The pigs largely came from two commercial pig farms located in Southeastern Spain. That posed its own difficulties.
"Most of the pigs used in this study were housed in farms used for meat production, rather than research purposes," Belmonte explained. "We had to convince the farmers to collaborate on this project. In addition, we cannot perform experiments whenever we want. For example, farms are typically closed during the summer due to hot weather."
The researchers persevered, injecting several different forms of human stem cells into pig embryos to see which would survive best. Those that did were "intermediate" human pluripotent stem cells. So-called "naïve" cells have unrestricted developmental potential, while "primed" cells have developed further.
"Intermediate cells are somewhere in between," Wu noted.
The human cells survived and formed the human-pig chimera embryos, which were allowed to develop for three to four weeks.
"Once the implants were removed," Belmonte said, "the [host] sows were euthanized and incinerated."
Cautions and Concerns
One reason the sows were incinerated was to eliminate any chance of the human cells escaping into an adult pig's body. Belmonte assured that the chances of that happening are "extremely unlikely," in any case.
"Ethically there have been some concerns raised about generating animal chimeras with human cells, particularly in cases where the cells might contribute extensively to the brain or to the germ cells," Janet Rossant, a senior scientist and chief of research emeritus at the Hospital for Sick Children, told Seeker. "This possibility needs to be carefully assessed in each specific experimental protocol and steps put in place to try to restrict unwanted contributions to tissues other than the ones required."
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In a PLOS Biology paper, Insoo Hyun of Case Western Reserve University said that the primary worry "is that in the process of biologically humanizing a research animal, scientists may end up also morally humanizing the resulting chimera, especially if there is acute human/non-human chimerism of the central nervous system."
The good news is that the human contribution to the pig-human chimeras was "low," Wu said.