Since the first human embryonic stem cell (hESC) line was generated by Thomson et al. (in Science 282:1145-1147, 1998), hundreds of hESC lines have been reported by different labs, providing resources for basic research and regenerative medicine as well. However it has been widely recognized that hESC lines varied on their properties, in terms of gene expression profile, epigenetic modify profile, and differentiation tendency. Generation of more hESC lines will largely enhance our knowledge of hESCs innate character. In this current work, we reported the generation of HN4, a hESC line derived from grade III IVF human embryo by using a mixture of human foreskin fibroblast (HFF) and mouse embryonic fibroblast (MEF) as feeder layers, and a whole-mechanical method in inner cell mass (ICM) isolation. HN4 satisfied the criteria of hESCs pluripotency, with high expression of hESC surface markers (SSEA-3, SSEA-4, TRA-1-60, TRA-1-81), transcription factors (OCT-4, NANOG, REX-1), and alkaline phosphatase. It is able to differentiate to three germ layer derivatives when cultured in vitro, or in teratoma formation. Moreover, it displayed promising potential in neural differentiation under a proper culture condition, suggesting the advantage of HN4 in further investigation. Additionally, the whole-mechanical protocol for ICM isolation facilitates hESC line generation for its ease to handle.