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Scientists create human/pig chimera

Human/animal chimeras can offer insights into early human development and disease onset and provide a realistic drug-testing platform. They may also someday provide a process for growing human cells, tissues, and organs for regenerative medicine. For now, however, they are helping scientists understand how human stem cells grow and specialize.


"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.

"It's like when you try to duplicate a key. The duplicate looks almost identical, but when you get home, it doesn't open the door. There is something we are not doing right,"
says Izpisua Belmonte. "We thought growing human cells in an animal would be much more fruitful. We still have many things to learn about the early development of cells."

The work was published January 26 in Cell.

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.


Other researchers had already created a rat/mouse chimera in 2010. That chimera was a mouse with pancreatic tissue formed from rat cells.


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.


As the organism matured, rat cells filled in where mouse cells could not, forming functional tissues of the organism's heart, eye, or pancreas.

But rat cells also grew to form a gall bladder in the mouse, even though rats stopped developing a gall bladder themselves over 18 million years when the two species separated evolutionarily.


"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."


The team then introduced humans' cells into cow and pig embryos as hosts as the size of those animals' organs more closely resembles humans than do mice.
They encountered many logistical problems, but the scientific challenge was "what kind of human stem cell" could survive in a cow or pig embryo.


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."


Pigs and humans about five times more distant evolutionarily than mice and rats. But, pigs also have a gestation period that is about one-third as long as humans, so the researchers needed to introduce human cells with perfect timing to match the developmental stage of the pig.


"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.


The human cells survived and formed a human/pig chimera embryo. Embryos were implanted in sows and developed for between three and four weeks.


"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.


One concern with the creation of human/animal chimeras is that the chimera will be too human. For instance, researchers don't want human cells to contribute to the formation of the brain.


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.

Abstract
Highlights
ĽNaive rat PSCs robustly contribute to live rat-mouse chimeras
ĽA versatile CRISPR-Cas9 mediated interspecies blastocyst complementation system ĽNaive rodent PSCs show no chimeric contribution to post-implantation pig embryos
ĽChimerism is observed with some human iPSCs in post-implantation pig embryos

Summary
Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that na´ve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

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

Cell (@CellCellPress), the flagship journal of Cell Press, is a bimonthly journal that publishes findings of unusual significance in any area of experimental biology, including but not limited to cell biology, molecular biology, neuroscience, immunology, virology and microbiology, cancer, human genetics, systems biology, signaling, and disease mechanisms and therapeutics. Visit: http://www.cell.com/cell. To receive Cell Press media alerts, contact press@cell.com.
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Jan 30, 2017   Fetal Timeline   Maternal Timeline   News   News Archive   



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
to allow easy access of an injection needle delivering human iPS cells.
Image Credit: Courtesy of Juan Carlos Izpisua Belmonte

 


 


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