FIG. 2.

Derivation of a hES cell line from a blastocyst developed from a frozen–thawed embryo. The images show the successful derivation of the Chula2.hES line from a poor-quality intact blastocyst developed from an 18-year- frozen embryo (A). The zona pellucida of the blastocyst was removed, and the entire blastocyst was plated on feeder cells. Three days after plating, an outgrowth of ICM (indicated by the star) surrounded by a TE was observed (B). The ICM was mechanically removed from the TE and plated onto new feeder cells. Seven days after removing the TE, an outgrowth of putative hES-like cells was observed (C), which later generated the stable hES cell line Chula2.hES (D). The morphologies of the colonies and individual Chula2.hES cells on three different feeder cell densities (E). A density of 35,000 cells/cm² was selected for further propagation. Scale bars=100 μm, hES, human embryonic stem; TE, trophectoderm.