Figure S3.

Derivation of ESCs, Related to Figure 4

(A) Generation and isolation of ICM from embryo fragments. Eight-cell-stage embryos are split into five blastomere and three blastomere portions. The 3/8th fragment is cultured in 2i for 24 h to the early blastocyst stage, followed by a further 48 h culture in N2B27+2i+LIF. Extraembryonic tissue is removed from the 3/8th blastocyst by immunosurgery, whereupon the ICM consisting only of EPI cells is cultured on laminin in N2B27+2i+LIF for 3–5 days to form an ES colony. After sufficient growth, the colony is disaggregated with trypsin and plated on laminin to establish an ESC line. The remaining 5/8th embryo fragment is transferred to a surrogate female at the early blastocyst stage.

(B) Growth of ES colony derived from a 3/8th blastocyst over 3 days. Scale bars: 50 μM.

(C) Efficiencies of ES colony formation and ESC derivation from whole embryos, 3/8th blastocysts, and quarter embryos. Fourteen ES colonies were derived from 17 3/8th blastocysts, corresponding to 82% efficiency. Six ESC lines were established from six of these colonies, and the remaining colonies were analyzed for Nanog and Oct4 expression.

All error bars indicate standard error.