“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

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.

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.”

How to create new blood vessels?

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

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

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

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

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

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.

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

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

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;113:1005-14 Control Day 5 Day 10 Reporter Probe Dose Intravenous Injection: Rabbit =10 X Rat! Patient = 150 X Rat!

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

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

Using Interventional Radiology to Guide Reporter Probe Injections

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

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

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-0399-00

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.

Trimodal Imaging MRI c-arm CT US