ES cell lines can be established nondestructively from single blastomeres, and still retain their pluripotency. The inhibition of endogenous differentiation and activation of proliferation in preimplantation embryos is helpful in establishing ESC lines.

Studies have demonstrated that media containing 2i (PD0325901 and CHIR99021) dramatically increases the efficiency of establishing mESCs¹. ACTH 1-24, adrenocorticotropic hormone fragments 1-24 also improved the survival/proliferation of mESCs, and enhanced the derivation of mESCs from single blastomeres². When used together, ACTH2i synergistically enhanced the derivation of mESC lines from ICR and C57BL/6J 0.5-4.5 days postcoitum denuded intact zygotes, embryos, or isolated blastomeres from 2- to 8-cell embryos when cultured in small volume droplets in KSR ESC medium with feeders. This method is easy to operate and highly efficient³.