A breakthrough was made in stem cell cloning pigs

Cloned piglets from stem cells that have undergone three gene edits. (Infographic/Figure)

Livestock stem cells have great application prospects in basic research in life sciences, cell culture artificial meat production and breeding of excellent varieties. Since the success of mouse embryonic stem cells in 1981, scientists have tried to establish a stable, long-term passage of large animal embryonic pluripotent stem cell lines, but have not been successful. Recently, Professor Han Jianyong's team of China Agricultural University has made major breakthroughs in many units in the United Nations, and successfully established a pig embryonic stem cell line with the largest number of livestock stem cell transmissions in the world (more than 260 generations) and can carry out continuous gene editing operations, and the first stem cell clone pig with multiple rounds of gene editing in the world, overcoming the international problem of building a multi-functional stem cell system in the superodermal layer of pig embryonic. On November 30, 2021, the research results were published online in the internationally renowned academic journal Cell Research.

Livestock embryonic stem cells are established

Embryonic stem cells are a class of pluripotent stem cells isolated from the early stage of animal embryonic development, which have the characteristics of infinite proliferation, self-renewal and multidirectional differentiation in vitro culture. Theoretically, embryonic stem cells can differentiate into any cell type in an animal body and develop into a complete individual. Human embryonic stem cells are mainly used in regenerative medicine and tissue replacement after injury or disease, while livestock embryonic stem cells can be used in the preparation of genetically modified animals, organ transplantation and livestock breeding.

In 1981, Martin Evans et al. of the University of Cambridge in the United Kingdom isolated the first mammalian embryonic stem cells from mouse early embryos. In addition to some applications in the field of basic research in the life sciences, the most eye-catching application of mouse embryonic stem cells is in the preparation of gene knockout mice. In 1989, American scientists Oliver Smith and Mario Cappage successfully bred the first batch of knockout mice independently using mouse embryonic stem cells, creating a new era of animal functional gene research and medical animal model research. In 2007, the three scientists were jointly awarded the Nobel Prize in Physiology or Medicine. However, mammalian embryonic stem cells are very difficult to isolate and establish a department, until 1998, researchers at the University of Wisconsin-Madison in the United States isolated and obtained a second mammalian embryonic stem cell, that is, human embryonic stem cells, which set off a research boom in embryonic stem cell therapy.

Compared with mouse and human embryonic stem cells, the research path of livestock embryonic stem cells is more tortuous. After forty years of exploration, scientists at home and abroad have made very limited progress in the field of livestock embryonic stem cell research. The so-called livestock embryonic stem cells cultivated are either difficult to meet the standards of mouse and human embryonic stem cells, often only have some characteristics of embryonic stem cells, or the number of passages is small, and it cannot withstand tossing, cannot tolerate multiple transgenic and gene editing operations, and is difficult to prepare healthy and surviving gene-modified animals.

It has been passed down steadily more than two hundred times

Pigs are both important carnivorous livestock and important laboratory animals, and are widely used in basic research, human disease models, xenotransplantation and other fields. In view of the difficulty of establishing a pluripotent stem cell system of livestock embryonic superoderm, the number of passages is short, and it is difficult to withstand multiple gene editing operations and other international problems, han Jianyong's team of China Agricultural University, together with Sichuan Agricultural University, Institute of Zoology of the Chinese Academy of Sciences, Beijing Institute of Genetics of the Chinese Academy of Sciences, Northeast Agricultural University and other units, has carried out joint scientific and technological research with pigs as model organisms, and achieved major breakthroughs in the field of livestock embryonic stem cells.

Starting from the molecular mechanism of early embryonic pluripotent development regulation, the researchers overcame the single-cell isolation technology of early pig embryonics, first mapped the high-quality single-cell transcriptome map of pig embryos on days 0-14 (complete pre-implantation stage), successfully analyzed the molecular mechanisms of changes in the pluripotency state (initial, intermediate and excited states) of the pig superoderm, and found that the proliferation and maintenance of the ectoderm of pig embryos require the presence of activator A and fibroblast growth factor 2 (FGF2). It is also necessary for the self-renewal of mouse and human embryonic stem cells.

Based on the above research, the researchers speculate that the 8th-10th day of pig embryonic development is an ideal stage for the formation of pluripotent stem cells. They developed a medium that can significantly maintain the state of pluripotent stem cells, created a porcine mesoderm pluripotent stem cell culture system, and successfully used the embryonic mesoderm cells on day 10 to establish 15 embryonic stem cell lines, which can maintain the pluripotency state of the mesoderm and have typical stem cell characteristics. These embryonic stem cells can be stably passed on for more than 240 generations without any differentiation, and the maximum number of passages can reach 260 generations, which is the largest number of large animal stem cell lines reported in the world, which solves the problem of the small number of passages of livestock embryonic stem cells.

The researchers conducted a multi-omics joint analysis of porcine mesoderm pluripotent stem cells, in which the genome structure of porcine mesoderm pluripotent stem cells was constructed by high-throughput chromatin conformation capture (Hi-C) sequencing technology, and it was found that the loose chromatin structure of porcine mesoderm pluripotent stem cells was an important reason for their strong cell differentiation ability. The researchers also identified 75 transcription factors that are highly correlated with the function of porcine mesodermal pluripotent stem cells such as maintaining pluripotency, laying an important foundation for the subsequent study of the regulatory mechanism of porcine pluripotent stem cells.

In order to test whether the established porcine mesoderm pluripotent stem cell line supports tolerance to multi-round gene manipulation, the researchers performed three consecutive gene manipulations on the porcine ectodermal pluripotent stem cell line, the first gene manipulation was to randomly integrate the green fluorescent protein gene into the stem cell genome by gene transfection, and the second gene operation was to use CRISPR/Cas9 to insert foreign genes into the designated location of the genome. The third gene manipulation used a single-base editor to exchange bases C to T for a gene that affects coat color. After three rounds of genetic manipulation, the stem cells remain in good shape. Next, the researchers used the stem cell line that had undergone three gene editing as donor cells, and through nuclear transfer technology, obtained gene-edited cloned pigs derived from pig ectodermal pluripotent stem cells and were born and survived, which solved the international problem that livestock embryonic stem cells are difficult to withstand long cycles and continuous multiple gene editing.

Broad application prospects

For the first time, the study established a pig embryonic stem cell line that can be stably passed more than 240 times, and successfully obtained cloned pigs that have undergone multiple gene editing operations, which has a wide range of potential uses in basic life science research, cell culture meat production, medical animal models, livestock stem cell breeding and other fields.

In the field of basic research in life sciences, as a new and stable embryonic pluripotent stem cell line, porcine mesoderm pluripotent stem cells have successfully established a line, creating a new direction for livestock pluripotent stem cell research. Compared with mice, the embryonic development of pigs is more similar to that of humans, and the embryonic stem cells of pigs are a more ideal cellular model for studying human embryonic development. Through the comparison of pluripotent stem cells of different species such as pigs, humans and mice, the differences and uniformity between species maintained by stem cell pluripotency can be found, which provides a theoretical basis for regenerative medicine research such as xenotransplantation.

In the field of cell culture meat, porcine mesoderm pluripotent stem cells can be used as seed cells for future cell culture and other cell-based functional products. At present, most of the seed cells developed by cell culture meat are progenitor cells of muscle and fat cells, and do not have the ability to proliferate for long-term passage, which has become a technical bottleneck for large-scale production of cell culture meat in vitro. Porcine mesoderm pluripotent stem cells can be stable passage in vitro for a long time, through large-scale culture in vitro, a large number of initial cells are obtained, and then muscle cells and fat cells are obtained through directional induction differentiation, which can solve the technical problems of cell culture meat such as short passage time of "seed cells".

In the field of medical model animal research, porcine mesoderm pluripotent stem cells are combined with gene editing and can be used to construct human disease models or xenotransplantation. One of the main limitations of the current use of swine somatic cell nuclear transplantation is that donor cells can usually only support one genome edit, it is difficult to obtain multi-gene editing clones of livestock at the same time, and it is often necessary to use repetitive cloning and re-isolation of fibroblasts to achieve multiple rounds of gene editing, short cycles, and low efficiency. Experiments have confirmed that porcine mesodermal pluripotent stem cells have a good tolerance to gene editing, and can tolerate at least three consecutive gene editing, which will greatly reduce the time and cost of preparing polygen-edited cloned pigs.

In the field of breeding of excellent breeds of livestock, porcine mesoderm pluripotent stem cells can achieve stem cell breeding by directed induction of differentiation into sperm and oocytes. Embryonic stem cells can induce the entire process of gamete development in vitro to form functional sperm or oocytes. Combining stem cells with technologies such as genome sequencing and genome selection, and then obtaining offspring embryos through in vitro germ cell induction and in vitro fertilization, combined with genome selection technology, the mass production and accurate selection of excellent livestock embryos can be completed in the laboratory, and livestock stem cell breeding can be realized, which can significantly shorten the generation interval and improve the selection intensity, quickly obtain 30-40 times the genetic gain, and achieve rapid genetic improvement of important economic traits.

However, from the successful establishment of pig embryonic stem cells to the industrial application of cell breeding meat and stem cell breeding, further in-depth research is needed, focusing on solving problems such as efficiency and cost.

Southern Weekend Contributing Writer Tombo