes formed grafts in 9 out of 10
hearts demonstrated by H&E staining,
immunohistochemical staining and GFP epifluorescence.
 Cells that demonstrated GFP epi-fluorescence
also stained positive (co-localized) for the
muscle marker alpha-actinin and exhibited
cross striations (sarcomeres).
 Furthermore cells that stained positive for the
antibody to GFP (immuno-histochemistry) also
stained positive for the muscle marker
sarcomeric actin.
A
E
B
C
F
D
G
Transplanted hESC-CMs form stable grafts in vivo in nude rat (4 weeks post-transplantation) A. Low power image of GFP
epi-fluorescence (green) in a heart engrafted with hESC-derived cardiomyocytes. B-D. GFP epi-fluorescence (green; panel
B, alpha-actinin immune reactivity (red, rhodamine-conjugated secondary antibody; panel C) and a merged image showing
both signals (panel D)
Transplanted hESC-CMs form stable grafts in vivo in nude rat (4 weeks posttransplantation). Green cells represent the transplant. Note their sarcomeres and
smaller diameter compared to native host myocardial cells on the right.
Results
At 4 weeks after transplantation,
engrafted hESC-derived cardiomyocytes expressed connexin 43,
suggesting the presence of nascent
gap junctions between donor and
host cells.
A
B
C
D
E
F
At 4 weeks engrafted hESC-CMs expressed connexin 43 in nude rat
Results
 No significant inflammatory response was
observed in the myocardium of nude rats
that received hESC-derive cardiomyocytes at
4 weeks after transplantation.
 In contrast, hESC-derived cardiomyocytes
injected into immune competent SpragueDawley rats resulted in an overt lymphocytic
infiltrate typical of rejection.
A
C
B
D
hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an
overt lymphocytic infiltrate typical of rejection at 4 weeks after transplantation.
Improvements in LVEF in Infarct Models
Receiving h-ESCs
Author
Reference
Model
LV Function
Results
Kofidis et al.
European J. Cardiothoracic Surgery 2005
Mouse MI Model
MRI-3 weeks
EF-control 44%
EF cells 58% (p = ?)
Author
Reference
Model
LV Function
Results
Laflamme et al.
Nature Biotech 2007
Rat MI model
MRI - 4 week
EF-control 42-45%
EF-cells + cocktail 50% (p=0.05)
Improvements in LVEF in Infarct Models Receiving
ESCs
Author
Reference
Model
LV Function
Results
Caspi et al.
JACC 2007
Rat MI Model
Echo 30-60 days
FS controls 20 → 10% from post MI to day 60
FS cell treated 22 → 25% (P<0.005)
Author
Reference
Model
LV Function
Results
VanLaake et al.
Stem Cell Research 2007
Mouse MI model
MRI 4 weeks , 12 weeks
4 week controls EF ~ 15%; cells EF ~ 25%
(p<0.05)
12 weeks controls, cells EF ~ 18-20% (p = NS)
Van Laake LW et al. 2007;Stem Cell Research 1:9-24
Van Laake LW et al. 2007;Stem Cell Research 1:9-24
Left ventricular ejection fraction (%)
80
67.6 ± 1.4
70
60.9 ± 1.4
60
50
40
hESC
Saline
Kearns-Jonker M, Dai W, Kloner RA
Laflamme MA et al., Nature Biotechnology 2007;9:1015
In the Laflamme, Murry study, in order to obtain successful
grafts of cardiomyocytes derived from human embryonic
stem cells, a complex pro-survival cocktail was required in
their model consisting of:
1. Activin A, bone morphogenetic protein 4 (Yields higher
2.
3.
4.
5.
6.
7.
differentiation to cardiomyocytes)
Matrigel
A cell-permeant peptide from Bcl-XL to block
mitochondrial death pathways
Cyclosporine A to attenuate cyclophilin D-dependent
mitochondrial pathways
Pinacidil (opens KATP channels to mimic
preconditioning)
IGF-1 (insulin growth factor-1) to activate Akt pathways
Caspase inhibitor ZVAD-fmk
In Kofidis et al. study
(European J Cardiothoracic Surgery 2006)
1. Allopurinal + uricase and
2. Ibuprofen pretreatment of the rats (i.e.
treating the host tissue)
Enhanced differentiation of transplanted
hESC engraftment in mice and resulted
in better restoration of myocardial
function
Kofidis T et al. Cardio-Thoracic Surg 2006;29:50-55
In Kloner studies & Laflamme and Murry
studies:
─ Anti-apoptotic proteins did not enhance the graft
(Kloner)
─ Overexpression in grafted cells of the
antiapoptotic proteins Bcl-2 and Akt alone did
not enhance grafts (Murry et al.)
─ Use of broad spectrum immunosupressive and
antiinflammatory cocktail (including steroids) did
not enhance the graft (Murry et al.)
─ In Kloner in-vitro studies, FGF (fibroblast growth
factor) enhanced human embryonic graft size
and myofilament content.
Physiological function and transplantation of
scaffold-free and vascularized human cardiac
muscle tissue.
KR Steven et al. PNAS 2009; 106:16568-73,
1. Patches of enriched cardiomyocytes did not demonstrate
significant survival following in vivo implantation.
2. The authors created prevascularized patches from
cardiomyocytes, endothelial cells (both human umbilical
vein and h-ESC derived endothelial cells) and fibroblasts.
3. These vascularized patches actively contracted, could
be electrically paced, and exhibited passive mechanics
similar to intact myocardium.
4. Implanting these patches into in vivo skeletal muscle or
heart resulted in larger viable grafts (10x) than
cardiomyocytes alone. The preformed human
microvessels connected with rat host coronary
vasculature.
5. The authors concluded that addition of vascular and
stromal elements to human myocardial grafts
enhanced graft size and survivability.
Tissue Engineering of Vascularized Cardiac
Muscle From Human Embryonic Stem Cells
Caspi O. et al. Circ. Res. 2007; 100:263-272.
The authors formed synchronously contracting engineered
3D human cardiac tissue grafts formed from human
embryonic stem cells with an endothelial vessel network
onto a scaffold composed of a porous sponge. Addition
of embryonic fibroblasts reduced endothelial cell death
and improved endothelial cell proliferation.
Review of studies of hES- derived
cardiomyocytes implanted into rodent infarct
models suggest:
1.
2.
3.
4.
5.
6.
There is some degree of cell survival.
Cells do thicken infarcted wall.
There is improvement in LV function at least
initially. EF improves initially by ~ 5 - 14%. One
study showed late loss of effect.
Rejection occurred in immune competent rats in
our studies.
Cells can be shipped over long distances and
survive.
Evidence of early gap junctions between
transplanted cells & host.
Nelson TJ et al. Repair of acute myocardial
infarction by human stemness factors induced
pluripotent stem cells. Circulation 2009;120:408416
Fibroblasts were transduced with stemness
factors OCT 3/4, SOX 2. KLF 4 and C-MYC and
converted into an embryonic stem cell-like
phenotype
Fibroblasts reprogrammed by stemness factors
acquired the potential to repair acute myocardial
infarction induced in mice
Nelson TJ et al. Circulation 2009;120:408-416
Nelson TJ et al. Circulation 2009;120:408-416
Nelson TJ et al. Circulation 2009;120:408-416
Nelson TJ et al. Circulation 2009;120:408-416
iPS Programmed Without a C-MYC Yield
Proficient Cardiogenesis for Functional Heart
Chimerism
iPS Programmed Without a C-MYC Yield Proficient Cardiogenesis for
Functional Heart Chimerism.
Martinez-Fernandez A, et al. Circ. Res. 2009; 105:648-656.
Original nuclear reprogramming included use of oncogenes, such as cMyc to dedifferentiate the cell. The concern here is cancer.
In this study transgenic expression of 3 human stemness factors SOX2,
OCT4, KLF4 reset murine fibroblasts to pluripotent state. These 3factor induced pluripotent stem cells (ips) clones demonstrated
cardiac differentiation including expression of cardiac markers and
in vitro excitation-contraction.
Thus 3-factor ips bioengineered without C-MYC could achieve
cardiogenesis.
We are currently studying the effects of cotransplantations of h-ESCs with MSCs alone or
MSCs that are genetically engineered to overexpress heme-oxygenase.
Conclusions
Human - embryonic stem cell derived
cardiomyocytes and induced pleuripotent stem
cell-derived cardiomyocytes can form viable
cardiac grafts that can contribute to improved
cardiac performance. More longer term studies
are needed as well as better approaches to long
term cell survival. Long term data on teratomas
are needed.