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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 19811,2; 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).
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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 stem-cell lines are in the process of being established.4 Key properties include continuous passage; clonability; chromosome stability; the expression of a variety of molecular, biochemical, and antigenic markers; and the ability to differentiate into various types of tissue and to form tumors (teratomas) after being grafted into immunocompromised animals.
It is not known how many human embryonic stem-cell lines exist in laboratories throughout the world, but in the United States, much attention has focused on the lines that were derived before August 9, 2001, at 9 p.m., since only research performed with the use of these lines is eligible for federal funding. There are 15 such lines in the National Institutes of Health (NIH) Registry. Most of these lines are expensive, and the Material Transfer Agreements for some of them contain stringent requirements.
The article by Cowan et al. in this issue of the Journal (pages 1353–1356), which reports the derivation of 17 new lines of human embryonic stem cells, is therefore an important contribution. In a tour de force, Douglas Melton of this research group mustered resources from the Howard Hughes Medical Institute, the Juvenile Diabetes Research Foundation, and Harvard University in order to produce new human embryonic stem-cell lines that are more readily available to investigators, although research using these lines is ineligible for NIH funding at present. The lines were derived with the use of trypsin for passaging and were grown in the presence of penicillin and streptomycin, all of which should make for easier handling in the laboratory. (Most human embryonic stem-cell lines currently available are intolerant of antibiotics.) These cell lines express the accepted embryonic stem-cell markers and demonstrate differences in doubling times — ranging from 24 to 72 hours. The shorter doubling times may well be important, given the limitations that slow growth rates place on experiments.
The lines were established and are grown on layers of mouse feeder cells, so their usefulness in humans may be limited,5 although the Food and Drug Administration has not ruled out the clinical use of cell-based therapies derived from lines grown on feeder layers from other species. Perhaps when the critical factor or factors produced by the feeder layers have been identified, we can count on the same team to derive new cell lines that can be grown without the need for feeder layers. Of course, it is only through research involving embryonic stem-cell lines that such factors may be discovered — once again underscoring the importance of the contribution made by Cowan et al.
Dr. Gearhart reports that Johns Hopkins University holds in escrow for him stock in the Geron Corporation. He also reports having received lecture fees from Johnson & Johnson.
Source Information
From the Institute for Cell Engineering, Johns Hopkins University, Baltimore.
This article was published at www.nejm.org on March 3, 2004.
References
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