Tumor Suppressor Genes Provide Clues to Reprogramming

Researchers are finding ways to make more induced pluripotent stem cells, faster. The catch: it requires turning off some of the cell's key tumor suppressors—a risky proposition for cells researchers hope to someday transplant into patients.

Manuel Serrano and colleagues at the Spanish National Cancer Research Centre in Madrid reported in the August 27 Nature that the Ink4/Arf locus, which encodes three tumor suppressors, acts as a barrier to reprogramming differentiated cells into induced pluripotent stem (iPS) cells (Li et al., 2009: PMID19668188). Knocking down that barrier, via gene knockouts or RNA interference, resulted in higher yield from reprogramming.

"This could be critical for the reprogramming of difficult cells, such as those from old individuals," Serrano wrote in an e-mail to StemBook. Current protocols with the Yamanaka factors Oct4, Sox2, Klf4 and cMyc (Takahashi and Yamanaka, 2006 PMID16904174) induce pluripotency in 0.01-0.2% of cells, so improving efficiency will be key in making iPS cells a commercially and medically viable option.

Another ongoing effort is to swap the genetic changes in the current iPS protocols for small molecules that will induce pluripotency without permanently altering the cell's DNA. Researchers have come up with a few alternatives to Sox2 expression, and drugs that temporarily inhibit barriers such as Ink4/Arf expression may follow soon.

The Ink4/Arf locus acts on two anti-tumor pathways: the paralogs p16-Ink4a and p15-Ink4b inhibit cell cycling upstream of the tumor suppressor Rb, and p19-Arf indirectly promotes activity of the tumor suppressor p53. All three are markers of senescence that increase with age (Krishnamurthy et al., 2004 PMID15520862). Ink4a appears to be the most important of the trio in preventing cancer and reprogramming in humans, while Arf is the key factor in mice.

Senior author Serrano, first author Han Li, and colleagues found that all three genes were downregulated in mouse embryonic stem and iPS cells. Compared to mRNA levels in differentiated embryonic fibroblasts, Ink4a, Ink4b and Arf mRNAs were reduced by approximately 80%.

The researchers surmised that if reduced expression of the Ink4/Arf locus was crucial to generation of iPS cells, artificially silencing the genes might improve reprogramming efficiency. Indeed, reprogramming mouse embryonic fibroblasts from Ink4a/Arf knockout animals resulted in 15-fold more iPS cells than wild-type cells, based on staining for the iPS marker alkaline phosphatase. With newborn keratinocytes as the starting material, the increase rose to 100-fold. The reprogramming was also faster, with alkaline phosphatase activity rising just three days after adding Yamanaka factors to the Ink4a/Arf knockout embryonic fibroblasts, as opposed to after day four for wild-type cultures. With human fibroblasts, silencing Ink4a with small hairpin RNA resulted in up to sixfold higher efficiency of reprogramming. RNA interference for Arf, less important in human cells, made no difference.

Serrano's paper was one in a series of five Nature letters implicating the p53 and Rb pathways as barriers to reprogramming. Another group confirmed that reduced expression of p19-ARF improved reprogramming yield and speed in mouse fibroblasts (Utikal et al., 2009 PMID19668190). Others showed that p53 limits reprogramming of cells with DNA damage (Marión et al., 2009 PMID19668189), and that knocking out p53 improved efficiency of reprogramming (Kawamura et al., 2009 PMID19668186), with induced pluripotency in up to 10% of transduced cells (Hong et al., 2009 PMID19668191).

A 2008 report, from scientists who screened several factors to discover improvements to the iPS protocol, also described silencing of p53 boosting reprogramming. When combined with augmented expression of UFT1, a transcriptional repressor that interacts with OCT4 and SOX2, p53 RNA interference resulted in more than 200-fold higher efficiency reprogramming (Zhao et al., 2008 PMID18983962).

"Reprogramming triggers protective, 'anti-reprogramming' responses with a striking parallelism to the protective responses that prevent oncogenic transformation," Serrano wrote. The key to efficient reprogramming, then, may be to convince cells to lift their restrictions on cell division for a short while, and then reactivate those anti-cancer barriers upon differentiation.

The link between reprogramming and cancer makes sense, said Ned Sharpless of the University of North Carolina, who was not involved in the recent flurry of papers. "A lot of these stimuli to reprogramming look like oncogenes," he noted. "Some aspects of the cancer transformation may look a little like reverting to a less-differentiated state." For example, alterations of myc, one of the four Yamanaka factors, are frequently implicated in cancer.

While other pathways may also be involved in blocking reprogramming, Sharpless suspects the p53 and Rb systems are the most crucial. He reasoned that the same pathways stall cell division in any cultured cell line, perhaps in response to the highly unnatural environment of a plastic dish at 20% oxygen, submerged in artificial media and fetal calf serum.

Ultimately, researchers want to put reprogrammed cells back into the people they came from, and scientists must consider their target patient population. "When the stem cell revolution comes, most of these people are going to be older," Sharpless said. Since the Ink4/Arf locus increases its expression with age, it could be a more significant barrier in the very cells doctors will most want to reprogram.

Accordingly, Serrano and colleagues looked at the impact of the Ink4/Arf locus in older cells. When they compared skin fibroblasts from two-month-old mice to the same cells from mice aged two years or more, they found approximately double the levels of Ink4a, Ink4b, and Arf mRNA in the older animals. Not surprisingly, older cells produced half as many iPS cells as younger fibroblasts. Treating those older cells with shRNA for Ink4a and Arf brought the efficiency up to young-cell levels.

This research provides some clues as to how to build a better reprogramming protocol. But there's a huge caveat: "Messing with p53 is dangerous, because its absence (even if transient) may facilitate the emergence of apparently normal iPS carrying genetic aberrations," Serrano wrote.

The best route, Sharpless suggested, would be to supplement culture media with drugs that inhibit p53, Ink4a/b or Arf for the minimum time necessary. Some relevant inhibitors, such as pifithrin for p53, already exist. Other small molecules, such as the histone deacetylase inhibitor valproic acid (Huangfu et al., 2008 PMID: 18568017) and DNA methyltransferase inhibitor 5-aza-cytidine (Mikkelsen et al., 2008 PMID: 18509334), have already been shown to improve reprogramming rates.

Ultimately, doctors will want to avoid all genetic changes in cells that will be returned to patients, and scientists are making progress with drugs that mimic the Yamanaka factors themselves. For example, researchers can induce pluripotency without the transgene Sox2 if they add the small molecules BIX-01294, a histone methyltransferase inhibitor, and Bayk8644, a calcium channel agonist, to the culture (Shi et al., 2008 PMID: 18983970).

In a new paper, posted online by Cell Stem Cell October 8, researchers from the Harvard University laboratories of Kevin Eggan and Lee Rubin in Cambridge, Massachusetts, report on another small molecule that can stand in for Sox2 expression (Ichida et al., 2009 not in PubMed yet; DOI: 10.1016/j.stem.2009.09.012). The researchers screened 200 drugs for compounds that allowed reprogramming with only Oct4, Klf4, and cMyc transgenes. The drug they have christened RepSox, for Replacement of Sox2, blocks Tgf-β signaling and induces expression of Nanog, a stem cell transcription factor.

Sharpless predicts that researchers will succeed in improving reprogramming efficiency with small molecules that act on the p53 and Rb pathways within six months.

References

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