For the first time, researchers have used embryonic stem cells to create a chimeric monkey whose body consisted of the cells of two genetically different individuals. The chimeric monkey was born alive and survived for ten days before being euthanized due to breathing problems. In further attempts, the added stem cells did not integrate into the organism or the surrogate mother's pregnancy ended in a miscarriage. In the future, the researchers want to improve the process in order to gain further insights into the possibilities of genetic engineering and to develop viable chimeras for medical research.
Chimeras are organisms made up of genetically different cells - either from the same species or from different species. In mice and rats, researchers have already succeeded in creating chimeric individuals in which a fertilized egg cell was combined with pluripotent stem cells from an animal of the same species. These experiments are considered evidence that pluripotent stem cells are capable of developing into the various tissues of a viable organism. In other species, however, attempts to create chimeras have so far failed - partly because it is difficult to precisely match the pluripotent stem cells to the developmental stage of the host embryo.
Green fluorescent stem cells
A team led by Jing Cao from the Chinese Academy of Sciences in Shanghai has now succeeded for the first time in creating a viable chimeric monkey. “This is a long-awaited goal in this area,” says Cao’s colleague Zhen Liu. “This research has implications not only for understanding naïve pluripotency in other primates, including humans, but also for genetic engineering and species conservation. In particular, this work could help us develop more precise monkey models for studying neurological diseases as well as other biomedical studies.”
To create the chimeras, the team first took stem cells from seven-day-old blastocyst embryos of long-tailed macaques. In order to be able to later track the extent to which these cells are integrated into a new organism, the researchers inserted a gene for a green fluorescent protein. They then injected the stem cells into four- to five-day-old long-tailed macaque embryos at the morula stage. At this stage, the cells that emerged from the fertilized egg begin to differentiate into an inner and an outer cell mass.
Low success rate
Cao and his team initially cultured the chimeric embryos created in this way under various conditions in the laboratory, where they found that the added stem cells were able to reproduce at different rates depending on the external conditions. On the other hand, conditions that were favorable for the proliferation of stem cells ensured that the embryos died more often at an early stage of development. Over the course of several experiments, the researchers found a balance between proliferation of the injected stem cells and survival of the early embryo.
Using this procedure, the team created 91 blastocysts, 74 of which fluoresced green, meaning they had a sufficient proportion of transferred stem cells. The researchers implanted these chimeric blastocysts into 40 macaque surrogate mothers, twelve of whom actually became pregnant. However, in only two cases did this result in a chimeric embryo. One of them was stillborn after about half the usual gestation period, one was born at term and alive. In five other live-born monkeys, the transferred stem cells did not multiply any further. “What was striking was the high rate of miscarriages in our chimera experiments,” report the researchers. “This also affected embryos in which the injected stem cells did not multiply. The death of these stem cells may also have had a negative effect on the embryo.”
Chimeric cells in all tissues
In the live-born chimeric macaque, the stem cells marked in green had spread throughout the body. His skin and eyes had a greenish color, and in internal organs such as the brain, heart, kidneys, liver, testicles and gastrointestinal tract, the researchers found that, depending on the tissue, 21 to 92 percent of the cells were on the injected stem cells decreased. “In this study, we have provided clear evidence that naive monkey pluripotent stem cells have the ability to differentiate in vivo into all the different tissues that make up a monkey body,” says Cao’s colleague Miguel Esteban. “This study deepens our understanding of the developmental potential of pluripotent stem cells in primates.”
However, the chimeric monkey's health was poor. “After surviving for ten days, his body temperature dropped sharply and he developed respiratory failure,” report the researchers. “Therefore, he was euthanized by a veterinarian for a detailed analysis.” Further investigations did not reveal a clear cause, but showed that the original and injected cells had different epigenetic changes in DNA methylation. “Although we cannot conclude that different DNA methylation levels were the cause of the miscarriages and poor health of the chimeric monkeys, this is a real possibility,” the researchers write. In future work, they want to investigate in more detail which mechanisms underlie the survival of embryos in order to optimize the process and improve the efficiency of chimera generation.
Source: Jing Cao (Chinese Academy of Sciences, Shanghai, China) et al., Cell, doi: 10.1016/j.cell.2023.10.005