Scientists from Oregon Health and Science University, Portland, U.S.A., achieved for the first time the generation of human embryonic stem cells by transferring the nucleus of a skin cell from specific patients into a human oocyte. The reprogramming of differentiated human cells into embryonic totipotent cells, which can create any type of cell or organ, can revolutionize medicine and organ transplantation but also re-opens a big ethical debate. The study, published online in the journal Cell yesterday (May 15th) will be freely available until June 20th.
In 1962 in a laboratory at Oxford when the young John Gurdon saw his experiments of nuclear transplantation, encouraged by his mentor Michail Fischberg, in frogs to finally succeed. He had the nucleus (the organelle of eukaryotic cells enclosing the genetic material) of a frog oocyte removed and he injected the nucleus of a somatic cell of a tadpole. At that time, there was a big debate whether the DNA was a subject to permanent modifications during differentiation and whether these could be reversed. Gurdon saw his transplanted egg to grow into a normal tadpole. That was the beginning of nuclear transplantation and the idea that cells can be reprogrammed into their embryonic totipotent state (the state where a cell is able to become any cell of the organism). The simple translation of this experiment is that in the cytoplasm of oocytes exist factors that can reset the state of transplanted somatic nuclei back to the embryonic state.
The belief that we could take a nucleus from a patient’s skin cell, transplant it into an oocyte and make any tissue or organ was created (therapeutic cloning). In theory, this would eliminate the issue of transplant rejection as the organ would come from your own cells. Together with this belief, there was the fear that humans would clone (make identical copies of) humans for organ harvesting or even slavery (human cloning).
Dolly’s (the sheep) birth in 1996 was a major scientific breakthrough. It was the first mammal to be cloned from a somatic cell, after 277 failed attempts. Later, Cumulina, the first cloned mouse, lived a healthy life and died of natural causes (contrary to Dolly who was euthanized early due to respiratory problems, after giving birth to four lambs). Several other cloned mammals followed such as cow, goat, pig or deer.
Cloned animals are the result of therapeutic cloning as scientists are researching ways to make animals produce medicines, such as blood clotting proteins in the case of Dolly, into their milk and treat human diseases, such as haemophilia.
Of course, there were several claims by controversial doctors that they have succeeded to clone the first humans but with no substantial base or proof. Others had their scientific publications disgracefully retracted due to faked data in 2004 and 2005.
Based on the numerous failed attempts, it seemed that the generation of human embryonic cells would not be possible. The problem was that transplanted human oocytes stopped dividing at the 8-cell stage, a premature step before the generation of a blastocyst (150-cell stage). Scientists could not find the right conditions to culture them as they were using conditions for other primates, a strategy which has been proven unsuccessful. Even Professor Ian Wilmut, the “father “of Dolly, abandoned cloning efforts in 2007.
In 2006, the induced pluripotent stem (iPS) cells came to the horizon by Takahashi and Yamanaka (Yamanaka and Gurdon shared the Nobel Prize in Physiology or Medicine in 2012). By adding just 4 transcription factors (proteins that regulate gene transcription), skin cells are reprogrammed and later can be differentiated into most types of cells. There are no ethical issues with iPS cells as there is no need for oocyte or embryo generation. And since then, the technique is being used by several researchers to treat many diseases. However, iPS cells are not fully characterized yet and accumulating evidence suggests that there are several drawbacks and have significant differences from ES cells.
Now, Mitalipov and his colleagues found the right conditions to create and maintain human embryonic stem cells. They have first shown that the reason why human oocytes cannot be activated is coming from problems in an organelle in the cell called spindle, which helps chromosomes separate during cell replication.***
They have tested several possible modifications in the culture conditions of rhesus macaque embryos. They utilized an inactivated (Sendai) virus to help the oocyte merge with the somatic cells. The researchers later found that somatic cell nuclear transfer (SCNT) and blastocyst formation is promoted when the oocyte is exposed to a small electric pulse and to an organic compound (trichostatin A – TSA) which blocks certain enzymes (deacetylases). They also managed to improve the quality of the human embryo and its cells’ spindles. They additionally treated human embryos with caffeine, which inhibits other types of enzymes (phosphatases), and the embryos successfully developed into blastocysts with remarkable efficiency and reproducibility.
Mitalipov cancelled his holiday plans when he saw the SCNT colonies grow in his culture dish. “I was happy to spend Christmas culturing cells,” he says. “My family understood.”
The ES cells matched the cells of the donor (with few exceptions in the mitochondrial DNA (mtDNA) which come from the oocyte) and can subsequently create any tissue or organ of the patient and treat several diseases such as Parkinson’s, Alzheimer’s etc. However, these changes in mtDNA should not be considered as negative, they can be exploited to correct mtDNA mutations and treat metabolic diseases.
Several women donated oocytes, whose quality was detrimental to the cloning success. Some somatic nuclei came from skin cells of a patient with Leigh syndrome but also from other healthy cells, showing the reproducibility of the method. But what cloning history has taught us is to be patient until other labs can reproduce the experiments. The results seem convincing and solid despite the fact that the publication had been accepted by the journal in just 3 days after submission (usually it takes 3-6 months after rigorous peer review). Head-to-head comparisons of iPS cells and SCNT cells derived from the same donor are possibly the next step. “These results,” says George Daley, a stem-cell scientist at Children’s Hospital Boston in Massachusetts, “will be fascinating.