Date: 19th April 2021
Developing tools to advance our understanding of early human development, disease pathology and aging is a formidable challenge. The ability to grow cells of one species within another, creating an interspecies chimera, will offers us a unique and powerful research tool to advance biomedical research. It will provide a platform for drug evaluation, will address the unmet clinic need for transplantable organs, and is likely to accelerate regenerative medicine. Now, researchers have grown human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo, demonstrating the hEPSCs can survive, proliferate and differentiate into several cell lineages.
In 2020 there were 39,000 organs transplants in the US alone however, with 107,000 still on the waiting list, and 17 people a day dying whilst waiting for an organ, demand for organs far outweighs supply. The drive to find alternatives to organ donors is therefore high, and one such strategy under investigation is human-animal chimeric organs. This could see human-animal hybrids being designed as organ incubators, whereby human cells are inserted into animal host with the animal subsequently acting as a surrogate for the ‘human’ organ.
The first steps toward producing transplantable human organs using large animals is already underway. In 2017, Juan Carlos Izpisua Belmonte from the Salk Institute in California and his international team created the first human interspecies chimera, inserting human pluripotent stem cells (hPSCs) into pig blastocytes although efficiency of human cell contribution was low. Human-sheep hybrids have also been created, but again the level of human contributed cells was far too low to consider this close to creating viable transplant-ready organs.
Now, Izpisua Belmonte’s lab and researchers from Kunming University of Science and Technology, China, have injected fluorescently tagged hEPSCs cells into macaque embryos, they show these cells survived and integrated with better relative efficiency than in the previous experiments in pigs. The human cells could differentiate into several lineages, and showed distinct transcriptomic profiles revealing enhanced and novel pathways in the chimeric cells.
The team started generating the human-monkey chimeric embryos by injecting 25 fluorescently tagged hEPSCs cells into early blastocycts from cynomolgus monkeys at 6 days post fertlisation (dpf) and cultivating them ex vivo for 19 days after injection. Immunofluorescence (IF) studies using antibodies specific for several embryonic and extra-embryonic lineages, revealed they contributed to several main lineages including the epiblast (EPI), hypoblast (HYP) and extra-embryonic mesenchyme cells (EXMC). However, of the 132 chimeric embryos originally containing human cells, by 9dpf only half contained fluorescent cells and a further progressive decline was observed, such that by 13dpf only ~1/3 were positive in the embryonic disc for human cells.
To delve deeper into the molecular communication pathways between the two species of cells the team analysed the chimeric transcriptome. Here they found that the monkey cells within the chimeric embryos segregated into distinct cell-type clusters – EPI, HYP, and TE – the human HYP- and TE-like cells clustered with EPI-like cells. This suggested that the host cells exhibited a more faithful lineage segregation than the human cells, but that hEPSCs could differentiate into several peri- and early post-implantation cell types.
Transcriptional kinetics of chimeric human and monkey cells showed that the monkey embryonic microenvironment exerted influence on the transcriptional states of human cells and vice versa. Cell-cell interactions between the two species of cells were reinforced, leading to a strengthening of pathways such as MAPK and PI3K-AKT, and stimulated new signalling pathways such as WNT signalling pathway.
Conclusions and future applications
The team here have demonstrated the huge potential of human pluripotent stem cells to incorporate into a closely related primate embryo, developing and differentiating into different cell types. Such advances will offer us an invaluable opportunity to study and exploit interspecies communication which will give us a better understanding of for example the aging process, or how diseases arise and progress. Importantly it will give us an unprecedented insight into the earliest stages of human development, which would otherwise be inaccessible.
Of course this type of work will always raise ethical questions, and is one that Izpisua Belmonte and the team are very much aware. They do not intend to implant any hybrid embryos into monkeys and their ultimate aim is to better understand how cells of different species communicate with each other in the embryo during its early growth phase, and to and determine which ones are critical to the success of this process.
It is hoped that these human-monkey chimeras studies will lay the foundation to translate back into creating more efficient chimeric organisms that are more evolutionary distant to humans, such as livestock animals like pigs or cows, or more traditional experimental organisms such as rats. Ultimately, improving the integration of human cells into more suitable hosts, will accelerate the development of transplantable organs. Furthermore, current advances in organoid technology should be used to support this type of work, reducing the need to push the ethical boundaries.
One limitation that will be challenging to overcome will be ability to direct the chimeric cells down specified lineages. Here for example there was no control of which cells developed into which tissue. This will be a critical milestone for achieving transplantable organs or tissues. Although, initially it is likely that maintaining higher numbers of chimeric embryos with a high number of human cells will be the priority, here the rapid plummet in embryo numbers suggests that this is still far from optimised.
However, this work is likely to increase our understanding of early human development and primate evolution, and allow us to develop new strategies to improve human chimerism and support regenerative medicine translation therapies.
For more information please see the press release from the Salk Institute
Tan, T., et al. (2021). “Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.” Cell 184(8): 2020-2032 e2014.
https://doi.org/10.1016/j.cell.2021.03.020
