“Seeing” Stem Cells Helps in Fight Against Peripheral Arterial Disease

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 willdiscuss 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

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

12. Bioluminescence Imaging Oxyluciferin Luciferin Luciferase ATP Light High Bioluminescence Signal Cells expressing luciferase Low

13. 10 8 6 4 2 0 10 8 6 4 2 0 10 8 6 4 2 0 10 8 6 4 2 0 10 8 6 4 2 0 ×103 ×106 ×106 ×105 ×105 Bioluminescence imaging of Embryonic Stem Cells A P/s/cm2/sr P/s/cm2/sr P/s/cm2/sr P/s/cm2/sr P/s/cm2/sr Cao F, et al. Circulation 2006;113:1005-14 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. BLI Immediately post injection Pure alginate caps PFOB encapsulated transfected MSCs How to Administer Reporter Probe? DynaCT – 24 hrs post

16. Using Interventional Radiology to Guide Reporter Probe Injections

17. Immediately post injection C-arm CT Targeting Pure alginate caps PFOB encapsulated transfected MSCs 24 hrs post injection 48 hrs post injection PFOB encapsulated transfected MSCs PFOB encapsulated transfected MSCs

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. Mark Pittenger Randall Young Christine Lorenz Tina Ehtati Steve Shea Wesley Gilson Robert Krieg Aravind Arepally Brad Barnett Jeff Bulte Lawrence Hofmann Gary Huang Acknowledgments NIHR01-HL63439, R01-HL73223, K08 EB004348, R21-HL89029, R01-EB007825, and MD-SCRFII-0399-00

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