A team led by scientists from The Scripps Research Institute
has developed a method that dramatically improves the efficiency of creating
stem cells from human adult tissue, without the use of embryonic cells. The
research makes great strides in addressing a major practical challenge in the
development of stem-cell-based medicine.
The findings were published in an advance, online issue of
the journal Nature Methods on October 18, 2009.
The new technique, which uses three small drug-like
chemicals, is 200 times more efficient and twice as fast as conventional
methods for transforming adult human cells into stem cells (in this case called
"induced pluripotent stem cells" or "iPS cells").
"Both in terms of speed and efficiency, we achieved
major improvements over conventional conditions," said Scripps Research
Associate Professor Sheng Ding, Ph.D., who led the study. "This is the
first example in human cells of how reprogramming speed can be accelerated. I
believe that the field will quickly adopt this method, accelerating iPS cell
research significantly."
In addition to its significant practical advantages, the
development of the technique deepens the understanding of the biology behind
the transformation of adult human cells into stem cells.
Tackling Major
Challenges
The hope of most researchers in the field is that one day it
will be possible to use stem cells — which possess the ability to develop into
many other distinct cell types, such as nerve, heart, or lung cells — to repair
damaged tissue from any number of diseases, from Type 1 diabetes to Parkinson's
disease, as well as from injuries. The creation of iPS cells from adult cells
sidesteps ethical concerns associated with the use of embryonic stem cells, and
allows the generation of stem cells matched to a patient�s own immune system,
avoiding the problem of tissue rejection.
The creation of human iPS cells was first announced in
December 2007 by two labs, one in Japan
and another in Wisconsin.
In both cases, the teams used viruses to insert multiple copies of four genes
(eg. c-Myc, Oct4, Sox2, Klf4) into the genome of skin cells. These four genes
then produced transcription factors turning on and off other genes, and pushing
the cell to "dedifferentiate" into stem cells.
While the work was a major breakthrough, it left two major
challenges for the field to solve before iPS cell therapy could be considered
of any potential practical use. The first involved safety, since the technique
relied on potentially harmful genetic manipulation, and worse yet, the insertion
of two known cancer-causing genes (c-Myc and Oct4). The second problem was the
length and inefficiency of the iPS cell process, which had a success rate of
roughly one in 10,000 cells and took about four weeks from start to finish.
Ding and colleagues essentially solved the first problem,
the reliance on genetic manipulation, earlier this year in a paper published in
Cell Stem Cell (Volume 4, Issue 5, May 8, 2009). In the paper, the researchers
demonstrated that they could use purified proteins to transform adult cells all
the way back to the most primitive embryonic-like cells, avoiding the problems
associated with inserting genes.
In the current paper, the team makes major strides in
solving the second problem, efficiency.
A Focus on Natural
Processes
In developing the improved method, Ding drew on his
knowledge of biology. He decided he would focus his efforts on manipulating a
naturally occurring process in cells, in particular in a type of adult cell
called fibroblasts, which give rise to connective tissue.
This naturally occurring process — called MET (mesenchymal
to ephithelial cell transition) — pushes fibroblasts closer to a stem-cell-like
state. If he could manipulate such a fundamental process to encourage MET and
the formation of stem cells, Ding reasoned, such a method would be both safer
and more direct than hijacking other aspects of biology, for example those
directly involved in cancer.
"People have studied this mechanism for 10 to 20
years," said Ding. "It is a fundamental mechanism."
Ding and colleagues tested a number of drug-like molecules,
looking for those that inhibited the TGFb (transforming growth factor beta) and
the MEK (mitogen-activated protein kinase) pathways, which are known to be
involved in the MET process. The researchers identified the most active
compounds, then looked at their effects on stem cell creation when used singly
and in combination.
The researchers found two chemicals — ALK5 inhibitor SB43142
and MEK inhibitor PD0325901 — used in combination were highly effective in
promoting the transformation of fibroblasts into stem cells.
"This method is the first in human cells that is
mechanism-specific for the reprogramming process," said Ding.
And the two-chemical technique bested the efficiency of the
classic genetic method by 100 times.
Efficient, Fast, Safe
But the researchers thought they might be able to do even
better.
Attempting to increase the efficiency of the process even
further, the team decided to enlist another natural pathway, the cell survival
pathway. After screening a library of compounds targeting this pathway, the
team focused on a novel compound called Thiazovivin.
The researchers found that a technique using Thiazovivin in
combination with the two previously selected chemicals, SB43142 and PD0325901,
beat the efficiency of the classic method by 200 times.
In addition, while the classic method required four weeks to
complete, the new method took two weeks.
In addition to its virtues of speed and efficiency, Ding
emphasizes that the safety profile of the new method is highly promising. Not
only is the method based on natural biological processes, he said, but also the
type of molecules used have all been tested in humans.
In addition to Ding, the article, "A Chemical Platform
for Improved Induction of Human iPS Cells," was authored by Tongxiang Lin
(first author), Rajesh Ambasudhan, Xu Yuan1, Wenlin Li, Simon Hilcove, Ramzey
Abujarour, Xiangyi Lin, and Heung Sik Hahm of Scripps Research, and Ergeng Hao
and Alberto Hayek of The Whittier Institute for Diabetes, University of
California San Diego.
The research was supported by the National Institutes of
Health and Fate Therapeutics.
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