New Technique Creates True Embryonic Stem Cells Without Using Embryos

Researchers from the Massachusetts General Hospital Cancer Center have turned back the clock on adult cells, reprogramming them, in mice, to an authentic embryonic state. If successfully translated to humans, the technique could one day enable researchers to study the biology of disease in a Petri dish or allow physicians to cure disease or regenerate damaged tissue using embryonic stem cells (ESCs) that are genetically matched to the patient. Moreover, since this technique does not rely on embryos, it skirts the ethical concerns associated with therapeutic cloning.

The research team, led by senior co-authors Konrad Hochedlinger, PhD, of the Cancer Center and the Massachusetts General Hospital Center for Regenerative Medicine, and Kathrin Plath, PhD, of the University of California at Los Angeles, refined a technique developed last year by scientists at Japan’s Kyoto University and extended their initial findings. The Kyoto team had found four genes which, when inserted into mouse skin cells, reprogrammed them to become pluripotent (like ESCs, with the potential of making any cell type in the body); however, these so-called induced pluripotent stem (iPS) cells showed limited potential. This year, Hochedlinger and colleagues devised a way to create iPS cells that are indistinguishable from embryonic stem cells – the holy grail of regenerative medicine.

"The main focus of our study was to characterize the epigenetic state of iPS cells,” says Hochedlinger, whose research was published in the June 7, 2007 issue of Cell Stem Cell. Epigenetic refers to the chemical changes to DNA that do not alter its sequence but regulate the pattern of genes that are expressed in a given cell. Epigenetic changes are necessary for cells to gradually differentiate from embryonic stem cells into the various cell types they are fated to become: nerve cells, heart cells, liver cells and so forth. As the organism develops, proteins called transcription factors govern this process, turning on and off the genes that trigger development of all the tissues of the body. A key question of the study was whether reprogramming adult cells into iPS cells also resets the epigenetic changes in the cell from a differentiated gene pattern to an embryonic one. That question becomes critical for potential therapeutic applications of iPS cells, because epigenetic abnormalities can result in diseases like cancer.

“We looked at epigenetic modifications at three levels: individual genes, an entire chromosome, and across the genome,” says Hochedlinger. “We concluded that the epigenetic state of directly reprogrammed skin cells in our study was identical to ESCs. That was remarkable because we only introduced four genes or factors, which were sufficient to reset the entire epigenome.”

Cancer Center researchers were able to achieve more complete reprogramming than the Kyoto team by devising a new approach for isolating iPS cells. The technique uses two markers that separate ESCs from unreprogrammed cells. “Our selection procedure allowed the adult cells to revert all the way back to an embryonic state,” explains Hochedlinger.

To test the developmental potential of their iPS cells, researchers inserted the iPS cells into a developing mouse embryo. They were successful in creating a chimera -- a mouse with DNA from the original embryo and DNA from the iPS cells – thereby demonstrating pluripotency of the iPS cells. In addition, the mice passed the iPS DNA on to their offspring. Until now, the only way to derive ESCs has been through the controversial and complex process of therapeutic cloning, or nuclear transfer: removing the nucleus of an egg and replacing it with the nucleus of an adult cell, stimulating the egg to divide into an early-stage embryo, and then removing the ESCs, thereby destroying the embryo. The direct reprogramming approach is a far simpler technique that does not involve eggs or embryos, thus bypassing the ethical and political barriers associated with nuclear transfer.

Converting the technique to humans, however, poses significant challenges. “First, we don’t even know if the same four genes can do the job in humans,” cautions Hochedlinger. “Most likely, additional genes would be necessary to reprogram human adult cells into iPS cells. The second challenge is the delivery system for these genes.” To insert them into adult cells, researchers have been using retroviruses which can activate cancer-causing genes.

If researchers can overcome these hurdles, says Hochedlinger, human iPS cells could be used to repair damaged tissue, replenish bone marrow after a transplant, study the progression of disease, and screen drug compounds. Direct reprogramming could also test whether malignancy can be reversed and epigenetic abnormalities eliminated. “This approach now allows us to study the biochemical events that must take place to convert an adult cell to an ESC. That was impossible before.”