Human embryonic stem cell (hESC) lines have been derived from the inner cell mass of blastocysts. Five hESC lines have been derived from 32 discarded blastocysts in Taiwan, and these lines have since been continuously cultured on mitotically inactivated mouse embryonic fibroblasts as feeder in the hESC medium for more than 44 passages and underwent freezing/thawing processes. All of five hESC lines expressed characteristic undifferentiated hESC markers such as SSEA-4, TRA-1-81, alkaline phosphatase, TERT, transcription factors POU5F1 (OCT4), and NANOG. The hESC lines T1 and T3 possess normal female karyotypes, whereas lines T4 and T5 are normal male, but line T2 is male trisomy 12 (47XY,+12). The hESC lines T1, T2, T3, and T5 were able to produce teratomas in SCID mice, and line T4 could only form embryoid bodies in vitro. Global gene expression profiles of single colonies of these five hESC lines were analyzed using Affymetrix human genome U133 plus 2.0 GeneChip. The results showed that 4,145 transcripts, including 19% of unknown functions, were detected in all five hESC lines. Comparison of the 4,145 genes commonly expressed in the five hESC lines with those genes expressed in teratoma produced by hESC line T1 and placenta revealed 40 genes exclusively expressed in all five hESC lines. These 40 genes include the previously reported stemness genes such as POU5F1 (OCT4), NANOG, TDGF1 (CRIPTO), SALL4, LECT1, and BUB1 responsible for self-renewal and pluripotent differentiation. The global gene expression analysis also indicated that the TGFβ/activin branch components inhibin BC, ACVR2A, ACVR1 (ALK2), TGFBR1 (ALK5), and SMAD2 were found to be highly expressed in undifferentiated states of these five hESC lines and decreased upon differentiation. The epigenetic states and expression of 32 known imprinted genes in these five hESC lines and/or differentiated derivatives were also investigated. In short, the hESC nature of these five hESC lines is supported by the undifferentiated state, extensive renewal capacity, and pluripotency, including the ability to form teratomas and/or embryoid bodies; and these cell lines will be useful for research on human embryonic stem cell biology and drug development/toxicity testing. The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.