Virus-free Embryonic-like Stem Cells Made From Skin Of Parkinson's Disease 
Patients
ScienceDaily (Mar. 8, 2009) - Whitehead Institute researchers have developed 
a novel method to remove potential cancer-causing genes during the 
reprogramming of skin cells from Parkinson's disease patients into an 
embryonic-stem-cell-like state. Scientists then used the resulting induced 
pluripotent stem (iPS) cells to derive dopamine-producing neurons, the cell 
type that degenerates in Parkinson's disease patients.
This marks the first time researchers have generated human iPS cells that 
have maintained their embryonic stem-cell-like properties after the removal 
of reprogramming genes. The findings are published in the March 6 edition of 
the journal Cell.
"Until this point, it was not completely clear that when you take out the 
reprogramming genes from human cells, the reprogrammed cells would actually 
maintain the iPS state and be self-perpetuating," says Frank Soldner, a 
postdoctoral researcher in Whitehead Member Rudolf Jaenisch's laboratory and 
co-author of the article.
Since August 2006, researchers have been reprogramming adult cells into iPS 
cells by using viruses to transfer four genes (Oct4, Sox2, c-Myc and Klf4) 
into the cells' DNA. Although necessary for reprogramming cells, these 
genes, the known oncogene c-Myc in particular, also have the potential to 
cause cancer. In addition, the four genes interact with approximately 3000 
other genes in the cell, which may change how the cell functions. Therefore, 
leaving the genes behind in successfully reprogrammed cells may cause 
unintended alterations that limit the cells' applicability for therapeutic 
use, for drug screens or to study disease in cell culture.
In the current method, Whitehead researchers used viruses to transfer the 
four reprogramming genes and a gene coding for the enzyme Cre into skin 
cells from Parkinson's disease patients. The reprogramming genes were 
bracketed by short DNA sequences, called loxP, which are recognized by the 
enzyme Cre.
After the skin cells were reprogrammed to iPS cells, the researchers 
introduced the Cre enzyme into the cells, which removed the DNA between the 
two loxP sites, thereby deleting the reprogramming genes from the cells. The 
result is a collection of iPS cells with genomes virtually identical to 
those of the Parkinson's disease patients from whom original skin cells 
came.
Removing the reprogramming genes is also important because of those genes' 
effect on an iPS cell's gene expression (a measure of which genes the cell 
is using and how much it's using those genes). When the researchers compared 
the gene expressions of human embryonic stem cells to iPS cells with and 
without the reprogramming factors, iPS cells without the reprogramming genes 
had a gene expression closer to human embryonic stem cells than to the same 
iPS cells that still contained the reprogramming genes.
"The reprogramming factors are known to bind to and affect the expression of 
3,000 genes in the entire genome, so having artificial expression of those 
genes will change the cell's overall gene expression," Dirk Hockemeyer, who 
is also a co-author of the Cell article. "That's why the four reprogramming 
genes can mess up the system so much. From now on, it will be tough for 
researchers to leave the reprogramming genes in iPS cells."
Jaenisch says that the process to remove the reprogramming genes is very 
successful, when compared with earlier experiments. "Other labs have 
reprogrammed mouse cells and removed the reprogramming genes, but it was 
incredibly inefficient, and they couldn't get it to work in human cells," he 
says. "We have done it much more efficiently, in human cells, and made 
reprogrammed, gene-free cells."
After removing the reprogramming genes, the Jaenisch researchers 
differentiated the cells from the Parkinson's disease patients into 
dopamine-producing nerve cells. In Parkinson's disease patients, these cells 
in the brain die or become impaired, causing such classic Parkinson's 
symptoms as tremors, slowed movement, and balance problems.
Because the cells reside in the patients' brains, researchers cannot easily 
access them to investigate how the disease progresses at the cellular level, 
what kills the cells, or what might prevent cellular damage. Therefore, the 
ability to create patient-specific iPS cells, derive the dopamine-producing 
cells, and study those patient-specific cells in the lab could be a great 
advantage for Parkinson's disease researchers.
Although the initial results are extremely promising, Jaenisch acknowledges 
that the process is far from over. "The next step is to use these 
iPS-derived cells as disease models, and that's a high bar, a real 
challenge. I think a lot of work has to go into that."

Rayilyn Brown
Director AZNPF
Arizona Chapter National Parkinson Foundation
[log in to unmask] 

----------------------------------------------------------------------
To sign-off Parkinsn send a message to: mailto:[log in to unmask]
In the body of the message put: signoff parkinsn