1. “Seeing” Stem Cells Helps in Fight Against Peripheral Arterial Disease
Dorota A Kedziorek, MD, Piotr Walczak, MD, PhD, Yingli Fu, PhD, Frank Wacker, MD,
Dara L Kraitchman, VMD, PhD
Johns Hopkins University
School of Medicine
Russell H. Morgan Department of Radiology and Radiological Science
Baltimore, MD

2. Presenter Disclosure Information
I will discuss off label use and/or investigational use of contrast agents in my presentation.
Financial Relationships to disclose:
Bayer Schering Pharma AG
Boston Scientific Corporation
Surgi-vision, Inc.

3. Peripheral Arterial Disease (PAD)
PAD affects ~8-12 million Americans.
1 in 5 patients with critical limb ischemia have severe enough disease to be ineligible for conventional medical or surgical revascularization therapy.
Gene, protein, and cellular therapies offer an alternative means to attack the response to occlusion by “therapeutic arteriogenesis.”

4. How to create new blood vessels?

5. Problems with Current Cell Therapy
Cannot “see” where cells are injected.
Needles marking injection sites

6. Problems with Cell Therapy
Cannot “see” where cells are injected.
Large number of cells die after administration

7. Solution: Place Cells in “Seaweed Bubble”
APA ≡Alginate-poly-L-lysine-alginate
Lim and Sun, Science 1980
Biocompatible
- Provides surface for cell adhesion
Selective permeability
- Blocks antibody and cellular destruction – good for transplanted donor cells
- Permits diffusion of nutrients and waste products.
IgG, IgM
O2, Glu
VEGF, PGE2,
IL-8, IL-6, HGF, etc.
Courtesy: Ming Chen

8. What If Imaging Visible Capsules Were Possible?
“Liquid Oxygen”
Trimodal Imaging Agent
Bromine – X-ray
Perfluorocarbon – U/S
19F - MRI

9. Imaging Sensitivity to PFOB Caps
capsules/dot: 5 10 25
c-arm CT
19F MRI
2 cm
Fu et al., SCMR 2009

10. X-ray Monitoring of PFOB Caps Delivery in a Rabbit PAD Model
Immediately after injection
Five weeks post-injection
PFOB Caps
Unlabeled
In this anterior-posterior x-ray fluoroscopic image, 7 PFOB caps injections are clearly seen, but the unlabeled caps were not visualized. DSA constructed CT images showed the persistence of PFOB caps up to 5 weeks.

11. Can see the PFOB Cap, but is the cell alive?

12. Bioluminescence Imaging
Oxyluciferin
Luciferin
Luciferase
ATP
Light
High
Tagged with a reporter gene that encodes one of the light-generating enzymes, in our case FIREFLY LUCIFERASE. This protein in the presence of oxygen and ATP oxidates the substrate – luciferin. During this reaction part of the chemical energy is released as a light
Bioluminescence Signal
Cells expressing luciferase
Low

13. Bioluminescence imaging of Embryonic Stem Cells
P/s/cm2/sr
P/s/cm2/sr
P/s/cm2/sr
P/s/cm2/sr
P/s/cm2/sr 10 8 6 4 2
×103 10 8 6 4 2
×105 10 8 6 4 2
×105 10 8 6 4 2
×106 10 8 6 4 2
×106
Cao F, et al. Circulation 2006;113:
Control
Day 5
Day 10
Reporter Probe Dose Intravenous Injection:
Rabbit =10 X Rat!
Patient = 150 X Rat!

14. Animal study #1 Immediately post injection 24 hrs post injection
PFOB encapsulated transfected MSCs
“Naked” transfected MSCs
“Naked” transfected MSCs

15. How to Administer Reporter Probe?
DynaCT – 24 hrs post
PFOB encapsulated transfected MSCs
Immediately post injection
Pure alginate caps
BLI

16. Using Interventional Radiology to Guide Reporter Probe Injections

17. C-arm CT Targeting Immediately post injection 24 hrs post injection
Pure alginate caps
PFOB encapsulated transfected MSCs
24 hrs post injection
48 hrs post injection
PFOB encapsulated transfected MSCs
PFOB encapsulated transfected MSCs
C-arm CT to guide the caps
Cell are alive

18. Seaweed Plus Liquid Oxygen Plus Firefly “Brew”
Seaweed (Protanal®)
Happy Cells
FDA-approved agents in bubble
Better able to survive to create new blood vessels for PAD
Visible by X-ray for Interventional Radiologist to tailor therapy
Liquid
Oxygen
(Oxygent®): _
Firefly

19. R21-HL89029, R01-EB007825, and MD-SCRFII-0399-00
Acknowledgments
Aravind Arepally
Brad Barnett
Jeff Bulte
Lawrence Hofmann
Gary Huang
Mark Pittenger
Randall Young
Christine Lorenz
Tina Ehtati
Steve Shea
Wesley Gilson
Robert Krieg
NIH R01-HL63439, R01-HL73223, K08 EB004348,
R21-HL89029, R01-EB007825, and MD-SCRFII

20. Conclusions “Seaweed plus liquid oxygen plus firefly brew”:
Enhanced cell viability
Enables visualization with X-ray and Optical Imaging
C-arm CT provides a minimally invasive method to target the reporter probe for localized injections to our X-ray visible capsules to reduce dose of reporter probe.
First demonstration of X-ray visible stem cell therapeutic with ability to measure cell viability by imaging.

21. Trimodal Imaging
MRI
c-arm CT US