Efficient gene transfer of a simian immuno-deficiency viral vector into cardiomyocytes derived from primate embryonic stem cells.
BACKGROUND
Embryonic stem (ES) cells continually proliferate and can generate large numbers of differentiated cells. Genetic manipulation of transplantable cells derived from primate ES cells offers considerable potential for development research and regenerative cell therapy. However, protocols for efficient gene transfer into primate ES-cell-derived cells have not yet been established.


METHODS
Spontaneously contracting areas were derived from cynomolgus monkey ES cells. Features of cardiomyocytes in the area were analyzed according to gene expression (RT-PCR), morphology (immunostaining and electron microscopy), and function (intracellular calcium transience). Beating cells were transduced using a simian immunodeficiency virus (SIV) vector expressing enhanced green fluorescence protein (EGFP), then transplanted into ischemic rat myocardium.


RESULTS
Beating cells derived from monkey ES cells displayed gene expression, ultrastructural and functional properties of early-stage cardiomyocytes. Highly efficient (97% cardiac phenotype) and stable transduction of these ES-cell-derived cardiomyocytes was achieved using SIV vector without altering contractile function. In addition, transduced cardiomyocytes survived in the myocardium of a rat myocardial infarction model.


CONCLUSIONS
A lentiviral vector system based on SIV represents a useful vehicle for genetic modification of cardiomyocytes derived from primate ES cells, and can extend the application of primate ES cells to gene therapy.
Nagata
Mihoko
M
Department of Medicine, Jichi Medical School, Tochigi 329-0498, Japan.
Takahashi
Masafumi
M
Muramatsu
Shin-Ichi
S
Ueda
Yasuji
Y
Hanazono
Yutaka
Y
Takeuchi
Koichi
K
Okada
Koji
K
Suzuki
Yutaka
Y
Kondo
Yasushi
Y
Suemori
Masafumi
M
Ikeda
Uichi
U
Nakano
Imaharu
I
Kobayashi
Eiji
E
Hasegawa
Mamoru
M
Ozawa
Keiya
K
Nakatsuji
Norio
N
Shimada
Kazuyuki
K
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D017931:DNA Primers; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins; D008252:Macaca fascicularis; D008854:Microscopy, Electron; D009206:Myocardium / Q000166:cytology / Q000648:ultrastructure*; D039904:Pluripotent Stem Cells; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015302:Simian Immunodeficiency Virus; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium