The Chinese Academy of Sciences team cultivated human mid-stage kidneys in pigs, and human cells were as high as 70%
On September 20, the world's second pig-human heart transplant was successfully performed at the University of Maryland. In March 2022, David Bennett, the world's first patient to undergo a gene-edited pig heart transplant, survived for up to 60 days after surgery.
The xenotransplant organ protocol used is to genetically modify pigs to humans through gene-editing technology, thereby reducing rejection. This major progress was obtained after further 10 genetic modifications on pigs, based on the breakthrough made 21 years ago by Lai Liangxue, a researcher at the Guangzhou Institute of Biomedicine and Health of the Chinese Academy of Sciences.
Figure 丨Lai Liangxue (Source: Lai Liangxue)
At that time, sheep cloning technology was just beginning to take off. In 2002, Lai Liangxue, who was engaged in postdoctoral research at the University of Missouri Animal Center, became the "first person" of gene-editing modified cloned pigs, and successfully constructed a galactosidyltransferase gene knockout cloned pig model through gene knockout technology combined with cloning technology, which solved the problem of hyperacute rejection of pig organ transplantation into the human body in one fell swoop, which caused a sensation in the entire academic circle. The study, published in Science, was considered a major breakthrough in xenotransplantation by the National Research Resource Center, calling the study a "landmark advance."
21 years later, Lai Liangxue and his collaborators set another important "milestone" on the road to solving the shortage of donor organs based on large animals - the first successful cultivation of human intermediate kidney in a kidney-deficient pig model [2]. According to statistics, the proportion of human-derived cells in the middle kidney is as high as 70%.
"The most exciting moment was when we took the chimeric fetus out and saw through a fluorescence microscope that large patches of red fluorescence appeared at its waist. This means that after five years of repeated exploration and experimentation, it is not far from success. Lai Liangxue sighed.
Through a series of follow-up experiments to rule out factors such as false fluorescence, the research team confirmed that the fluorescence from the location of the pig's kidney was emitted by human-derived cells. After 28 days of development in kidney-defective embryos, these derived pluripotent stem cells were successfully embedded in the middle kidney of pigs, forming the middle renal tubule, giving the kidney that originally exhibited the defect a complete structure.
Photo丨Cover of the current issue of Cell Stem Cell (Source: Cell Stem Cell)
Recently, the research results were published in Cell Stem Cell as a cover paper. On the cover, it is a piglet flying in with a hundred treasures. Researcher Lai Liangxue said that pigs can fly is a very miraculous thing, but also a seemingly "impossible" thing, which also indicates that our research "will make the impossible possible".
Interestingly, the flying piglet held a gourd in its hand, and the "panacea" contained in the gourd was the induced pluripotent stem cells (iPSCs) used to build the kidneys in human sources. The new human-derived iPSCs obtained by the team are one of the key factors for the success of this research, and with the blessing of genetic modification technology and new culture systems, the differentiation potential and viability of these cells are greatly enhanced, and finally the pig "grows" functional parenchymal organs containing a large number of human-derived cells.
"We hope that the kidneys that are recreated in pigs can be used for the benefit of humans." Researcher Lai Liangxue pointed out that this technology can not only be used for the development of human organs and the study of developmental diseases, its "ultimate goal" is to obtain functional organs that can be used for human organ transplantation.
Let stem cells have high differentiation ability and strong survivability at the same time
"After going through constant exploration and failure, we have succeeded. Behind this seemingly accidental, it is inseparable from the team's solid foundation and accumulation of every step in the fields of gene editing, gene modification, embryo manipulation, stem cells and other fields. Lai Liangxue sighed.
To obtain cells with stronger heterochimeric ability, the research team solved two key questions. In the preliminary work, Miguel M. Miguel A. Esteban's team found that adding four small molecule compounds and leukemia inhibitors to stem cell culture medium can obtain stem cells with differentiation similar to early human embryonic cells.
The similarity between stem cells and early embryonic cells is crucial to their ability to eventually form various tissues and organs, and this culture system empowers stem cells with higher differentiation potential, providing an important guarantee for the smooth formation of target organs in pig fetuses.
In order to make human-derived cells survive better in xenomorphs, Pan Guangjin's research group explored the mechanism of interspecific difference disorders. They found that overexpression of the two genes, MYCN and BCL2, in human stem cells can effectively avoid mass apoptosis in xenomorphs caused by differences between species. The previous research results of the research group also proved that human cells overexpressing MYCN and BCL2 can generate human blood stem cells in vascular defective mice. In this way, under the "strong combination" of high differentiation ability and strong survivability, the researchers obtained cells with strong heterochimeric ability (4CL/N/B cells).
(Source: Cell Stem Cell)
On the other hand, the team used gene editing technology to create a new model of pigs with kidney defects. Specifically, the researchers cloned kidney defective pig models with the knockout of the key genes for kidney development SIX1 and SALL1 through novel gene-editing tools, combined with somatic cell nuclear transfer technology and embryo microinjection.
At the same time, the researchers used novel gene-editing tools to combine somatic cell nuclear transfer technology and embryo microinjection to obtain kidney-deficient pig models that were knocked out of the key genes for kidney development, SIX1 and SALL1, through cloning. The multigenic modified kidney defective pig model showed severe middle kidney development defect, and the posterior kidney was completely missing, which provided sufficient development space for human-derived cells.
Figure丨Humanized kidney cells (red fluorescence) in embryos compared with wild-type pig embryos (Source: Cell Stem Cell)
In addition, all aspects of the embryo compensation technology system will affect the development of chimeric embryos, which is also one of the key problems to be solved to achieve the in vivo reconstruction of human organ xenogenesis. In this regard, the collaborative team made many attempts and made many "detours" to finally determine the ideal technical solution for constructing chimeric embryos: injection of 3-5 human stem cells into reconstituted pig embryos that have developed to the mulberry embryo and early blastocyst stage.
At the same time, given that human cells and pig embryos require different culture conditions when cultured in vitro, the researchers also explored the culture conditions suitable for culturing chimeric embryos in vitro. Finally, an in vitro culture system suitable for culturing chimeric embryos was identified.
Combined with the above-constructed pluripotent stem cells with high differentiation potential, strong proliferation and anti-apoptotic ability, an optimized embryo compensation technology system, and a multigene modified kidney defective pig model. The research team eventually succeeded in regenerating the human kidney in pigs.
Related papers (Source: Cell Stem Cell)
Finally, a related paper, entitled "Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation", was published as the cover of the current issue in Cell Stem Cell [ 2]。
The first author of the paper is Wang Jiaowei, a postdoctoral fellow at the Guangzhou Institute of Biomedical and Health Research, Chinese Academy of Sciences, and the co-corresponding authors are researcher Lai Liangxue, researcher Dai Zhen, and Miguel J. Miguel A. Esteban and Pan Guangjin.
New protocols for obtaining donor organs
In China, there are about 300,000 people who need organ transplants for organ failure every year, and human organ donation alone is far from filling this gap. To this end, scientists have been exploring new ways to obtain donor organs. Finding the right organ donor means prolonging a patient's life and improving their quality of life.
Recreating human-derived organs that can be used for transplantation in animals through bioengineering is one of the ideal solutions to solve the shortage of donor organs, which is also known as "xenochimeric technology". The theoretical feasibility of this strategy has been verified in rodent-based studies more than a decade ago.
Since the gestation periods of mice and rats are similar, the embryonic development time is basically the same. Therefore, in the early stage, researchers first carried out research on the in vivo reconstruction of xenoorgans based on the two. A number of research results have proved that the strategy of xenochimerism can achieve interspecific reconstruction of various organs such as pancreas and liver in rats or mice.
In November 2019, the team of Academician Zhou Qi of the Institute of Zoology of the Chinese Academy of Sciences injected monkey embryonic stem cells into pig embryos, and finally cultivated 2 "chimera" pigs containing monkey cells in their bodies, resulting in the world's first "pig-monkey chimera". The study proved that primate cells can form chimerism in pigs, but monkey cells have a very limited chimera ratio in chimeras. One of the important reasons for this may be that the carrier pigs used in the study did not provide an organ niche vacancy.
In April 2021, Academician Ji Weizhi and collaborators of the Institute of Primate Translational Medicine of Kunming University of Science and Technology reported on Cell that they had successfully constructed the first "human-monkey chimeric embryo" [3]. The researchers injected human stem cells into primate cynomolgus monkey embryos, and after 20 days of in vitro culture, human cells accounted for 4% of the chimeric embryos. This study provides an important reference for studying the early development of human embryos.
Both humans and monkeys belong to primates, with a closer origin, and monkeys have a gestation period of 5 and a half months, which is less different from humans. But it is precisely for these reasons that implanting a human monkey chimeric embryo into a surrogate mother and eventually producing a chimeric fetus may lead to more serious ethical problems.
Therefore, uterine transfer of human monkey chimeric embryos is still not allowed internationally. In addition, the technical system for constructing monkey models with defective genes is not fully mature and the efficiency is still low. Therefore, it is currently not possible to obtain human organs that can be used for transplantation based on human-monkey chimera.
Figure 丨Cloned pig (Source: Lai Liangxue)
So, what are the advantages and differences of recreating human organs through pigs? From the point of view of the structure, size and genes of pig organs, it is similar to humans, but there are certain differences in some physiological characteristics, such as human body temperature is 37 ° C, and pig body temperature is 38.5 ° C, and such as human pig fetuses still have the problem of unsynchronized development, from the point of view of pregnancy time, there is a big difference in the embryonic development time of humans and pigs, the gestation period of humans is 10 months, and the gestation period of pigs is about 114 days.
It cannot be ignored that there will be a hidden concern about xenotransplantation - will the pig become human by embedding human cells in the pig?
Lai Liangxue said: "This is practically impossible to do technically, we just cultivate an organ in pigs. The ethical requirement is that human nerve cells, as well as human germ cells (sperm and eggs), cannot be present in pigs. ”
In this study, the researchers detected only a small number of human-derived nerve cells in the spinal cord of a 28-day gestational fetus, but not the formation of human germ cells. In the future, controversy will also be prevented to the greatest extent by knocking out genes that are key to the neurological and reproductive development of human cells.
From gene knockout to the "first person" to the first human kidney cultivation in pigs
After returning to China in 2007, Lai Liangxue was appointed as a professor at the College of Animal Husbandry and Veterinary Medicine of Jilin University and a researcher at the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. His research interests mainly include the construction and application of genetically modified large animal models, the development and optimization of new gene modification technologies and cloning technologies, and the isolation and culture of human and animal pluripotent stem cells.
In 2018, he collaborated with the team of Professor Li Xiaojiang of the Guangdong-Hong Kong-Macao Institute of Central Nervous Regeneration of Jinan University to successfully establish the first transgenic pig model of Huntington's disease in Cell [4]. In addition, he has built a variety of functional protein humanized pig models with important application value, which will be used to produce important medical protein preparations such as insulin, coagulation factors, and hemovenous proteins, with broad market prospects.
In this published study, Lai Liangxue and his team cultivated human kidneys in pigs. Based on this work, they have begun to reconstruct other vital organs such as the heart, pancreas, liver, lungs and other important organs in pigs. For the heart that is most difficult to cultivate, Lai Liangxue said: "The pancreas can survive even if it is only 70-80% underdeveloped. But the heart must be very well done to stay alive. ”
Theoretically, with the advancement of stem cell technology and the development of personalized medicine, it may be possible to achieve such a scenario in the future: if a patient wants to obtain an organ for transplantation, a piece of the skin can be taken to make it a stem cell, and then the relevant organ will grow in the pig. Lai Liangxue pointed out that this organ is taken from the patient himself and used in the same person, so it will greatly reduce the rejection reaction, and this path will be more superior.
He believes that the first problem that needs to be tackled in the future is how to extend the development time of chimeric fetuses containing human organs in pigs. Based on the solved problem of stem cell differentiation ability, so the possible increase in the future will not be too large. At the same time, however, he pointed out that more obstacles needed to be overcome in terms of interspecific differences. For example, differences in gene expression (affecting production capacity), etc.
On the other hand, how to solve the problem of synchronous development of humans and pigs needs to be further solved. "I believe that with the increase of state investment in this field and the participation of more partners, there will be greater breakthroughs in this area." Lai Liangxue finally said.
Resources:
1.Lai, L.,……,Randall S Prather. Production of α -1,3-Galactosyltransferase Knockout Pigs by Nuclear Transfer Cloning. Science 295(5557):1089-1092 ( 2002).
2.Wang,J.,……,Lai, L. Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation. Cell Stem Cell 30,9,1235-1245 (2023). https://doi.org/10.1016/j.stem.2023.08.003
3.Tan,T.,et al. Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo. Cell 184, 8, 2020-2032 (2021). https://doi.org/10.1016/j.cell.2021.03.020
4. Yan, S., ……, Lai, L., Li, X. A huntingtin knockin pig model recapitulates features of selective neurodegeneration in huntington's disease. Cell. 2018, 173(4): 989-1002.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9671127/
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