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Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.
In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1
A representative bioluminescence image of transplanted cells in the left ischemic hindlimb is shown in Figure 1. During acquisition of bioluminescence, the intensity will increase with time, and the maximum value obtained during the time course should be reported as the final value.
ESCs are a promising cell source for the treatment of tissue ischemia because of their plasticity of differentiation and their ability to give rise to cell lineages comprising all three germ layers, including endothelial cells. To overcome the ethical concerns associated with ESCs, induced pluripotent stem cells (iPSCs) may be an alternative pluripotent stem cell source that overcomes the ethical concerns. Besides ESCs, adult stem cells such as endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs) can also be used, but these cell types may have limited therapeutic effect in patients with PAD. Intramuscular delivery of cells is minimally invasive and easy to perform, and this mode of delivery is also amenable to the delivery of soluble factors or plasmids. However, for easier access of the transplanted cells to the vasculature, systemic delivery into the femoral artery or tail vein can be used instead of intramuscular injections.
Bioluminescence imaging offers the advantage of performing high-throughput and non-invasive tracking of cell survival. When combined with functional assays such as laser Doppler blood perfusion or histological analysis of neovascularization, these techniques together can allow researchers to assess the therapeutic effect of cell transplantation on the recovery of hindlimb ischemia.
In conclusion, we have demonstrated a simple and reproducible method for delivering and tracking of ESC-derived endothelial cells for treatment of hindlimb ischemia.
The authors thank Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, and the Stanford Small Animal Imaging Core for technical assistance. The authors also thank A.M. Bickford, Inc. for support of veterinary equipment. This research was supported by research grants from the National Institutes of Health (R01 HL-75774, R01 CA098303, R21 HL085743, 1K12 HL087746), the California Tobacco Related Disease Research Program of the University of California (15IT-0257 and 1514RT-0169 ), and the California Institute for Regenerative Medicine (RS1-00183).
N.H. is supported by a fellowship from the American Heart Association. can Heart Association.