341. Lentivirus Transduction of Murine Embryonic Stem Cells with Truncated Human Low-Affinity Nerve Growth Factor Permits Efficient Purification of Genetically Modified Cells without Loss of Stem Cell Characteristics

Abstract

The ability to genetically modify embryonic stem cells (ESC) expands the potential of ESC to correct hereditary disorders and deliver gene products for specific therapies. Integrating gene transfer vectors, such as lentivirus vectors, have been used to deliver genes to ESC, but the transduction efficiencies are far from 100% and selection of transduced cells using drug resistance is often unpredictable and toxic. A surface marker delivered by a lentivirus vector that could be employed in a magnetic-bead purification strategy would be a powerful tool in selecting transduced from untransduced ESC. To address these issues, we hypothesized that: (1) lentivirus transfer of the truncated human low-affinity nerve growth factor (LNGFR), a non-toxic, non-tumorigenic and non- functional protein expressed on the surface of transduced cells, could be used to isolate transduced ESC; (2) LNGFR expression would not alter the characteristics of the undifferentiated ESC; and (3) LNGFR expression would not interfere with the response of ESC to differentiation signals. To assess LNGFR as a purification strategy for ESC, we utilized a lentivirus vector with a human phosphoglycerate kinase 1 (PGK) promoter to drive expression of LNGFR. Murine ESC (C57BL/6 strain) were transduced with this vector and cultured on a murine embryonic fibroblast (MEF) feeder layer. Flow cytometry analysis (FACS) 48 hr after transduction revealed LNGFR expression in 44% of the ESC and 2 % in the corresponding mouse IgG1-PE control. On day 4, LNGFR+ ESC were purified with magnetic beads using an anti-LNGFR antibody. After single purification with magnetic beads, 95% of the transduced cells were LNGFR+. Several different assessments demonstrated that LNGFR expression did not alter the characteristics of the purified undifferentiated ESC. First, LNGFR+ cells plated onto a MEF feeder layer remained undifferentiated for 2 wk and the expression pattern of SSEA1 (FACS analysis), a marker for undifferentiated ESC, was identical in mock and LNGFR+ ESC (p=0.7). Second, embryoid body formation was identical in LNGFR+ and mock transduced ESC. Third, after subcutaneous administration of purified LNGFR+ or mock transduced ESC, teratomas, tumors arising from undifferentiated ESC, developed within 2 wk. Finally, to determine whether LNGFR expression would interfere with the response of ESC to differentiation signals, differentiation to endoderm was induced by Activin A. After 5 days, FACS analysis for CXCR4, a marker for definitive endoderm, showed similar levels of expression in LNGFR+ ESC and the control group (20.4% vs 21.2%), suggesting that LNGFR expression does not interfere with differentiation to endoderm. Together, these observations demonstrate that lentivirus- mediated transfer of LNGFR is a useful tool in purifying transduced ESC without deleterious or toxic effects on maintenance or differentiation.