Chinese scientists cultivate embryonic human kidneys in pigs for 28 days

·“ The long-term goal is to optimize the technology for use in human organ transplantation, but the work will be complex and could take years. Since organs are made up of many types of cells and tissues, growing a fully functional human-derived organ in pigs requires some additional steps. ”

Heterologous organ transplantation is an effective alternative to damaged or failed organs, but is limited by severe organ shortages. A breakthrough solution is to generate human organs in other large mammals through embryonic complementation, but it faces many challenges, not least poor integration of human cells with recipient tissues.

Past studies have shown that the generation of interspecific chimeras through the complementarity of embryos with pluripotent stem cells (PSCs) is a potential solution. Scientists have succeeded in generating organs such as the pancreas, thymus and kidneys between mice and rats. Pigs are similar to humans in physiology, organ size, and embryonic development, making them good "incubators" for generating human organs. However, solid organs have not been produced to date due to the poor overall contribution of human pluripotent stem cell-derived cells to pig tissue.

Researchers at the Guangzhou Institute of Biomedical and Health Research, Chinese Academy of Sciences, have successfully created chimeric embryos containing human and pig cells. After 28 days of transplantation into the surrogate sow, the developing human-derived kidney structure and tubular formation were normal. This is the first time scientists have been able to grow a physical human-derived organ in another species. On September 7, the paper was published in Cell Stem Cell, a journal owned by Cell Press.

"Our findings demonstrate the possibility of culturing humanized primitive organs in pigs with organ dysfunction, opening an exciting avenue for regenerative medicine and an artificial window into the study of human kidney development." The researchers wrote in the paper.

The researchers chose the kidney as a transplanted organ because the kidney is one of the earliest developing organs and is the most common transplanted organ in human medicine.

Integrating human stem cells into pig embryos has been a challenge because pig cells are more competitive than human cells, and pig cells and human cells have different physiological needs. Lai Liangxue, corresponding author of the paper and a researcher at the Guangzhou Institute of Biomedical and Health Research, Chinese Academy of Sciences, said: "Our method improves the integration of human cells and recipient tissues, so that human organs can be cultured in pigs. ”

Humanized kidney cells (red fluorescence) within embryos compared to wild-type pig embryos. Image source: Paper

The team's technology consists of 3 key components:

First, the researchers used CRISPR gene-editing technology to engineer single-celled pig embryos so that they lack two genes needed for kidney development, creating a vacancy in pig embryos so that human cells do not have to compete with pig cells.

Second, the researchers engineered human pluripotent stem cells (a type of cell with the potential to develop into any cell type) to make them easier to integrate and less likely to self-destruct by temporarily shutting down apoptosis (the autonomous, orderly death of genetically controlled cells to maintain an internal environment stability). They then cultured these cells in a special medium to transform them into "natural" cells that resembled cells from early human embryos.

Third, before implanting the developing embryos into surrogate sows, the researchers cultured the chimeras under optimal conditions that provide unique nutrients and signals for human and pig cells, which often have different needs.

The researchers transferred a total of 1,820 embryos to 13 sows. After 25 or 28 days, they terminated the pregnancy and extracted embryos to assess whether the chimera had succeeded in producing a human-derived kidney.

The researchers collected 5 chimeric embryos (2 25 days after implantation and 3 at 28 days after implantation) for analysis and found that they had structurally normal kidneys at developmental stages and were made up of 50%-60% human cells. In 25-28 days, the kidneys enter the middle renal phase (the second stage of renal development), where tubules and cell buds are formed, which will eventually develop into ureters that connect the kidneys and bladder.

The team also looked at whether human cells have an effect on other tissues throughout the embryo, particularly whether large numbers of human cells are found in the neural or germline tissues of piglets, which could have ethical implications. They found that the vast majority of human cells are located in the kidneys, while the rest of the embryo is made up of pig cells.

"We found that if a vacancy is created in a pig embryo, then human cells naturally enter these spaces." Dai Zhen, corresponding author of the paper and a researcher at the Guangzhou Institute of Biomedicine and Health of the Chinese Academy of Sciences, said, "We found only a few human nerve cells in the brain and spinal cord, and no human cells were found in the reproductive crest (when the embryo develops to 4-5 weeks, on both sides of the base of the mesentery on the back of the body cavity, and two bumps formed by epithelial hyperplasia of the body cavity appear on the left and right sides, collectively known as the urogenital crest), indicating that human pluripotent stem cells have not differentiated into germ cells." The researchers say this could be further prevented by knocking out more genes in human pluripotent stem cells, which could be tested in future studies.

Now, the researchers have optimized the conditions for growing human-derived kidneys in human-pig chimeras and hope to allow the kidneys to develop for longer. They also plan to grow other human organs in pigs, including the heart and pancreas.

The long-term goal, the researchers say, is to optimize the technology for use in human organ transplants, but the work will be complex and could take years. Since organs are made up of many types of cells and tissues, growing a fully functional human-derived organ in pigs requires some additional steps.

Further improving the survival rate of transplanted pig organs is not simple. In this study, the researchers created a vacancy for just one cell subpopulation, meaning that the kidneys contain porcine vascular cells that, if used for transplantation, could lead to immune rejection. In addition, significant physiologically significant interspecific differences may still lead to dysfunction.

The corresponding author of the paper, researcher Miguel A. "Because organs are not just made up of a lineage of cells, in order for everything in an organ to come from humans, we may need to 'engineer' pigs in more complex ways, which also presents some additional challenges," Esteban said. ”

The technique can also be used to study the development of human organs and developmental diseases. "This approach provides a window into human development before organs can be made for clinical use." "You can trace injected human cells and manipulate them to study how disease and cell lineages form," Esteban said. ”

In the paper, the researchers note that further research is needed to clarify whether integrated human cells are consistent with the developmental state of kidneys in wild-type pigs. Optimization of human pluripotent stem cells (exogenous factors, culture conditions, or injection procedures) has the potential to promote chimerism, but this may result in increased contributions to suboptimal lineages and therefore requires careful evaluation. For example, excessive contributions to extraembryonic tissues such as the placenta may increase the risk of immune rejection in developing embryos. To guide the overall optimization process, it would be valuable to use single-cell omics techniques to analyze the dynamics of donor cells within organogenesis disorders or wild-type host embryos at different developmental stages.

In 2016, the BBC reported on similar research: Pablo Ross, a reproductive biologist at the University of California, Davis (UCD), whose team tried to use CRISPR's gene-editing technology to delete DNA that can develop into the pancreas in a newly fertilized pig embryo through CRISPR's gene-editing technology, and implant iPS (induced pluripotent stem cells) into pig embryos. Let it develop into a pancreas in pigs that can be used for human transplantation.

The study sparked great ethical controversy at the time. A blog sponsored by the Center for Ethics at Emory University's Center for Neuroethics has posted that a small number of human stem cells may migrate and enter the developing brain of pig embryos, thereby giving the latter some human traits, although Ross insists that the probability of producing a human brain is very low.

The National Institutes of Health (NIH) declined to fund the study because of ethical concerns.