[Establishment and characteristics of clonal human embryonic stem cell lines].
OBJECTIVE
To establish clonal human embryonic stem cell lines and investigate their biological characteristics.


METHODS
Cells were derived from one inner cell mass of human blastocyst, multiplied for 20 passages, and then dissociated into single cell suspension by digestion with 0.5% trypsin. Single cell was picked up and plated into individual well of a 96-well plate containing feeder-layers directly under a dissection microscope. The outgrowth clones were passed by treatment with collagenase. Surface markers were detected by cytochemistry and histoimmunochemistry. Karyotypes were tested using standard G-banding techniques. The pluripotency was analyzed by inoculating cells into severe combined immunodeficient (SCID) mice.


RESULTS
Two clonal human embryonic stem cell lines were established. Cells of these two lines possess the characteristics and differentiating potencies: normal 46 XX karyotypes; expressing a series of surface markers such as: alkaline phosphotase, stage-specific embryonic antigen (SSEA)-4, tumor recognition antigen (TRA)-1-60, TRA-1-81 etc; and forming teratomas comprising derivatives of three embryonic germ layers such as neural tissue, cartilage, squamous epithelium and columnar epithelium when injected into SCID mice.


CONCLUSIONS
The two single cell-cloned human embryonic stem (hES) cell lines were derived in our laboratory. The cells possess stable biological characteristics of undifferentiated hES cells.
Peng
Hong-mei
HM
Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100083, China.
Chen
Gui-an
GA
D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D004622:Embryo, Mammalian / Q000473:pathology*; D053595:Embryonic Stem Cells / Q000473:pathology*; D006801:Humans; D007621:Karyotyping / Q000379:methods*; D055746:Stage-Specific Embryonic Antigens; D013234:Stem Cells / Q000473:pathology*
D055746:Stage-Specific Embryonic Antigens