PROGENITOR CELLS AND CELL-BASED THERAPIES Single cell analysis reveals phenotypically distinct sub- populations in putative endothelial progenitor cells Jerry Chen, MD, Michael Januszyk, MD, Micael Sorkin, MD, Geoffrey C Gurtner, MD, FACS Stanford University, Stanford, CA INTRODUCTION: Although preliminary work has already been performed on animal models and even human studies, the true clin- ical potential of EPCs is limited as multiple definitions and methods for isolation of EPCs exist. We hypothesize that current isolation techniques result in phenotypically distinct cell populations and inter-population heterogeneity. METHODS: EPCs were isolated from human peripheral blood by two widely utilized techniques. Primary cells were prospectively iso- lated using flow cytometry and the cell surface markers CD34, CD31, and AC133. EPCs were also isolated by plating mononuclear cells on fibronectin-coated plates and selecting for adherent cells after 7 days in culture. Gene expression of single EPCs was then quantified utilizing microfluidics technology. Cells were clustered into sub- populations based on similar gene expression patterns. RESULTS: Single cell analysis revealed several distinct and repro- ducible sub-populations within both FACS sorted and cultured EPCs. Cluster groups were defined by differential expression of genes including the SDF-1-alpha receptor CXCR4 and the stemness genes NANOG and c-Kit. Comparing gene expression between sorted and cultured EPCs demonstrated that these two populations have signif- icantly different profiles. CONCLUSIONS: Although there exists a wealth of literature pur- porting the properties of EPCs, there lacks a universally accepted definition for these cells. Utilizing single cell gene expression meth- ods, we show that what have been termed “EPCs” are in fact a largely heterogenous cell population. Additionally, different isolation tech- niques result in cell populations with unique phenotypes which could explain the wide array of contradictory results seen in the literature. Future investigation may reveal the truly angiogenic sub- population of cells. Cancer stem cell (CSC) vaccination induces anti-csc immunity by priming csc-reactive antibodies and cytotoxic t lymphocytes Martin U Egenti, MD*, Fang Zheng, MD, Max S Wicha, MD, PhD, Jeffery S Moyer, MD, Mark E P Prince, MD, Qiao Li, PhD, Alfred E Chang, MD, FACS University of Michigan, Ann Arbor, MI INTRODUCTION: Clinical trials utilizing immunotherapy to treat cancer have shown limited efficacy for patients with advanced dis- ease. The inability of these approaches to target cancer stem cells may represent a significant factor for treatment failures. In this study, we identified and isolated CSCs and used them as a source of antigen in a dendritic cell-based tumor vaccine. METHODS: CSCs were sorted from murine D5 (melanoma) and SCC7 (squamous cell cancer) tumor cells using ALDEFLUOR/ ALDH as a marker. Dendritic cells (DC) were pulsed with the lysate of CSCs to create a CSC-DC tumor vaccine. The immunogenicity of the CSCs was evaluated by vaccinating immunocompetent murine hosts, which were then challenged intravenously or subcutaneously with D5 and SCC7 tumor cells. Lung metastases and subcutaneous tumor growth were monitored. Serum and peripheral blood mono- nuclear cells (PBMCs) were also collected for assessment of systemic immunity. RESULTS: The lungs of the CSC-DC vaccinated hosts had signif- icantly fewer metastases when compared to non-CSC-DC vacci- nated hosts. The subcutaneous tumors were significantly smaller in the CSC-DC-vaccinated group. CSC-specific IgG was detected in the serum of hosts subjected to CSC-DC vaccination. These anti- bodies bound specifically to CSCs, and such binding resulted in CSC lysis in the presence of complement. In addition, CTLs generated from the PBMCs harvested from murine hosts subjected to CSC-DC vaccine selectively killed ALDH-high CSCs more effec- tively than ALDH-low non-CSCs (p0.05). CONCLUSIONS: CSC-based vaccination confers significant pro- tective anti-tumor immunity. This immunity was associated with the induction of CSC-specific IgG antibody and CSC-specific reactive T cells. In vitro differentiation of pluripotent stem cells from human amniocytes into definitive endoderm Shaun Michael Kunisaki, MD, Guihua Jiang, MS, Luis G Villa-Diaz, PhD, Kathy Sue O’Shea, PhD, Paul H Krebsbach, PhD University of Michigan, Ann Arbor, MI INTRODUCTION: Induced pluripotent stem (iPS) cells are somatic cells have been genetically reprogrammed into an embryonic stem (ES) cell-like state. The purpose of this study was to determine whether human fetal cells harvested during an amniocentesis could be used to generate iPS cells that could be subsequently differentiated into definitive endoderm, the developmental precursor tissue of the pancreas, liver, and lung. METHODS: After IRB approval, human amniocytes (n=3) under- went lentiviral reprogramming via ectopic expression of Oct4, Sox2, cMyc, and Klf4 on a feeder-free substrate. After three weeks, the resultant iPS cell colonies were evaluated in multiple assays with human ES cells (H9) as positive controls. Differentiation of iPS cells into the endodermal lineage was assessed by immunocytochemistry. RESULTS: Pluripotent stem cell colonies were successfully gener- ated from amniocytes based on morphology and alkaline phospha- tase expression. The iPS cells showed strong expression of all pluri- potency markers, including NANOG, Oct4, Sox2, SSEA3, SSEA4, Tra-1-60, andTra-1-81. Furthermore, the cells were capable of em- bryoid body formation and were successfully differentiated into defini- tive endoderm as shown by positive staining for alpha-fetoprotein, Sox17, and Foxa2. CONCLUSIONS: The amniotic fluid is a robust cell source for the generation of human iPS cells via lentivital reprogramming methods. Further differentiation of definitive endoderm may be useful as re- placement therapy in pediatric surgical patients with congenital dis- orders involving the pancreas, liver, or lungs. Moreover, the genera- S93 © 2012 by the American College of Surgeons ISSN 1072-7515/12/$36.00 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jamcollsurg.2012.06.247