In an article in Cell published on January 26, Salk Institute researchers report that their attempt to grow the first embryos containing cells from humans and pigs turned out to be more challenging than what they had anticipated. Human / animal chimeras can provide a realistic drug-testing platform and offer understandings into disease onset and early human development. In the future, they may also provide a means of growing human tissues, cells and organs that could be used for regenerative medicine. Currently, they are helping scientists better understand how human stem cells grow and specialize.
Juan Carlos Izpisua Belmonte, a professor in the Salk Institute of Biological Studies’ Gene Expression Laboratory and the lead investigator for the study, noted that although this is an important first step, their ultimate goal is to grow transplantable, functional organs or tissue. He admits that the team is still far away from that.
Although scientists have tried for decades to coax stem cells grown in Petri dishes to turn into fully functional specialized adult cells, they have not managed to do this. They are even further away from growing three-dimensional organs and tissues. Izpisua Belmonte likens the process to trying to duplicate a key. Although the duplicate looks almost identical to the original, it might not open the door when you try to use it. He notes that they are still doing something wrong. The team thought that growing human cells in an animal would prove to be more successful, but it turns out there are still many things to learn about the early development of cells.
Izpisua Belmonte and scientist Jun Wu created a rat / mouse chimera by introducing rat cells into mouse embryos as a first step, and then letting them mature. This was already achieved by other researchers in 2010 and that chimera was a mouse that had pancreatic tissue formed from rat cells.
Izpisua Belmonte and Wu expanded on that experiment using genome editing to coax the rat cells to grow in precise developmental niches in the mouse. They used CRISPR genome editing tools to accomplish this and deleted critical genes in fertilized mouse egg cells. They would for example delete a single gene critical for the development of an organ in a given cell, such as the pancreas, heart, or eye. They would then introduce rat stem cells into the embryos to see if they would fill the vacant niche. Wu explained the rat cells have a functional copy of the missing mouse gene, so they can replace mouse cells in occupying the developmental organ niches that are empty. As the organism grew, the rat cells filled in where mouse cells were not able to, forming the functional tissues of the organism’s pancreas, heart, or eye.
Rats and mice separated evolutionarily 18 million years ago and since then, rats stopped developing gall bladders. Even though this is the case, rat cells interestingly enough grew to form a gall bladder in the mouse. Wu believes that this means that the reason a rat does not generate a gall bladder is not because it can’t do so, but because the potential has been repressed by a rat specific developmental program that has evolved through millions of years.
As a next step, the team introduced human cells into an organism. Cow and pig embryos were used as hosts because the size of these animals’ organs are closer to humans than mice. Although many logistical challenges were encountered, the scientific challenge was finding out which type of human stem cell could survive in a pig or cow embryo.
Izpisua Belmonte admits that they underestimated the effort involved. As experiments with pig embryos are less difficult and cost less than cows, the team zoomed in on pigs. In order to complete studies of 1,500 pig embryos over a period of four years, contributions from over 40 people, including pig farmers, were received.
Humans and pigs are about five times more distant evolutionarily than rats and mice. Pigs’ gestation period is also about one third as long as what humans’ are, so the researchers had to introduce human cells to match the developmental stage of the pig with perfect timing. Izpisua Belmonte likened this to human cells entering a freeway going faster than the normal freeway and noted that if you have different speeds, accidents will happen.
Several different types of human stem cells were injected into pig embryos to see which would survive best. Intermediate human pluripotent stem cells survived the longest and showed the most potential to continue developing. Wu explained that naïve cells resemble cells from an earlier developmental origin with unrestricted developmental potential, while primed cells have developed further, but are still pluripotent. Intermediate cells are somewhere in between the naïve and primed cells in the development cycle.
The human cells survived and a human / pig chimera embryo was formed. The embryos were then implanted in sows and allowed to develop for approximately three to four weeks. Izpisua Belmonte noted that although this is long enough for the team to try to understand how the pig and human cells mix early on, it is not long enough to raise ethical concerns about mature chimeric animals.
Wu did notice that even when using the best performing human stem cells, the level of contribution to the chimera embryo was low, and for Izpisua Belmonte, this is good news, as this alleviates the concern with the creation of human / animal chimeras that the chimera will be too human. Researchers will for example not use human cells to contribute to the formation of the brain. In the research, the human cells did not become originators of brain cells that could possibly grow into the central nervous system.
The human cells were targeted at becoming precursors of other organs and developing into muscle cells in this research. At this point, all the researchers were interested in was whether human cells can contribute to the creation of human / animal chimeras, or not. Now that they know it is possible, their next challenge is to guide human cells into forming a specific organ in pigs, and to improve efficiency. To achieve this, the team used CRISPR to perform genome editing on the pig genome as they did with mice. This opens gaps that can be filled in with human cells. The work is ongoing.