Efficient gene delivery in differentiated human embryonic stem cells.
Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.
Kim
Jae-Hwan
JH
Cell and Gene Therapy Research Institute, Pochon CHA University, Gangwondo, Korea.
Do
Hyun-Jin
HJ
Choi
Seong-Jun
SJ
Cho
Hyun-Jung
HJ
Park
Kyu-Hyung
KH
Yang
Heung-Mo
HM
Lee
Sang-Hwa
SH
Kim
Dong-Ku
DK
Kwack
KyuBum
K
Oh
Sun-Kyung
SK
Moon
Shin-Yong
SY
Cha
Kwang-Yul
KY
Chung
Hyung-Min
HM
D000199:Actins / Q000235:genetics; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D002352:Carrier Proteins; D002454:Cell Differentiation; D002645:Chickens; D003587:Cytomegalovirus / Q000235:genetics; D016503:Drug Delivery Systems; D004622:Embryo, Mammalian / Q000166:cytology*; D004625:Embryo, Nonmammalian; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D008856:Microscopy, Fluorescence; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics; D000099176:Noggin Protein
D000199:Actins; D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D020648:Peptide Elongation Factor 1; D000099176:Noggin Protein; C485184:enhanced green fluorescent protein