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Scientists create human/pig chimera
"The ultimate goal is to grow functional and transplantable tissue or organs, but we are far away from that," says lead investigator Juan Carlos Izpisua Belmonte, a professor in the Salk Institute of Biological Studies' Gene Expression Laboratory. "This is an important first step."
But, efforts by Salk Institute researchers to grow the first embryos containing cells from humans and pigs proved more challenging than anticipated. Despite decades of work, scientists are still struggling to coax stem cells grown in Petri dishes to become fully functional — specialized adult cells, let alone three-dimensional tissues and organs.
As a first step, Izpisua Belmonte and Salk Institute staff scientist Jun Wu created a rat/mouse chimera by introducing rat cells into mouse embryos and letting them mature.
Izpisua Belmonte and Wu built on that experiment by using genome editing to flexibly direct the rat cells to grow in specific developmental niches in the mouse. To accomplish this, they used CRISPR genome editing tools to delete critical genes in fertilized mouse egg cells. In a given cell, they would delete a single gene critical for the development of an organ, such as the heart, pancreas, or eye. Then, they introduced rat stem cells into the embryos to see if they would fill the open niche. "The rat cells have a functional copy of the missing mouse gene, so they can outcompete mouse cells in occupying the emptied developmental organ niches," says Wu.
"This suggests that the reason a rat does not generate a gall bladder is not because it cannot, but because the potential has been hidden by a rat-specific developmental program," says Wu. "The microenvironment has evolved through millions of years to choose a program that defines a rat."
Cow embryos were more difficult and costly than pigs, so the team selected pigs. The effort required to complete studies of 1,500 pig embryos involved the contributions of over 40 people, including pig farmers, over a four-year period. "We underestimated the effort involved," says Izpisua Belmonte. "This required a tour de force."
"It's as if the human cells were entering a freeway going faster than the normal freeway," says Izpisua Belmonte. "If you have different speeds, you will have accidents."
Human cells that survived the longest and showed the most potential to continue to develop were "intermediate" human pluripotent stem cells. Called "naïve" cells, which resemble cells from an earlier point in human development with unrestricted potential; "primed" cells are further along in developement, but still pluripotent. "Intermediate cells are somewhere in between," says Wu.
"This is long enough for us to try to understand how the human and pig cells mix together early on without raising ethical concerns about mature chimeric animals," says Izpisua Belmonte.
Even using the most well-performing human stem cells, the level of contribution to the chimerized embryo was not high. "It's low," says Wu.
Izpisua Belmonte considers this good news.
In this study, the human cells did not become precursors of brain cells that can grow into the central nervous system. Rather, they were developing into muscle cells and precursors of other organs. "At this point, we wanted to know whether human cells can contribute at all to address the 'yes or no' question," he says. "Now that we know the answer is yes, our next challenge is to improve efficiency and guide the human cells into forming a particular organ in pigs."
To do this, researchers are again using CRISPR to perform genome editing on the pig genome as they did with mice. This process opens gaps that human cells can fill in. The work is in progress.
This research study was supported by The Fundación Séneca in Murcia, Spain, the Universidad Católica San Antonio de Murcia (UCAM), the Fundacion Dr. Pedro Guillen, the G. Harold and Leila Y. Mathers Charitable Foundation, and The Moxie Foundation.
Cell, Wu et al.: "Interspecies chimerism with mammalian pluripotent stem cells." http://www.cell.com/cell/fulltext/S0092-8674(16)31752-4
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Injection of human iPS cells into a pig blastocyst. A laser beam (green circle with a red cross inside) was used to perforate an opening to the outer membrane (Zona Pellucida) of the pig blastocyst