[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.