Newswise LA JOLLA, CA---- Stem cells are key to the promise of regenerative medicine: the repair or replacement of injured tissues with custom grown substitutes. Essential to this process are induced pluripotent stem cells (iPSCs), which can be created from a patient's own tissues, thus eliminating the risk of immune rejection. However, Shinya Yamanaka's formula for iPSCs, for which he was awarded last year's Nobel Prize, uses a strict recipe that allows for limited variations in human cells, restricting their full potential for clinical application.

Now, in this week's issue of Cell Stem Cell, the Salk Institute's Juan Carlos Izpisua Belmonte and his colleagues show that the recipe for iPSCs is far more versatile than originally thought. For the first time, they have replaced a gene once thought impossible to substitute, creating the potential for more flexible recipes that should speed the adoption of stem cells therapies.

Stem cells come in two types: embryonic stem cells (ESCs), which are immature cells that have never differentiated into specific cell types, and induced pluripotent stem cells, which are mature cells that have been reprogrammed back into an undifferentiated state. After the initial discovery in 2006 by Yamanaka that introducing four different genes into a mature cell could suffice for reprogramming the cell to pluripotency, most researchers adopted his recipe.

Izpisua Belmonte and his colleagues took a fresh approach and discovered that pluripotency (the stem cell's ability to differentiate into nearly any kind of adult cell) can also be accomplished by balancing the genes required for differentiation. These genes code for "lineage transcription factors," proteins that start a stem cell down the path to differentiate first into a particular cell lineage, or type, such as a blood cell versus a skin cell, and then finally into a specific cell, such as a white blood cell.

"Prior to this series of experiments, most researchers in the field started from the premise that they were trying to impose an 'embryonic-like' state on mature cells," says Izpisua Belmonte, who holds the Institute's Roger Guillemin Chair. "Accordingly, major efforts had focused on the identification of factors that are typical of naturally occurring embryonic stem cells, which would allow or further enhance reprogramming."

Despite these efforts, there seemed to be no way to determine through genetic identity alone that cells were pluripotent. Instead, pluripotency was routinely evaluated by functional assays. In other words, if it acts like a stem cell, it must be a stem cell.

That condition led the team to their key insight. "Pluripotency does not seem to represent a discrete cellular entity but rather a functional state elicited by a balance between opposite differentiation forces," says Izpisua Belmonte.

Once they understood this, they realized the four extra genes weren't necessary for pluripotency. Instead, it could be achieved by altering the balance of "lineage specifiers," genes that were already in the cell that specified what type of adult tissue a cell might become.

"One of the implications of our findings is that stem cell identity is actually not fixed but rather an equilibrium that can be achieved by multiple different combinations of factors that are not necessarily typical of ESCs," says Ignacio Sancho-Martinez, one of the first authors of the paper and a postdoctoral researcher in Izpisua Belmonte's laboratory.

The group was able to show that more than seven additional genes can facilitate reprogramming to iPSCs. Most importantly, for the first time in human cells, they were able to replace a gene from the original recipe called Oct4, which had been replaced in mouse cells, but was still thought indispensable for the reprogramming of human cells. Their ability to replace it, as well as SOX2, another gene once thought essential that had never been replaced in combination with Oct4, demonstrated that stem cell development must be viewed in an entirely new way.

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Salk Scientists Discover More Versatile Approach to Creating Stem Cells

Turning human cells into stem cells without changing their genes could lead to therapies that do not carry a risk of generating mutations.

Andrew Brookes/Corbis

Scientists have demonstrated a new way to reprogram adult tissue to become cells as versatile as embryonic stem cells without the addition of extra genes that could increase the risk of dangerous mutations or cancer.

Researchers have been striving to achieve this since 2006, when the creation of so-called induced pluripotent (iPS) cells was first reported. Previously, they had managed to reduce the number of genes needed using small-molecule chemical compounds, but those attempts always required at least one gene, Oct42, 3.

Now, writing in Science, researchers report success in creating iPS cells using chemical compounds only what they call CiPS cells1.

Hongkui Deng, a stem-cell biologist at Peking University in Beijing, and his team screened 10,000 small molecules to find chemical substitutes for the gene. Whereas other groups looked for compounds that would directly stand in for Oct4, Deng's team took an indirect approach: searching for small-molecule compounds that could reprogram the cells in the presence of all the usual genes except Oct4.

Then came the most difficult part. When the group teamed the Oct4 replacements with replacements for the other three genes, the adult cells did not become pluripotent, or able to turn into any cell type, says Deng.

The researchers tinkered with the combinations of chemicals for more than a year, until they finally found one that produced some cells that were in an early stage of reprogramming. But the cells still lacked the hallmark genes indicating pluripotency. By adding DZNep, a compound known to catalyse late reprogramming stages, they finally got fully reprogrammed cells, but in only very small numbers. One further chemical increased efficiency by 40 times. Finally, using a cocktail of seven compounds, the group was able to get 0.2% of cells to convert results comparable to those from standard iPS production techniques.

The team proved that the cells were pluripotent by introducing them into developing mouse embryos. In the resulting animals, the CiPS cells had contributed to all major cell types, including liver, heart, brain, skin and muscle.

People have always wondered whether all factors can be replaced by small molecules. The paper shows they can, says Rudolf Jaenisch, a cell biologist at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, who was among the first researchers to produce iPS cells. Studies of CiPS cells could give insight into the mechanisms of reprogramming, says Jaenisch.

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Stem cells reprogrammed using chemicals alone