Further studies on thermal treatment of two‐cell stage embryos to produce complete embryonic stem‐cell‐derived mice by cell‐aggregation methods

Abstract

Employing aggregation techniques with two embryonic sources, one from two-cell stage embryos treated by thermal stimulation and the other from mouse embryonic stem (ES) cells that had been obtained from a feeder layer, simple and most effective methods of producing a complete generation of mice from ES cells were explored. Although thermal treatment affected embryos at various developmental stages, the embryos at the two-cell stage of development were selected because of the remarkably reduced number of cells present in the inner cell mass (ICM) at blastocyst stage after thermal conditioning. Under these conditions, a combination of thermally treated host embryos and an aggregated ES cell-clump was found to produce a high rate of live newborns by natural delivery. That the newborns were completely derived from ES cells was checked by two criteria: microsatellite analysis and coat color analysis. Importantly, all of these mice were healthy and fertile. The aggregation techniques reported here might well be applied to other animal species whose ES cells form stable colonies on a feeder layer.

Conditions and Techniques for Mouse Embryonic Stem Cell Derivation and Culture

“…Mouse embryonic stem (ES) cells, typically derived from inner cell masses (ICMs) or corresponding earlier blastomeres or later epiblasts (develop to embryo proper), are an example of pluripotent cells that can self-renew and generate all types of body cells in vivo and in vitro, but cannot generate the extraembryonic trophoblast lineage [11][12][13][14]. Under some particular conditions, an ES cell-derived mouse with germline transmission can be generated routinely [15][16][17][18][19][20][21]. Multipotent cells, such as hematopoietic stem cells, can give rise all cell types within a particular lineage.…”