New human embryonic stem-cell lines--more is better.

It is rare that a field of scientific research can simultaneously represent a domain of fundamental discovery in human biology and potentially have major effects on human health and the quality of life. Human embryonic stem cells serve not only as a resource for basic research, but also as the starting material for the development of cell-based therapies. Formidable scientific challenges face those who aim to develop cell-based therapies. It will take the efforts of many scientists and clinicians in a variety of disciplines to bring this endeavor to fruition. Of immediate importance and concern is whether or not we have sufficient starting material for the initial phases of this research. Embryonic stem cells are derived from the inner cell mass of blastocysts, the preimplantation stage at which the developing mammalian embryo is implanted in the uterine endometrium (see Figure). The derivation of mouse embryonic stem cells was first reported in 1981 1,2 ; Thomson et al. reported the derivation of human embryonic stem cells in 1998, 3 and their general procedure has been used by others. To isolate human inner cell mass, cleaving preimplantation-stage embryos are cultured to the blastocyst stage; the blastocysts are treated with a weak acid to remove the acellular zona pellucida that covers the surface of the conceptus, and the trophoblast cells are lysed (through immunosurgery) in the presence of antihuman antiserum (usually antibodies to human red cells) and guinea pig complement. The isolated inner cell masses are then cultured, in the presence of culture medium with growth factors, on a feeder layer of mouse embryonic fibroblasts (after the feeder cells have been irradiated or treated with mitomycin to stop them from dividing). The need for feeder cells is an annoying obstacle to the convenient growth and efficient study of embryonic stem cells in the laboratory. Moreover, the use of mouse feeder cells raises the specter of the interspecies transfer of viruses. Therefore, one goal of research on embryonic stem cells is to find a way to derive and culture cell lines without the use of feeder layers. In the meantime, the use of feeder cells of human origin addresses the concern about the interspecies transfer of viruses, although intraspecies transfer remains possible. The slow growth rate of embryonic stem cells (most have a doubling time of 32 hours or more) is often the limiting factor in experiments using these cells. For this reason, initial passages are routinely performed by means of mechanical dissociation, which, though time-consuming, results in higher plating efficiency than dissociating the cells with enzymes. Some human embryonic stem-cell lines are maintained solely by means of mechanical dissociation, especially when large numbers of cells are required for an experiment. The defining characteristics of embryonic stemcell lines are in the process of being established. 4