microrna as a biological drug and recovery of myocardial ...figure 2. left, fibrin hcmp patch (2 cm...
TRANSCRIPT
Jianyi (Jay) Zhang, MD, PhD
Professor of Medicine, of EngineeringUniversity of Alabama - Birmingham
1
MicroRNA as a Biological Drug and Recovery of Myocardial Infarction
Exosome
UAB
Rebuilding the Failing Heart with Cell Therapy
Road Blocks to Overcome
1. Low engraftment rate
2. Increased arrhythmic potential
3. Potential Mechanisms of Actions
2
Roadblock 1: Low engraftment rate
• Strategies:
– hiPSC - HLA I/II KO to generate Universal Cell Lines
• In collaboration with Townes lab at UAB
– Local Delivery : Myocardial Tissue Equivalent Patch using hiPSC- tri lineage Cardiac Cells
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4
Roadblocks 2: Arrhythmias: Microenvironment of graft and
recipient heart
• Strategies:
– Deciphering the mechanisms of arrhythmias
• Ectopic center or reentry?
• Ca2+ and Action potential propagation passing the interface: Optical mapping and micro impedance
– hiPSC Gap Junction Protein Over Expression:Cx43
Roadblock 3
Unknown Potential Mechanisms of
Actions
• Strategy 3:
– Local Delivery : Myocardial Tissue Equivalent Patch using hiPSC- tri lineage Cardiac Cells
– The potential mechanisms of actions from the perspective of the regulations in myocardial perfusion, metabolism and function in the in vivo heart
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(A)
(D)
HNA
DAPI
Patch
Interface
(C)
Patch
cTnT
Ki67
PCs
DAPI
(F)
Patch
Host
Interface
Interface
hiPSC-CM TE graft 4 weeks after transplantation
Wendel J et al 2015 SCTM
IB4
DAPI H &E
(A) (B)
PatchPatch
Interface
(C)
Significant Angiogenesis Support the CM Grafts
(week 4)
Wendel J et al
Fabrication human cardiac
muscle patch
CM cluster Fabrication of
hiPSC-CM Patch
Zhang L Circ 2014
Fabrication of Larger and Thicker Human Myocardial Tissue Equivalent
hiPSC Derived tri- Lineage Cardiovascular Cells for Postinfarction
LV Remodeling
UAB
Figure 2. Left, Fibrin hCMP patch (2 cm x 4 cm) containing 10 million hiPSC-CMs, 5
million hiPSC-ECs, and 5 million hiPSC-SMCs. After 7 days in culure, the hCMP beats
regularly at rate of 100 beat/min. Right, Two rectangle fibrin hCMP were sutured on the
surface of a pig heart that exposed to 60 minutes of no flow ischemia reperfusion
Fabrication of Larger and Ticker
Myocardial Tissue Equivalent (MTE)
Electrical pacing of human cardiac MTE patch
Conduction velocity = 15.1 cm/s APD50 = 306 ms
APD80 = 353 ms
Optical mapping of Vm in cardiac patch
Rate dependence of APD and conduction velocity
240
260
280
300
320
340
360
380
400
500 700 900 1100 1300
AP
D (
ms)
CL (ms)
APD50
APD80
8
9
10
11
12
13
14
15
16
17
500 700 900 1100 1300
CV
(cm
/s)
CL (ms)
Dual mapping of ventricle and
implanted patch.
5 mm
A 10 ms 20 ms 30 ms
40 ms 50 ms 60 ms
Activa
tio
n T
ime
(m
s)
5 mm
RH
23
7
GC
aM
P6
Ventricle
CV = 60.5 cm/s
Patch
CV = 29.2 cm/sB
C
5
10
15
20
25
30
0
5
0
Day 1
Day 28
hcTnT DAPI Merged
hcTnT DAPI Merged
CM Maturation in vivo
Completely Noninvasive Cardiac MR Spectroscopy
at 7T/65CM Magnet
hESC/iPS
-VCs
hIPSC-
VCs +
CMs
MSCs
Patching the Heart: Myocardial Repair from Within or Outside
DE MRI TUNEL cTnT
CD31 SMA
BrdU CPC
Infarct size
reduction
Perfusion and
Chamber function
Metabolism
Wo
rkin
g h
yp
oth
esis
CinePerfusion
UAB
Acknowledgements:
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Collaborators :
Tranquillo, Garry, UMN
Kamp, Ge , UW-Madison
Townes, Rogers, Fast, Walcott; UAB
Bursac , Duke
NIH Grants :
NIH RO1s HL67828, HL 95077,
HL114120,
UO1 HL100407
Zhang lab
Acknowledgements:
22
Ø Cardiac Repair using
Stem Cells
Ø Myocardial energetics 31P MR spectroscopy
Collaborators :
Tranquillo, Garry, UMN
Kamp, Ge , UW-Madison
Townes, Rogers, Fast; UAB
Zhang lab
NIH Grants :
NIH RO1s HL67828, HL 95077, HL114120,
UO1 HL100407
N-MSCs HP-MSCs
Hypoxia
Preconditioning
CD4 CD8
Immune Modulation
Arrhythmia detection
PET IH
Telemetry
NHP Model (N=49)
Electromechanical
Stability (PES)
Intramyocardial
Injection
A Non-human Primates
Study
Molecular
Mechanism
Cell Tracing
Cell Survival
Cardiac
Metabolism
Cardiac Function
Evaluation
Circres JAN 2016
Potential Mechanisms of Actions
• Strategy 3:
– Local Delivery : Myocardial Tissue Equivalent Patch using hiPSC- tri lineage Cardiac Cells
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Resistance vessel density (week-4)
0
100
200
300
400
500
MI Patch Cell
Art
eri
ole
de
nsi
ty (
mm
-2)
p<0.05
p<0.05
CD31 SMA cTnT
MI
Patch
P+Cell
BZ myocardial energetics
Unidirectional ATP utilization rate:
ATP→ ADP + Pi
In vivo 31P MR spectroscopy*#
**#
*
Xiong Q et al Circulation . 2013
BZ myocardial Wall stress, FluxATP→Pi contractile function
2
LVSP radius
thickness
Laplace law:
wall stress (P)
-BZ
-BZ
-BZ
-BZ
Normal MI CELL
Over stretched myocytes in failing hearts
Murakami Y et al Circ 1999
31P spectra were acquired with a 3D ultra-short TE chemical shift
imaging (UTE-CSI) sequence in a normal adult mongrel dog
acquired on a Magnetom 7T scanner
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F-actin
cTnI
DAPI
Disaggregated neonatal rat cardiac cells seeded into fibrin gel
Fabrication of a Myocardial Tissue Equivalent
7 days 7 days
CX43
cTnT
DAPI
0
0.5
1
1.5
2
2.5
3
3.5
1 2 3 4
Peak F
orc
e G
en
era
ted
(m
N)
Stimulation Frequency (Hz)
Twitch Force Generation
0 500 1000 1500 2000 2500 3000 3500 4000 4500 50005.5
6
6.5
7
7.5
8
8.5
9
9.5
1 Hz
0 1000 2000 3000 4000 5000 60005.5
6
6.5
7
7.5
8
8.5
9
9.5
0 1000 2000 3000 4000 5000 6000
6
6.2
6.4
6.6
6.8
7
7.2
7.4
0 1000 2000 3000 4000 5000 60007.4
7.6
7.8
8
8.2
8.4
8.6
8.8
9
2 Hz
3 Hz
4 Hz
Forc
e (m
N)
Time (ms)
Patch
Myocardial Tissue Equivalent
Study Groups:1) Sham, (n=5)2) MI, (n=6) : Ligation Only3) TE CM-, (n=5): MI+ tissue equivalent constructed without CMs4) TE CM+, (n=7): MI + tissue equivalent containing CMs
Week 1 and 4 follow up with ECHO Wendel J TE 2014
Engraftment of Tissue Equivalent to the Host Myocardium
Host
Myocardium Patch
Host
Myocardium Patch
nonCM
CM
Sham
Host GraftInterface
nonCM
CM (D)
Host
50um100um
20um
cTnTF-actinDAPI
F-actin
0
20
40
60
80
100
CMpatch
nonCMpatch
MI only
Pe
rce
nt
of
LV a
nte
rio
r w
all
Infarct Size
*
*
9.4T-65cm magnet 7T-90cm magnet
Center for Magnet Resonance Research
Aa b c
d e f
[ADP] K x [(PCr) /(ATP)] -1
k
[PCr] [ADP] [ATP] [Cr]
Fig. 6. Number of proteins identified from heart tissue
homogenate using gene ontology annotation for cell
compartment analysis.
Myocardial Differential Protein Expression Profile Changesin response to Cell Patch Therapy
-1.0 0.0 1.0
No
rmal
MI
MI+
iPS
C
-VC
Regulation of
metabolic process
Cytoskeleton organization
Regulation of cell
morphogenesis
Electron transport chain
ATP Synthesis coupled
electron transport
Pro
tein
s u
p-r
eg
ula
ted
in
MI
Pro
tein
s d
ow
n-
reg
ula
ted
in
MI
AA
B
C
Recipient myocardial protein expression
profile changes
Phase contrast
Identify the grafted hiPSC-CMs (week-4)
GFP DAPI cTnT DAPI
Merged
Cell transplantation reduced apoptosis (day-3)
Patch only
TUNEL+ cTnI cTnI DAPI Merged
MI
P + Cell
Cell transplantation activated c-Kit+ CV progenitor cell (week-4)
0
300
600
900
1200
1500
MI Patch Cell
c-K
it +
CV
PC
de
nsi
ty (
cm-2
)
Patch
c-Kit cTnT DAPI
MI Patch Cell
c-KitDAPI
MI Cell
*
Summary
• The preliminary results suggest the capacity of a fibrin-based cardiac tissue equivalent to engraft 4 weeks after transplantation, which is accompanied by a reduction of infarct size, and improvement of LV chamber function.
• The mechanisms of the reduced infarct size are not clear, but are likely related to the cytokine related protective effect.
• The optimized synergetic effects of the cytokine signaling pathways are depend upon communications between myocytes and non-CM cardiac cells.
Identify the grafted hiPSC-SMCs (week-4)
GFP DAPI SMA DAPI
Bright fieldGFP SMA cTnI DAPI
Identify the grafted hiPSC-ECs (week-4)GFP cTnTDAPI
hCD31 DAPI
Bright field
hCD31 cTnI DAPI
42
EC SMC CM
Growth factor, cytokine Angiogenin 58136 14962.3 56303.5
Angiopoietin-1 1273 8030.25 3967.75
Angiopoietin-2 12206.3 790 1060
IL-6 46934.3 2234.5 5450.5
PDGF-BB 2140.25 306 25.75
VEGF 25.5 18 513.5
TGF-beta1 654.75 758.25 706.5
ChemokineGrowth regulated
protein 57706.5 3516.25 8524.25
IL-8 23679.8 2910.5 4585
MCP-1 65447 65447 65442.8
RANTES 1539 1510 96
MCP-3 1200.25 524.5 16919
Inhibitor of
metalloproteinases TIMP-1 13565.8 18635.5 13430.5
TIMP-2 7394.5 12428.3 8581
Angiogeic inhibitor Angiostatin 219 244.25 267.25
Endostatin 575 159.75 310.75
Protease MMP-9 731 196.75 163.25
Plasminogen activator u PAR 3236.5 2618.5 1101
Angiogeic receptor VEGF R2 700.75 150 185.5
VEGF R3 299.5 326.5 299.5
Tie-2 178 216.25 228.5
Angiogeic profile of EC and SMC conditioned medium
Aims• To develop an efficient hiPSC-CM selection protocol• To examine the efficiency of a patch and microspheres based
enhanced delivery of hiPSC - 3 lineage cardiovascular cells for myocardial repair using an immuno-suppressed porcine model of postinfarction LV remodeling:
- engraftment rate, - vascular density and myocardial perfusion,- myocardial protection and apoptosis
- tracking the endogenous CV PCs with BrdU• Using novel NMR technology to examine the myocardial
bioenergetics and ATP turnover rate in the in vivo hearts with orwithout cell transplantation
• The electrophysiology stability was examined by loop recorder andrecipient myocardial differential protein expression profile byproteomics
3D Porous PEGylated Fibrin patch for enhanced delivery of hiPSC 3-lineage cardiac cells
Zhang, G Tissue Engineering
2007
PEG
GM
Gelatin microsphere for IGF delivery
Cell engraftment rate (week-4)
Quantitative PCR (qPCR) for human Y chromosome
Dual immunostaining
26.76%
33.44%
39.80%
0%
10%
20%
30%
40%
50%
hiPSC-CMs hiPSC-ECs hiPSC-SMCs
Pe
rce
nta
ge
0%
5%
10%
15%
Total cell engraftment rate
Pe
rce
nta
ge
8.97±1.8%