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Saturday, March 7, 2009

A novel way to make human stem cells from skin without the risk of disease caused by genetic manipulation

Stem Cell Research Takes Another Step Forward

By Michael Smith, North American Correspondent, MedPage Today
Published: March 05, 2009
Reviewed by Zalman S. Agus, MD; Emeritus Professor University of Pennsylvania School of Medicine

CAMBRIDGE, Mass., March 5 -- For the second time in a week, researchers are reporting a novel way to make human stem cells from skin without the risk of disease caused by genetic manipulation.

Using skin fibroblasts from people with Parkinson's disease, Rudolf Jaenisch, M.D., of the Whitehead Institute and colleagues used now-established methods to create what are called induced pluripotent stem cells.

The new wrinkle, they reported in the March 6 issue of Cell, is that they were then able to remove all traces of the factors used to reprogram the skin cells.

The report comes only days after two collaborating groups -- one in Great Britain and one in Canada -- reported on a way to generate such cells without using viruses to insert the reprogramming factors. (See: Two Teams 'PiggyBAC' Stem Cell Research)

Skin cells can be a fertile source of pluripotent cells, as several research groups have shown. But the method -- using a virus to carry four genes into skin cells -- is thought to carry the risk of disease. (See: Adult Skin Cells Reprogrammed into Stem Cells for Disease Research)

The four genes -- c-Myc, Klf4, Oct4 and Sox -- reprogram skin cells to act as stem cells, but the virus itself is considered a potential danger, since it could disrupt the DNA of a patient's cell and possibly cause cancer. In addition, c-Myc is an oncogene and some of the other genes involved can also cause cancer.

There are also concerns that the viral vector may affect what genes are on or off in the newly created stem cells, so that the use of viruses is a major limitation of current technology, Dr. Jaenisch said in a statement.

To get around that limit, he said, "we used a modified virus you can excise. After they've done their job, you can get rid of them."

The researchers took skin fibroblasts from five patients with idiopathic Parkinson's disease and two healthy volunteers. Using either all four of the reprogramming factors or a subset lacking c-Myc, they employed a lentivirus to carry the genes into the cells.

The trick was that the lentivirus had been modified so that it could be found and excised by an enzyme called Cre recombinase.

After a line of stem cells had been created, they could be treated with the enzyme to establish a daughter line that retained no trace of the original reprogramming factors or the viral vector, the researchers said.

Both sets of cell lines -- with and without the reprogramming factors -- were shown to have the genetic markers of pluripotency and could be made to grow into dopaminergic neurons, Dr. Jaenisch and colleagues said.

The establishment of in vitro lines of neurons derived from Parkinson's patients should be a boon to research on the disease, Dr. Jaenisch and colleagues said.
"Such in vitro models could be utilized for large-scale genetic or drug-based screens since large numbers of (stem cells) can be generated and robustly differentiated into dopaminergic neurons," they said.

But the new method of getting rid of the transforming factors is also important, since one aspect of the experiments showed that they affect the genetic profile of the new stem cell.

Dr. Jaenisch and colleagues used genome-wide expression analysis to see what genes were active in human embryonic stem cells, induced pluripotent stem cells, and the induced stem cells without the transforming factors.

They found 271 genes whose expression was significantly different (at P<0.05)>
In contrast, they found, only 48 genes were differentially expressed between embryonic stem cells and the factor-free pluripotent cells -- a reduction of 80%.
"The vector-free cells are much more closely related to embryonic stem cells than to the parental cells," Dr. Jaenisch said. The finding "argues that even low vector expression somehow changes the transcriptional profile of cells."

The factor-free stem cells may also form a source of neurons for transplant, the researchers said, especially since they were derived from patients regardless of age.
The research was supported by the Life Science Research Foundation, the National Institutes of Health, the Howard Hughes Medical Institute, Udall Parkinson's Disease Center of Excellence, and the Michael Stern Foundation.
Dr. Jaenisch is an adviser to Stemgent, which has obtained a license from MIT to distribute some of the reagents used in this paper.


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1 comment:

Denver Refashionista said...

In some ways this stuff has always given me the heebie jeebies (think Frankenstein), on the other hand it may hold all the answers to making us all whole again and so I look to science with increased interest.