Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Characterization of the Endothelial Progenitor Cell from Adult Tissue using Vav/Cre RFP-GFP Murine Model and Single Cell Microfluidics
Melanie Rodrigues, PhD, Robert C. Rennert, BA, Sarah Bishop, MD, Michael Januszyk, MD, Zeshaan Maan, MD, Michael Sorkin, MD, Dominik Duscher, MD, Geoffrey C. Gurtner, MD.
Stanford University, Stanford, CA, USA.

PURPOSE:
Endothelial progenitor cells (EPCs) from circulating blood have been shown to form blood vessels and are touted to aid in therapeutic neovascularization. However, EPCs are inadequately characterized and current isolation relies on intricate in vitro culture techniques rather than a defined surface-marker profile, limiting their immediate utilization.
METHODS:
To characterize EPCs, our lab developed a transgenic mouse, vav-cre RFP-GFP, with cells of the hematopoietic lineage expressing GFP and non-hematopoietic cells, expressing RFP. In parallel, we established a microfluidics-based single cell transcriptional analysis technique to identify cell surface markers for these elusive cell-populations. Combining these novel techniques in a parabiotic model of neovascularization, we aimed to isolate unique EPC populations and determine their origin, hierarchy and cell surface characteristics.
RESULTS:
Parabiosis of vav-cre RFP-GFP to a severe combined immunodeficiency (SCID) mouse containing an ischemic flap on its dorsal surface repeatedly drew in GFP+ but not RFP+ cells from the vav-cre mouse. GFP+ Lin- cells, considered putative progenitor cells from the vav cre were isolated on a single cell level from the flap of the SCID mouse. Single cell microfluidic PCR on these cells revealed three distinct stem cell/ progenitor sub-populations of hematopoietic origin entering the ischemic neovascularization site: a circulating hematopoietic stem cell population c-kit+, CD34-, Sca1 low, Myc+, a stem cell population of hematopoietic origin CD34+ Sca1+ Klf4+ Cxcl12+, and most importantly a putative endothelial progenitor cell population, expressing both stem cell genes Sca1, CD34, Klf4 as well as endothelial genes. These cells were traced to the bone marrow and were found to be present at the neovascularization site even after 4 weeks of injury. Cryosections of the ischemic flap on the SCID mouse confirmed the incorporation of these stem/ progentior cells at regions of neovascularization.
CONCLUSION:
We have identified a circulatory endothelial precursor cell derived from the bone marrow, which incorporates into blood vessels at sites of neovascularization.


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