microrna as a biological drug and recovery of myocardial ...figure 2. left, fibrin hcmp patch (2 cm...

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

3

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

5

(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:

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

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

24

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

29

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%