Stem Cell Research Opens a Second Front for the Cloning Sheep Technology Route

The Leap from Sheep to Monkey

Without the two papers published on November 20, Shoukhrat Mitalipov would have been the most watched stem cell research scientist during this period.

On November 14, this scientist at the Oregon National Primate Research Center in the United States published a paper in Nature, announcing that he had successfully obtained cloned rhesus monkey embryos. This was the first time scientists had cloned viable embryos from a primate. Of course, in 2004, Hwang Woo-suk of South Korea once announced that the research team he led had obtained the first cloned human embryos, but as people later learned, this achievement was fabricated. Cloned embryo research in primates remains a major challenge, and many scientists believe it is different from cloning cattle and sheep.

Mitalipov's achievement is outstanding. He used the traditional somatic cell nuclear transfer technique, transplanting rhesus monkey skin cells into egg cells, culturing this cell into an embryo, and extracting embryonic stem cells from it. This is not very different from the technique the British used to create Dolly the sheep in 1996, but Mitalipov improved some steps, allowing this technique to be achieved in monkeys (although the success rate is still not high).

After 11 years, stem cell research scientists have finally, with difficulty, achieved the leap from sheep to monkey, making people realize that human cloned embryo technology may soon emerge. Pluripotent embryonic stem cells can be extracted from cloned embryos and used to treat some major diseases such as Alzheimer's disease, which is of great significance. But inevitably, a new round of ethical debate about human cloning has begun again.

A Landmark Advance

However, just one week later, these debates were like a balloon that had been poked, deflating to one side. On November 20, Shinya Yamanaka of Kyoto University in Japan and James Thomson of the University of Wisconsin in the United States published heavyweight papers in Cell and Science respectively, announcing that they had used genetic modification to transform human somatic cells into embryonic stem cell-like cells, almost comparable in function to embryonic stem cells.

The fact that nearly identical results were obtained simultaneously in two laboratories made the possibility of fraud extremely small. It can be said that the stem cell research field suddenly had a completely new way to obtain embryonic stem cells: no need to use human egg cells, no need to cultivate and destroy human embryos, and no need to exhaust every method to create human-animal hybrid cells. All previous ethical debates could cease, and this was undoubtedly a landmark advance.

After learning this news, the "father of Dolly the sheep," Ian Wilmut, a professor at the University of Edinburgh in Scotland, Britain, immediately announced that he would abandon using somatic cell nuclear transfer technology to study embryonic stem cells. He believed the new method was the key to treating difficult diseases in the future.

Robert Lanza, chief scientist at Advanced Cell Technology in the United States, praised the work, saying its "biological value is equivalent to the Wright brothers' first flight."

Facing Multiple Obstacles

Perhaps the happiest person about this breakthrough was U.S. President Bush. For more than six years, despite heavy criticism, Bush had consistently opposed human embryonic stem cell research, believing that the process of obtaining embryonic stem cells involves "creating" and "killing" life. Bush had used his veto power to prohibit federal funding for embryonic stem cell research, and later made a slight concession in 2001, agreeing that scientists could use federal funds to study already existing human embryonic stem cell lines. Bush firmly believed that one day scientists would find a way to obtain embryonic stem cells without destroying embryos, and now this prediction had finally come true.

However, it was precisely because of multiple obstacles that embryonic stem cell research had been progressing slowly. On the one hand, scientists found it difficult to obtain large amounts of funding to research human embryonic stem cells, and on the other hand, because the success rate of this research was not high and large numbers of human eggs were needed, research costs were enormous. While these difficulties obstructed progress in stem cell research, they also forced scientists to start thinking of other ideas.

One method was to let animals replace humans as "cloning Trojan horses."

Esmail Zanjani, a professor at the University of Nevada, had for many years worked on cultivating human cells in sheep embryos. He injected human bone marrow stem cells into sheep embryos and once obtained sheep with as much as 15% human cells. Through this method, it might be possible to use sheep to cultivate human organs and overcome the problem of transplant rejection. But opponents held strongly skeptical attitudes toward this, and the prospect of human-sheep hybrids also frightened people. In addition, many researchers transplanted human somatic cells into animal egg cells (such as those of rabbits and sheep), using factors in animal egg cells to reprogram human somatic cell nuclei and cultivate embryos. However, human-animal hybrid cells were likewise very difficult for people to accept, and they also faced many technical problems.

Opening a Second Front

The methods of Yamanaka and Thomson can be said to open a new path.

Somatic cells are cells that have already differentiated very maturely, equivalent to having their fate already settled. But Yamanaka and Thomson used retroviruses as vectors to transfer four key genes with different functions into somatic cells, causing them to recombine with the original genes, and unexpectedly enabled the somatic cells to return to the "origin of life," becoming multifunctional stem cells with the characteristics of embryonic stem cells. People called these cells induced pluripotent stem cells (iPS), and they were as if they had lost their memory, forgetting their previous identity and starting over "from zero."

This method seems astonishingly simple, yet even one year earlier no one would have dared say it could succeed.

The first glimmer of dawn appeared in August 2006. At that time, Yamanaka first reported that his research group used modified retroviruses as "transport vehicles" to introduce four transcription factor genes into mouse tail-tip fibroblasts, and the new "skin cells" obtained in this way had the characteristics of stem cells - they could differentiate into other types of cells. However, they still differed considerably from embryonic stem cells and could not be cultured into mouse embryos. Nevertheless, this was a very important first step, breaking people's "superstition" about embryonic stem cells. Many people regarded this as the maiden flight of the "stem cell Wright airplane."

The publication of this paper directly gave rise to an "arms race" in the stem cell research field. The first reaction of many scientists was "how is this possible," and some large laboratories immediately began related work.

In June 2007, Yamanaka's laboratory and institutions such as the Harvard Stem Cell Institute and the Whitehead Institute for Biomedical Research in the United States successively published further research work. This time, scientists still used these 4 genes, but obtained truly pluripotent stem cells, and these could enter the germ line - when these skin cells were implanted into early fetal mice, they could develop like true embryonic stem cells and participate in forming all parts of the fetal mouse body. It can be said that the conditions had ripened.

Then came these two new papers more than four months later. Yamanaka extracted cells from the facial skin of a 36-year-old woman and the connective tissue of a 69-year-old man, while Thomson chose fetal skin cells and the foreskin cells of a newborn. However, in this "research race to the beachhead," Thomson's group gained a slight upper hand, because Yamanaka's group still used the original four genes (Oct3/4, Sox2, c-Myc and Klf4), while Thomson chose genes with two differences (Oct4, Sox2, Nanog and Lin28). These genes were like switches that opened the cell's "path to rejuvenation." However, the c-Myc gene was an inferior switch, and its introduction might bring side effects such as cancer.

Yu Junying's Contribution

The fierce research collision made the scientists participating in this competition all tense. Yu Junying said that in the past two years she had worked more than ten hours a day in the laboratory, often giving up holidays. Yu came from Zhuji, Zhejiang, China. She graduated from the Department of Biology at Peking University in 1997, and that same year went to the University of Pennsylvania in the United States to pursue a doctorate. She joined Thomson's laboratory in 2003, and currently serves as an assistant researcher in the laboratory.

Yu Junying is the first author and co-corresponding author of the paper published by Thomson's group in Science, which also shows that she undertook the main experimental work. She wrote in an email that at the time "I independently proposed the experimental approach. This was an extremely labor-intensive plan, and I believed this was the only way to study the reprogramming problem. I did not expect anyone would do the same work in mice. Because mouse experimental research cycles are shorter and methods are relatively simple, the Japanese research group was able to seize the initiative. Fortunately, we did not fall behind in research on human cells."

In July of this year, Yu Junying finally completed this part of the work. She was extremely excited, but the first celebration she thought of was going home and having a good sleep. Yu Junying hoped that later she would have time to read books, watch television, and go to nearby Milwaukee to watch a Yi Jianlian game. But biological research competition is quite fierce and time-consuming, and she had to immediately devote herself to the next stage of research.

A New Starting Point

However, the new discovery is not perfect. The success rate of stem cell transformation is very low, and because retroviruses are used as gene vectors, they trigger oncogene activity. Among the mice cultivated at Kyoto University, 20% developed tumors, and if applied to humans it could similarly induce cancer.

Yu Junying also told Southern Weekly that improving the method of gene transfer is the focus of the next step, and it is still necessary to further prove the similarity between induced pluripotent stem cells and embryonic stem cells, but she felt the two "do not differ much in clinical application," and "induced pluripotent stem cells should be able to completely replace embryonic stem cells."

Yu Junying believes that "the application of this technology in the medical field is still in its initial stage." For organ transplantation, "the current method only solves the problem of immune rejection," and the remaining problems are the same as those faced by embryonic stem cell research. Therefore, "it is still too early to discuss using these stem cells to cultivate human organs and perform transplantation. The first step in application will be in drug testing for some disease models."

But this achievement will become a new starting point in the stem cell research field. Next, many laboratories will use this as a basis for more breakthrough research. Once the related technical problems are solved, traditional human embryonic cloning technology, because of ethical problems and high cost, will "no longer be necessary."