Nuclear reprogramming: converting specialized cells into early stem cells

Figure 1. Nuclear reprogramming by transfer of an adult cell nucleus. In experiments pioneered by Gurdon and subsequently extended by others, the nucleus of a specialized adult cell is transferred to an enucleated egg. The resulting zygote can give rise to embryonic stem cells, a partially developed animal (tadpole or fetus), or a fertile adult.

Dormant, but ready for rejuvenation

Biologists have long wondered about the process by which descendants of a single fertilized egg specialize — or differentiate — to construct an adult animal. Once a skin, muscle, or brain cell commits to its fate, it does not revert to a ‘totipotent’ state, with the potential to become any type of cell. A cell destined to become muscle need not retain its ability to fire neuronal messages, nor must a brain cell remember how to soak up nutrients in the intestine.

This apparently irreversible differentiation could arise from either of two distinct processes. In one, championed by August Weismann in the late 1800s, cells cast off genes as they progress down a particular specialization route; in the other, cells retain their complete collection of genes, but turn them on and off as needed. The first scenario would preclude the possibility of changing one type of mature cell into another because the cell would no longer contain the genetic wherewithal to perform all possible functions. This line of reasoning produced a clear experimental test: Replace an egg’s nucleus with that of a specialized cell and assess whether the resulting cell could develop into a complete animal. If so, a nucleus from a fully differentiated cell retains a complete genome, capable of directing all types of specialization.

In 1952, Robert Briggs and Thomas King conducted this experiment with frogs. They transferred nuclei from very early embryos — at the so-called blastula stage — into eggs from which they had removed the nuclei. If cells discarded genes when they embarked on the path from zygote to fully developed frog, these nuclei would lack genes necessary to build an animal. Briggs and King found that approximately a third of the transplanted early embryonic nuclei produced normal tadpoles. In contrast, when they repeated the experiment with cells from later-stage embryos, the percentage of transplanted nuclei that gave rise to embryos and animals dropped precipitously. The researchers concluded that permanent nuclear changes occur as cells specialize.

In 1956, Gurdon began his graduate work at the University of Oxford with developmental biologist Michail Fischberg. Captivated by the observations of Briggs and King, but cognizant that technical limitations rendered their interpretations debatable, Gurdon repeated their experiments in a different frog, Xenopus laevis, which is easier to work with than the one Briggs and King had used, Rana pipiens. In each study, Gurdon destroyed the nucleus from the recipient cell with UV light and then injected a donor cell’s nucleus.

Crucially, Fischberg had discovered a naturally occurring ‘marker’ that allowed Gurdon to distinguish the donated from the recipient nucleus. Nuclei from donor cells carried one rather than two copies of a structure called the nucleolus. This innovation allowed Gurdon to verify whether embryos and animals arose from the transferred nucleus or the failure to inactivate the original one. That proof became essential because, in 1958, his results contradicted those of Briggs and King. He showed that nuclei from cells in late embryonic stages can give rise to apparently normal adult frogs. Many scientists initially doubted Gurdon’s results, because they found it hard to believe that a graduate student could overturn the wisdom established by two esteemed developmental biologists. But Gurdon’s experimental design was so robust, eventually his data convinced the skeptics.

Gradually, he extended his results by using donor cells from increasingly specialized cells and older animals (see Fig. 1). For example, he showed that nuclei from a particular type of fully specialized frog skin cell could give rise to tadpoles, with muscle and nerve cells. In 1966, he generated fully developed, fertile frogs from tadpole intestinal cell nuclei. The observation that these nuclei could give rise to healthy animals that themselves produced offspring showed that a completely differentiated nucleus was indeed totipotent: It could direct the formation of all cells, including eggs and sperm, necessary to compose a completely functioning adult.

Gurdon therefore established that the vast majority of the body’s cell types retain their genomes as they specialize. Furthermore, the right conditions can awaken genes that have turned idle during development. His discoveries ignited the entire field of nuclear reprogramming and allowed all subsequent work to unfold.

One question remained. Although Gurdon had raised tadpoles from frog cells and generated reproductively competent frogs from fully specialized tadpole cells, he had never produced a fertile adult from the nucleus of an adult cell. In 1997, Keith Campbell and Ian Wilmut (Roslin Institute, Scotland) created Dolly the sheep by transferring into an enucleated egg the nucleus from a mammary gland cell that had been removed from a pregnant ewe. Dolly eventually gave birth to her own lambs, thus closing the experimental gap.

Four factors, a plethora of possibilities

The nuclear reprogramming work raised the possibility of crafting medically useful banks of replacement cells for tired or injured tissue. But such prospects raised challenges. Obtaining human eggs is ethically fraught and technically difficult. Furthermore, cells or tissues derived by such a strategy would be vulnerable to attack by the recipient’s genetically unrelated immune system.

In 1999, Shinya Yamanaka began to wonder whether he could devise a nuclear-reprogramming method that would circumvent these hurdles. He knew that the late Harold Weintraub had shown in 1988 that a single gene could convert fibroblasts, a type of connective tissue cell, into muscle cells. If nuclei from fully differentiated cells could be genetically re-set, Yamanaka reasoned, and if one gene could force a certain cell type to behave like another, perhaps he could reprogram adult cells to an embryonic state by adding to them a small number of factors.

As he devised his scheme, he relied heavily on knowledge from the field of embryonic stem (ES) cells, which naturally possess the ability to specialize into any type of adult cell. In the early 1980s, Martin Evans (Lasker Basic Medical Research Award, 2001) figured out how to isolate such cells from mice, grow them in the laboratory, and make animals that are completely composed of their descendants. More than a decade later, James Thomson (University of Wisconsin) took the next step toward potential medical applications when he isolated ES cells from humans.

Yamanaka and colleagues, including graduate student Yoshimi Tokuzawa, compiled a list of 24 genes that had been implicated in maintaining mouse ES cells in an uncommitted state in culture, reasoning that these genes were good candidates for converting fully differentiated cells, such as skin fibroblasts, into ones with multiple possible fates. As a first step, he and his colleagues set up an experimental system that would allow embryonic fibroblasts to survive only if they behaved like ES cells.

In a technical tour de force, Yamanaka and another student, Kazutoshi Takahashi, tested whether the 24 genes, added together, could convert mouse fibroblasts into stem cells that could, in turn, morph into different types of specialized cells. He delivered active versions of these genes, using retroviruses engineered for the purpose. This method generated ES-like cells in 2006 and Yamanaka named them induced pluripotent stem (iPS) cells. He then whittled the group of 24 genes to four — Oct3/4, Sox2, c-Myc, and Kif4 — that, in combination, triggered iPS cell production from embryonic and adult skin fibroblasts. iPS cells resembled ES cells in their appearance and growth characteristics. Furthermore, they displayed molecules characteristic of ES cells. When injected into immunodeficient mice, the iPS cells grew into neural tissue, cartilage, muscle, and forebears of cells that line the intestinal tract — representing all classes of tissue that give rise to a complete body. Therefore, iPS cells displayed properties of ES cells in animals as well as in test tubes.

Figure 2. The generation of induced pluripotent stem cells from adult fibroblast cells to create iPS cells. *For a full description of the figure, please see legend at the end of the essay.

Yamanaka’s discovery that a mere four factors could erase the specialization process galvanized the research community. Multiple labs raced to verify and expand his findings — and to overcome safety concerns. For instance, one of the four factors, c-Myc, causes unrestrained growth under some circumstances. In addition, the retroviruses that deliver the reprogramming genes land in chromosomes, where they can trigger aberrant activity of resident genes and promote cancer formation. Furthermore, the gene-activity profiles of the first-generation iPS cells were similar, but not identical, to those of ES cells — and chemical marks called methyl groups on the DNA did not exactly reflect those on ES cells either. This type of comparison between iPS and ES cells will continue to be crucial as scientists develop reprogramming techniques.

The following year, Yamanaka adjusted his strategy to isolate cells that more closely resemble ES cells. Blastocysts injected with the new, improved iPS cells — but not with the original ones — developed into mice; various organs in the animals carried tissues of iPS origin, indicating that the cells could mature down many physiological paths. Simultaneously, Rudolf Jaenisch (Massachusetts Institute of Technology) and Konrad Hochedlinger (Massachusetts General Hospital) reported similar findings. The Yamanaka experiments were especially notable in that some of his mice grew up to parent offspring that contained iPS-derived cells (see Fig. 2). This achievement proved that iPS cells can give rise to all cell types — including sperm and eggs — required for normal adult life.

Only a year after he produced the first iPS cells from adult mouse skin cells, Yamanaka generated iPS cells from adult human skin cells, employing human versions of the same four genes that he had used in the mouse work. Simultaneously, George Daley (Children’s Hospital, Boston) and James Thomson achieved similar successes. Yamanaka goaded iPS cells to specialize into neural or cardiac cells in culture. He then wanted to assess whether human iPS cells would differentiate in a live animal. He injected them into immunocompromised mice, where they specialized into the body’s main cell types, as the mouse iPS cells had done earlier.

Now, many laboratories worldwide are refining human iPS cell technology and investigating how closely the cells resemble natural ES cells, activities that are necessary to translate nuclear reprogramming to the clinic. Scientists are defining the minimal number of reprogramming genes necessary, identifying them, and trying to replace the introduced genes with chemicals. Yamanaka and others already have produced iPS cells without retroviruses and active c-Myc, innovations that should reduce the risk of cancer. Learning how reprogramming occurs in iPS cells may ultimately reveal new insights into the workings of ES cells.

Nuclear reprogramming has raised the possibility of changing easily accessible adult cells, such as those from skin, into tissue that might repair damage caused by a wide variety of injuries and diseases — without destroying embryos. Eventually, researchers would like to make patient-specific iPS cells. Because the same individual would donate the precursors of such cells and receive them as therapy, the cells would not spark an immune attack. iPS cells provide a way to study how some disorders progress from the earliest stages of a cell’s development through full specialization, and they offer the opportunity to assess drug effectiveness and toxicity for a given person before subjecting that individual to treatment.

The conceptual and technical breakthroughs spearheaded by Gurdon and Yamanaka have unleashed previously unimagined strategies for combating diseases and probing normal development as well as pathological processes. They have launched an era in which scientists can reverse the clock to fashion cells that possess all possible fates from those that have arrived at a single one.

by Evelyn Strauss

*Figure 2. The generation of induced pluripotent stem cells from adult fibroblast cells. To create iPS cells, Yamanaka took cells from an adult mouse’s tail and grew them in petri dishes. Then he added various combinations of genes that had been implicated in maintaining ES cells in culture. Using a special selection system, he allowed cells to grow only if they behaved like ES cells. Four genes did the trick: Oct 3/4, Sox2, Kif4,and c-Myc. After injecting these so-called iPS cells into a normal blastocyst, he introduced the resulting early embryo into a normal female mouse. Offspring included chimeric animals — those with cells that descended from the iPS cells as well as from the normal blastocyst. Some of these mice grew up to parent offspring that contained iPS-derived cells. Yamanaka thus had shown that iPS cells could give rise to all cell types — including sperm and eggs — required for normal adult life.

Key publications of John Gurdon

Gurdon, J.B., Elsdale, T.R., and Fischberg, M. (1958). Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei. Nature 182, 64-65.

Gurdon, J.B. (1962). The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J. Embryol. Exp. Morph. 10, 622-640.

Gurdon, J.B. and Uehlinger, V. (1966). “Fertile” intestine nuclei. Nature. 210, 1240-1241.

Gurdon, J.B., Laskey, R.A., and Reeves, O.R. (1975). The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs. J. Embryol. Exp. Morph. 34, 93-112.

Gurdon, J.B. and Byrne, J.A. (2003) The first half-century of nuclear transplantation. Proc. Natl. Acad. Sci. USA. 100, 8048-8052.

Gurdon, J.B. (2006) From nuclear transfer to nuclear reprogramming: the reversal of cell differentiation. Ann. Rev. Cell Dev. Biol. 22, 1-22.

Key publications of Shinya Yamanaka

Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126, 663-676.

Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature. 448, 313-317.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131, 861-872.

Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N., and Yamanaka, S. (2008). Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26,101-106.

Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T., and Yamanaka, S. (2008). Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science. 321, 699-702.

Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., and Yamanaka, S. (2008). Generation of mouse induced pluripotent stem cells without viral vectors. Science. 322, 949-953.