Download S. Nunes de Vasconcelos

Tissue engineering strategies for cardiovascular regeneration
Sara S. Nunes de Vasconcelos, Ph.D.
Scientist
Div. of Experimental Therapeutics
Toronto General Research Institute
University Health Network
Overview
Cardiovascular diseases
 Leading cause of death globally (WHO)
Myocardial infarction
 Myocardial infarction (MI) – death of
cardiomyocytes and loss of function
 Adult cardiomyocytes – low proliferation
Cardiac tissue regeneration:
Ischemia
 Re-establish the vasculature
 Replace lost cardiomyocytes
Human stem cell-derived cardiomyocytes – high potential for application in
regenerative medicine
Stem cells: hES and iPS cells
Human ES and iPSC - unlimited self renewal
- differentiate into any cell type
iPSC
ESC
Cell Stem Cell , Volume 10, Issue 1, Pages 16-28 (DOI:10.1016/j.stem.2011.12.013)
hESC and hiPSC cardiomyocyte differentiation
Concentration gradients
hPSC growth
Feeder
depletion
aggregation
Stage 1
Stage 2
Stage 3
BMP4
BMP4
bFGF
Act.A
VEGF
DKK1
VEGF
DKK1 (?)
bFGF
d0
d1
d4
Hypoxia d0-d12
d8
Human iPSC-derived cardiomyocytes
Cardiomyocyte = 69% (green)
Control = 2.5%(black)
Yang et al, Circ. Res. 2014.
hESC and hiPSC-derived cardiomyocytes
•
Characteristics
Sarcomere structure - absence of H zones, I bands
and M lines
High proliferation rates: 17%, EBs day 37; 10%, EBs
day 21-35;
Expression of the “fetal gene program” (ANF, BNP
and α-MHC).
Immature electrophysiological properties
Low force of contraction – 0.1-11.8±4.5mN/mm2
(adult 50mN/mm2)
Immature action potentials and Ca2+ handling
properties - immature sarcoplasmic reticulum
Automaticity
Maturation platform
•
Extracellular matrix cues (tension, col I, laminin)
•
3D tissues
•
Self assemble
•
Soluble factors (VEGF, bFGF)
•
Electrical stimulation (pacing)
•
Variable size
Cardiac biowire platform
Adapted from Nature Reviews, 2006.
Nunes et al, Nature Methods 2013.
Cardiac biowires
Optical mapping
Nunes et al, Nature Methods 2013.
Electrical field stimulation regimen
Low frequency ramp up regimen (3Hz)
Day 0
Pre-culture
Cell seeding
Day 7
E. Stimulation (3-4V/cm)
1Hz 1.83 2.66
3
3
3
Day 14
3Hz
Analysis
Stimulation chamber
Biowire
High frequency ramp up regimen (6Hz)
Day 0
Pre-culture
Day 7 E. Stimulation (3-4V/cm) Day 14
Electrode
Cell seeding
1Hz 1.83 2.66 3.49 4.82 5.15 6Hz
Analysis
Controls: Non-stimulated biowires, starting population EBd20 and age matched EBs (EBd34)
Cardiomyocytes display more mature sarcomere structure
Nunes et al, Nature Methods 2013.
Electrical pacing improves cardiomyocyte impulse
propagation
Nunes et al, Nature Methods 2013.
Relative gene expression
(normalized to housekeeping)
Electrical pacing improves cardiomyocyte
electrophysiology
20
KCNJ2/Kir2.1
15
10
5
0
EBd20 EBd34 CTRL
3 Hz
6 Hz
Nunes et al, Nature Methods 2013.
Electrical pacing improves calcium handling properties
Non-stimulated
control (n=18)
3Hz (n=11)
6Hz (n=19)
p value
(control vs.
6Hz)
Amplitude (F/F0)
2.50 ± 0.13
2.64 ± 0.18
2.93 ± 0.12*
0.017
Rising slope (F/F0/s)
5.29 ± 0.55
5.55 ± 0.66
7.36 ± 0.64*
0.025
Time to peak (s)
0.511 ± 0.046
0.496 ± 0.044
0.403 ± 0.037*
0.049
τ-decay (s)
0.591 ± 0.058
0.521 ± 0.057
0.419 ± 0.043*
0.022
Time to base (s)
1.461 ± 0.137
1.310 ± 0.147
1.142 ± 0.091*
0.035
*denotes statistical significance vs. non-stimulated control (mean ± SEM).
Nunes et al, Nature Methods 2013.
Calcium handling: SR protein expression
Unpublished data.
Relative gene expression
(normalized to housekeeping)
Culture in biowire downregulates fetal gene program
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
NPPB/BNP
1.2
NPPA/ANF
1.2
1
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0
EBd20 EBd34 CTRL
3 Hz
6 Hz
MYH6/α-MHC
EBd20 EBd34 CTRL
3 Hz
6 Hz
EBd20 EBd34 CTRL
3 Hz
6 Hz
Nunes et al, Nature Methods 2013.
Cardiomyocyte proliferation and automaticity
Cardiomyocyte proliferation (%)
25
**
20
*
15
#
&
10
5
0
EBd20 EBd34 Control
3Hz
6Hz
30%
39%
85%
Nunes et al, Nature Methods 2013.
Vascularized cardiac tissues
•
Cardiomyocyte maturation – vasculature
•
Regenerative medicine - implants
I
II
III
Adapted from Nature Reviews, 2006.
Cardiac tissue regeneration
Cardiac regeneration:
 Replace lost cardiomyocytes
 Survival
Time course study showing death of transplanted
cardiomyocytes into a rat myocardial infarction model after MI.
Zhang et al. J. Mol. Cell. Cardiol. (2001).
Microvessel fragment-based neovascularization
Advantages:
•
•
•
•
•
•
•
Maintain vessel shape - 3D arrangement
All the relevant cell types – EC and PVC
Connect to host circulation
Form a hierarchical vasculature in vivo
Robust PVC coverage
Long-lasting - over 6 months post implantation
High potential for application in regenerative medicine
Week 2
Week 4
Nunes et al, Microcirculation 2010; Microvascular Research. 2010; PlosOne 2011.
Microvasculature works as a metabolic interface between
the host circulation and implanted hepatocytes
Liver tissue mimics
Nunes et al, Nature Scientific Reports. 2013.
Can microvessels promote the survival of stem cellderived cardiomyocytes in vivo?
Cardiac tissue mimics – 30 days post implantation
Unpublished data.
Stem cell-derived cardiomyocyte survival is extensive
Cardiomyocyte area
(cTNT staining, normalized to 1h)
hESC-CM + adipose-derived microvessels
Implanted for 30 days
1.2
1
0.8
0.6
0.4
0.2
0
1h
6 weeks
Unpublished data.
Conclusions
Human stem cell-derived cardiomyocytes in Biowires remodel in a manner consistent with
enhanced maturation, displaying:
•
Increased cell area/size
•
Decreased proliferation
•
Reduced intrinsic firing rates
•
Improved electrophysiological and calcium handling properties
•
Downregulation of fetal gene program
•
Not adult phenotype
•
Neovasculatures from adipose-derived microvessels support extensive survival of
hESC-derived cardiomyocytes
Acknowledgments
Wafa Altalhi (graduate student)
Adrian Lee (summer student)
Elena Bajenova
• Radisic lab (U. Toronto)
Jason Miklas
Boyang Zhang
Carol Laschinger
• Laflamme lab (U. Washington)
Benjamin VanBieber
• Keller lab (McEwen Centre)
Mark Gagliardi
Nicole Dubois
• Nanthakumar lab (U. Toronto)
Stephane Masse
Patrick Lai
• Backx lab (U. Toronto)
Jie Liu
Rooz Sobbi
• Coles lab (Hospital for Sick Children)
Shabana Aafaqi
Microvessel-derived vasculatures recruit PVCs and
display specific AV identity
A
a
d
coverage
perivascular
%%
coverage
smooth musclecell
B
Week 4
Week 2
b
c
100
80
60
40
20
0
0c
3c
8c
10c
cultured
angiogenic
days
Days
14i
28i
implanted
post-angiogenic
Human ESC-cardiomyocytes respond to Isoproterenol
treatment similarly to adult cardiomyocytes
Day 0
Culture in biowire
Day 7
Drug treatment
Day 14
Analysis
Force of contraction (mN/mm2)
EB d20
0.6
0.5
p < 0.001
0.4
0.5Hz
0.3
* *
0.2
1Hz
0.1
0
Control
ISO
Unpublished data.
Isoproterenol treatment induces the ‘fetal gene
program’ and promotes hypertrophy
aMHC
2
1.2
1
ANF
1.5
Ratio of normalized gene expression
0.8
1
0.6
0.4
0.5
Area (µm2)
0.2
0
0
EBd20 EBd34 CTRL
EBd20 EBd34 CTRL
ISO
20
2.5
ISO
BNP
15
1.5
10
1
5
0.5
260.0 ±46
ISO
351.9 ±47
Table 1: Measurements performed on single
cardiomyocytes dissociated from biowires at the
end of cultivation. Cell area (μm2), average ±s.e.m.,
n= 3.
bMHC
2
Control
0
EBd20 EBd34 CTRL
0
EBd20 EBd34 CTRL
ISO
ISO
Unpublished data.
Human iPSC-derived cardiomyocyte maturation
Fig. 2: Schematic representation of cardiac development. Approximate human and murine gestational ages are indicated above
the drawings and active transcription factors at respective stage are listed below. A schematic cross section through an early
embryo is shown in the first panel to indicate the bilaterally symmetric cardiac structures composed of an endocardial tube (En)
separated by an extracellular matrix (ECM) from the myocardial precursors (M). Bilateral dorsal aortae (DAo) are indicated. The
second panel shows a frontal view of the midline cardiac tube. Gene expression analysis reveals early specification of chambers
including right ventricle (RV) and left ventricle (LV) and atrium. Panels 3 and 4 represent frontal and left lateral views, respectively,
of a looped heart tube. The pro-epicardial organ (ProE) is located posteriorly and gives rise to epicardial cells (Ep, shown in brown)
that migrate over the ventricles, as indicated by arrows. Panels 5 and 6 depict vascular remodeling. The aortic arches in panel 5
(numbered) are populated by neural crest cells (NC, arrows) and are color coded to match the mature arterial segments indicated
in panel 6. TA, truncus arteriosus. RSC, right subclavian artery. RCC, right carotid artery. LCC, left carotid artery. LSC, left subclavian
artery. DA, ductus arteriosus. Ao, aorta. PA, pulmonary artery. Taken from Epstein and Buck (Pediatr Res 48: 717–724, 2000).
Human stem cell-derived cardiomyocyte applications
Yang et al, Circ. Res. 2014.
Myocardial infarction
Ischemia
Electrical pacing improves calcium handling properties
Nunes et al, Nature Methods 2013.