A method for single-cell sorting and expansion of genetically modified human embryonic stem cells

Genetic modification of human embryonic stem (hES) cells is essential for studies of gene function and differentiation. The expression of transgenes may direct tissue-specific differentiation and aid in the identification of various differentiated cell types. Stable genomic integration of transgenes is optimal because hES cell differentiation can span several days to weeks and include numerous cell divisions, and establishing homogeneous modified cell lines will facilitate research studies. Herein we provide a method for producing and expanding hES cell lines from single cells that have been isolated by fluorescence-activated cell sorting (FACS) following genetic modification by lentivirus vectors. Using this method, we have established enhanced green fluorescent protein (eGFP)-expressing hES cell lines that are pluripotent, contain a diploid chromosomal content, and stably express eGFP following more than 2 months of routine culture and in vivo differentiation.