High-efficiency derivation of human embryonic stem cell lines using a culture system with minimized trophoblast cell proliferation

Chuti Laowtammathron, Pimjai Chingsuwanrote, Roungsin Choavaratana, Suphadtra Phornwilardsiri, Ketsara Sitthirit, Chidchanok Kaewjunun, Orawan Makemaharn, Papussorn Terbto, Supaporn Waeteekul, Chanchao Lorthongpanich, Yaowalak U-pratya, Pimonwan Srisook, Pakpoom Kheolamai, Surapol Issaragrisil

Due to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells (hESCs) have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy. The first hESC line was generated in 1998 by culturing inner cell mass (ICM) cells isolated from human blastocysts using an immunosurgery technique. Since then, many techniques including mechanical ICM isolation, laser dissection, and whole embryo culture have been used to derive hESC lines. However, the hESC derivation efficiency remains low, usually less than 50%, and it requires a large number of human embryos to derive a significant number of hESC lines. Due to a shortage of and restricted access to human embryos, a novel approach with better hESC derivation efficiency is badly needed to decrease the number of embryos used. We hypothesized that the low hESC derivation efficiency might be due to extensive proliferation of trophoblast (TE) cells which could interfere with ICM proliferation. We therefore developed a methodology to minimize TE cell proliferation by culturing ICM in a feeder-free system for 3 days before transferring them onto feeder cells. This minimized trophoblast cell proliferation (MTP) technique could be successfully used to derive hESCs from normal, abnormal, and frozen-thawed embryos with better derivation efficiency of more than 50% (range 50-100%; median 70%). We successfully developed a better hESC derivation methodology using the "MTP" culture system. This methodology can be effectively used to derive hESCs from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies. With this methodology, large-scale production of clinical-grade hESCs is feasible.