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Scientists Reprogram Mature Cells to Become Embryonic Stem Cells

June 8th, 2007 Medgadget Editors Genetics

By now you have probably read elsewhere in the media that scientists under Dr. Rudolf Jaenisch from the Whitehead Institute created embryonic stem cells in mice without destroying any embryos. The press statement from MIT explains how the research was conducted:

In August 2006, researchers at Kyoto University reported that by activating four genes in a mouse skin cell, they could reprogram that cell into a pluripotent state resembling an embryonic stem cell. However, the resulting cells were limited when compared with real embryonic stem cells, and the Kyoto team was unable to generate live mice from these cells.
The Jaenisch team decided to replicate this experiment, while refining certain technical aspects. This group included Jaenisch lab postdocs Marius Wernig, Alexander Meissner and Tobias Brambrink, MIT graduate student Ruth Foreman, Manching Ku, a research fellow from Bradley Bernstein’s lab at Massachusetts General Hospital, and Konrad Hochedlinger, formerly of the Jaenisch lab and now at Massachusetts General Hospital.
Using artificial viruses called vectors, the team activated the same four genes in a batch of mouse skin cells. These genes, Oct4, Sox2, c-Myc and Klf4, are called transcription factors, meaning that they regulate large networks of other genes. While Oct4 and Sox2 are normally active in the early stages of embryogenesis, they typically shut down once an embryo has developed beyond the blastocyst stage.
“We were working with tens of thousands of cells, and we needed to devise a precise method for picking out those rare cells in which the reprogramming actually worked,” says Wernig. “On average, it only works in about one out of 1,000 cells.”
To test for reprogramming, the team decided to zero in on Oct4 and another transcription factor called Nanog. These two hallmarks for embryonic stem cell identity are only active in fully pluripotent cells. The trick would be to figure out a way to harvest Oct4- and Nanog-active cells from the rest of the population.
The answer came in the form of a laboratory technique called homologous recombination. Here, the scientists took genetic material known to be resistant to the toxic drug neomycin and spliced it into the genomes of each cell right beside Oct4 and Nanog. If Oct4 and Nanog switched on, the drug-resistant DNA would also spring into action. The researchers then added neomycin to the cells. Only those fully reprogrammed cells with active Oct4 and Nanog survived.
Next, the team ran these cells through a battery of tests, seeing if they could discover any substantial differences between these cells and normal embryonic stem cells.
“In all tests…there were no molecular markers distinguishing these two groups,” says Meissner.
But definitive proof would only come through demonstrating that these cells could actually develop into any kind of body tissue and cell type. The researchers approached this question in three ways.
First, they fluorescently labeled these reprogrammed cells and injected them into early-stage embryos, which eventually gave rise to live mice. While these mice consisted of both the reprogrammed cells and the natural cells from the original embryo, the fluorescent tags indicated that the reprogrammed cells contributed to all tissue types in the mouse, everything from blood to internal organs to hair color.
Next, they bred these mice and found lineages of the reprogrammed cells in the subsequent generation, proving that these new cells had contributed to the germ line.
Finally, the team took advantage of another lab technique that involves creating a genetically abnormal embryo whose cells all consist of four chromosomes, rather than two. Because of this aberrant formation, the embryo can only form a placenta and cannot develop into a full-term fetus. The researchers injected the reprogrammed cells into this embryo and then implanted it in a uterus. Eventually live late-gestation fetuses could be recovered–created exclusively from the reprogrammed cells.
“This is the most stringent criteria anyone can use to determine if a cell is pluripotent,” says Jaenisch.

Press release and Videos: Scientists create embryonic stem cells without destroying embryos …
Nature: Simple switch turns cells embryonic

Medgadget Editors

Medical technologies transform the world! Join us and see the progress in real time. At Medgadget, we report the latest technology news, interview leaders in the field, and file dispatches from medical events around the world since 2004.

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