Exp Mol Med.  2005 Feb;37(1):36-44.

Efficient gene delivery in differentiated human embryonic stem cells

• Kim JH
• Do HJ
• Choi SJ
• Cho HJ
• Park KH
• Yang HM
• Lee SH
• Kim DK
• Kwack K
• Oh SK
• Moon SY
• Cha KY
• Chung HM

Abstract

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.

Keyword

differentiation; embryoid body; human embryonic stem cell; promoter; RT-PCR; transfection

MeSH Terms

Actins/genetics
Animals
Bone Morphogenetic Proteins/genetics
*Cell Differentiation
Chickens
Cytomegalovirus/genetics
Drug Delivery Systems
Embryo/*cytology
*Gene Therapy
Green Fluorescent Proteins/genetics/*metabolism
Humans
Immunoenzyme Techniques
Microscopy, Fluorescence
Peptide Elongation Factor 1/genetics
Pluripotent Stem Cells/*cytology
Promoter Regions (Genetics)/*genetics
Research Support, Non-U.S. Gov't
Reverse Transcriptase Polymerase Chain Reaction
Transcription, Genetic/genetics