MicroRNA as a Biological Drug and Recovery of Myocardial ...Figure 2. Left, Fibrin hCMP patch (2 cm...
Transcript of MicroRNA as a Biological Drug and Recovery of Myocardial ...Figure 2. Left, Fibrin hCMP patch (2 cm...
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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
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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
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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
3
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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
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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
5
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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
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IB4
DAPI H &E
(A) (B)
PatchPatch
Interface
(C)
Significant Angiogenesis Support the CM Grafts
(week 4)
Wendel J et al
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Fabrication human cardiac
muscle patch
CM cluster Fabrication of
hiPSC-CM Patch
Zhang L Circ 2014
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Fabrication of Larger and Thicker Human Myocardial Tissue Equivalent
hiPSC Derived tri- Lineage Cardiovascular Cells for Postinfarction
LV Remodeling
UAB
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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)
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Electrical pacing of human cardiac MTE patch
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Conduction velocity = 15.1 cm/s APD50 = 306 ms
APD80 = 353 ms
Optical mapping of Vm in cardiac patch
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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)
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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
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Day 1
Day 28
hcTnT DAPI Merged
hcTnT DAPI Merged
CM Maturation in vivo
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Completely Noninvasive Cardiac MR Spectroscopy
at 7T/65CM Magnet
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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
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Acknowledgements:
21
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
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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
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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
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Potential Mechanisms of Actions
• Strategy 3:
– Local Delivery : Myocardial Tissue Equivalent Patch using hiPSC- tri lineage Cardiac Cells
24
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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
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BZ myocardial energetics
Unidirectional ATP utilization rate:
ATP→ ADP + Pi
In vivo 31P MR spectroscopy*#
**#
*
Xiong Q et al Circulation . 2013
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BZ myocardial Wall stress, FluxATP→Pi contractile function
2
LVSP radius
thickness
Laplace law:
wall stress (P)
-BZ
-BZ
-BZ
-BZ
Normal MI CELL
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Over stretched myocytes in failing hearts
Murakami Y et al Circ 1999
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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
29
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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)
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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
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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
*
*
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9.4T-65cm magnet 7T-90cm magnet
Center for Magnet Resonance Research
Aa b c
d e f
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[ADP] K x [(PCr) /(ATP)] -1
k
[PCr] [ADP] [ATP] [Cr]
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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
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Recipient myocardial protein expression
profile changes
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Phase contrast
Identify the grafted hiPSC-CMs (week-4)
GFP DAPI cTnT DAPI
Merged
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Cell transplantation reduced apoptosis (day-3)
Patch only
TUNEL+ cTnI cTnI DAPI Merged
MI
P + Cell
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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
*
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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.
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Identify the grafted hiPSC-SMCs (week-4)
GFP DAPI SMA DAPI
Bright fieldGFP SMA cTnI DAPI
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Identify the grafted hiPSC-ECs (week-4)GFP cTnTDAPI
hCD31 DAPI
Bright field
hCD31 cTnI DAPI
42
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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
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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
![Page 45: MicroRNA as a Biological Drug and Recovery of Myocardial ...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.](https://reader033.fdocuments.net/reader033/viewer/2022060522/605102bde5156147ce6ce7b4/html5/thumbnails/45.jpg)
3D Porous PEGylated Fibrin patch for enhanced delivery of hiPSC 3-lineage cardiac cells
Zhang, G Tissue Engineering
2007
PEG
GM
![Page 46: MicroRNA as a Biological Drug and Recovery of Myocardial ...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.](https://reader033.fdocuments.net/reader033/viewer/2022060522/605102bde5156147ce6ce7b4/html5/thumbnails/46.jpg)
Gelatin microsphere for IGF delivery
![Page 47: MicroRNA as a Biological Drug and Recovery of Myocardial ...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.](https://reader033.fdocuments.net/reader033/viewer/2022060522/605102bde5156147ce6ce7b4/html5/thumbnails/47.jpg)
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%