1. Stem Cells Build New Blood Vessels to Treat Peripheral Arterial Disease Dorota A Kedziorek, MD, Yingli Fu, PhD, Piotr Walczak, MD, PhD, Tina Ehtiati, PhD, Steffi Valdeig, Gary Huang, Jeff W.M. Bulte, PhD, Lawrence V. Hofmann, MD, Frank K. 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. Siemens Healthcare USA

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. Normal signals to build new vessels are in foot, but problem higher in the leg.

4. How to create new blood vessels?

5. Problem with Current Cell Therapy Large numbers of cell are administered but fail to engraft due to: Poor oxygen/nutrient supply Inflammatory cytokines Survival signals by cell-cell contact lost Zhang, Murry et al., J Mol Cell Cardiol 33, 907–921 (2001)

6. Solution: Place Cells in “Seaweed Bubble” Courtesy: Ming Chen  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. Lim and Sun, Science 1980 APA ≡ APA ≡Alginate-poly-L-lysine-alginate VEGF, PGE 2, IL-8, IL-6, HGF, etc. IgG, IgM O 2, Glu

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

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

9. C-arm CT Flat panel detector 16 second digital subtraction angiogram (DSA) acquisition

10. MRI & CT of Seaweed Bubble Cells 19 F MRIC-arm CT ComparisonRegistration Error Post-mortem/CT2.83 ± 0.85 mm CT/MRI0.32 ± 0.14 mm

11. How to See Stem Cells in Bubble? X-ray-visible & “Firefly” Stem Cells Bioluminescence Signal High L ow

12. In Vivo Viability of MSCs Blue: Nucleus Green: Dying cell

13. 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

14. In Vivo Experimental Protocol Male New Zealand White Rabbits Harvest 72 hr2 wk Bone marrow aspirate Adherent “mesenchymal” stem cells isolation & culture Freeze MSCs for “off-the-shelf” use ♂

15. In Vivo Experimental Protocol Female New Zealand White Rabbits (n=21) Harvest Endovascular occlusion of the superficial femoral artery with pre-occlusion angiogram SFA Occlusion 72 hr2 wk ♀♀ Liddell et al., JVIR, 2005;16(7): 991-8.

16. In Vivo Experimental Protocol Harvest Six IM injections X-ray capsules X-ray Fluoroscopic documentation of: – X-ray Cap location – Collaterals by X-ray Angiogram XCap SFA Occlusion 72 hr2 wk Female New Zealand White Rabbits (n=21) XCaps + MSCs (n=5) Naked MSCs (n=5) Sham (n=6) XCaps + No MSCs (n=5) ♀

17. Female New Zealand White Rabbits (n=21) XCaps + MSCs (n=5) Naked MSCs (n=5) Saline Sham (n=6) XCaps + No MSCs (n=5) In Vivo Experimental Protocol Harvest SFA Occlusion XCap Histopathology 72 hr 2 wk

18. Example: Empty XCap Pre-occlusionDay 14 Post-occlusion

19. Example: Xcap with Stem Cells Pre-occlusionDay 14 Post-occlusion

20. Efficacy of Stem Cells at Day 14 Empty CapsuleXCap with Stem Cells

21. TIMI Frame Count @ 14 Days N=16 Time 0 5 10 15 Xcap Blank Xcaps Time (sec) P<0.002 Naked Cells P<NS P<0.01

22. Histopathology – Vessel Density CD31 Staining for Endothelium

23. Trimodal Imaging 19 F and 1 H MRI c-arm CT BLI

24. Conclusions An X-ray-visible capsule made of clinical grade components was developed that: 1.enabled targeting of injections where most needed using common interventional radiology X-ray equipment. 2.enabled determination of cell viability in vivo. 3.enhanced cell viability after administration. 4.improved therapeutic effect of creating new blood vessels for treatment of peripheral arterial disease.

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