Korean J Reprod Med.  2009 Dec;36(4):283-292.

Transduction of eGFP Gene to Human Embryonic Stem Cells and Their Characterization

• Kim YY
• Ku SY
• Park YB
• Oh SK
• Moon SY
• Choi YM

Abstract

OBJECTIVE
Human embryonic stem cells (hESCs) can proliferate indefinitely and differentiate into all kinds of cell types in vitro. Therefore, hESCs can be used as a cell source for cell-based therapy. Transduction of foreign genes to hESCs could be useful for tracing differentiation processes of hESCs and elucidation of gene function. Thus, we tried to introduce enhanced green fluorescent protein (eGFP) gene to hESCs, XX and XY cell lines in this study.
METHODS
Lentivirus containing eGFP was packaged in 293T cells and applied to hESCs to transduce eGFP. Expression of transduced eGFP was evaluated under the fluorescence microscope and eGFP positive population was analyzed by FACS. Expression of undifferentiation state markers such as Oct4, Nanog, SSEA4 and Tra-1-81 was examined by RT-PCR and/or immunofluorescence in eGFP-hESCs after transduction. In addition, the ability of eGFP-hESCs to form embryoid bodies (EBs) was tested.
RESULTS
eGFP was successfully transduced to hESCs by lentivirus. eGFP expression was stably maintained up to more than 40 passages. eGFP-hESCs retained expression patterns of undifferentiation state markers after transduction. Interestingly, disappearance of transduced eGFP was notably observed during spontaneous differentiation of eGFP-hESCs.
CONCLUSION
We established eGFP expressing hESC lines using lentivirus and showed the maintenance of undifferentiation haracteristics of these eGFP-hESCs. This reporter-containing hESCs could be useful for tracing the processes of differentiation of hESCs and other studies.

Keyword

Human embryonic stem cells; Enhanced green fluorescent protein; Lentivirus

MeSH Terms

Cell Line
Embryoid Bodies
Embryonic Stem Cells
Fluorescence
Fluorescent Antibody Technique
Green Fluorescent Proteins
Humans
Lentivirus
Green Fluorescent Proteins