We found women willing to contribute eggs on an anonymous basis for use in our research by placing advertisements in publications in the Boston area. We accepted women only between the ages of 24 and 32 who had at least one child. Interestingly, our proposal appealed to a different subset of women than those who might otherwise contribute eggs to infertile couples for use in in vitro fertilization. The women who responded to our ads were motivated to give their eggs for research, but many would not have been interested in having their eggs used to generate a child they would never see. (The donors were recruited and the eggs were collected by a team led by Ann A. Kiessling-Cooper of Duncan Holly Biomedical in Somerville, Mass. Kiessling was also part of the deliberations concerning ethical issues related to the egg contributors.)
We asked potential egg contributors to submit to psychological and physical tests, including screening for infectious diseases, to ensure that the women were healthy and that contributing eggs would not adversely affect them. We ended up with 12 women who were good candidates to contribute eggs. In the meantime, we took skin biopsies from several other anonymous individuals to isolate cells called fibroblasts for use in the cloning procedure. Our group of fibroblast donors includes people of varying ages who are generally healthy or who have a disorder such as diabetes or spinal cord injury¿the kinds of people likely to benefit from therapeutic cloning.
Our first cloning attempt occurred last July. The timing of each attempt depended on the menstrual cycles of the women who contributed eggs; the donors had to take hormone injections for several days so that they would ovulate 10 or so eggs at once instead of the normal one or two.
We had a glimmer of success in the third cycle of attempts when the nucleus of an injected fibroblast appeared to divide, but it never cleaved to form two distinct cells. So in the next cycle we decided to take the tack used by Teruhiko Wakayama and his colleagues, the scientists who created the first cloned mice in 1998. (Wakayama was then at the University of Hawaii and is now at Advanced Cell Technology.) Although we injected some of the eggs with nuclei from skin fibroblasts as usual, we injected others with ovarian cells called cumulus cells that usually nurture developing eggs in the ovary and that can be found still clinging to eggs after ovulation. Cumulus cells are so small they can be injected whole. In the end, it took a total of 71 eggs from seven volunteers before we could generate our first cloned early embryo. Of the eight eggs we injected with cumulus cells, two divided to form early embryos of four cells¿and one progressed to at least six cells¿before growth stopped.
WE ALSO SOUGHT TO DETERMINE whether we could induce human eggs to divide into early embryos without being fertilized by a sperm or being enucleated and injected with a donor cell. Although mature eggs and sperm normally have only half the genetic material of a typical body cell, to prevent an embryo from having a double set of genes following conception, eggs halve their genetic complement relatively late in their maturation cycle. If activated before that stage, they still retain a full set of genes.
Stem cells derived from such parthenogenetically activated cells would be unlikely to be rejected after transplantation because they would be very similar to a patient¿s own cells and would not produce many molecules that would be unfamiliar to the person¿s immune system. (They would not be identical to the individual¿s cells because of the gene shuffling that always occurs during the formation of eggs and sperm.) Such cells might also raise fewer moral dilemmas for some people than would stem cells derived from cloned early embryos.