This piece, by Elie Dolgin, builds on the more-general article now up on Nature News.
The tumor suppressor gene p53 is usually thought of as a master regulator that helps stave off cancer, but it’s also a major barrier to cellular reprogramming. Blocking the p53 pathway vastly improves the efficiency of transforming differentiated cells into induced pluripotent stem (iPS) cells and with fewer genes than the commonly used reprogramming recipes, new research shows. The findings, which should make it easier to derive patient-specific stem cells from any tissue, provide a bridge between tumour formation and cellular reprogramming that could force a rethink about cancer development.
The p53 pathway was first linked to reprogramming five years ago when Japanese researchers showed that mouse germ cells spontaneously formed embryonic-like stem cells in the absence of p531. Then last year, a Chinese team found that a combination of knocking down p53 with RNA interference and adding a little-studied transcription factor called Utf1 greatly increased human iPS cell numbers, but it was unclear why2. Now, five studies published online in Nature report that the p53 pathway alone acts to thwart cells from reverting back to a stem-like state. After disabling the pathway, the researchers saw their reprogramming rates soar more than 100-fold compared to most standard — and woefully inefficient — techniques.
“All of these papers add mechanistic information and corroboration that if you modify the p53 pathway somewhere along the way then it contributes to a very significant improvement in the derivation efficiency,” says George Daley of Children’s Hospital Boston, who was not involved in the research.
Using p53 knockout mouse cells and retroviruses containing the four most commonly used transcription factors — Oct4, Sox2, Klf4, and c-Myc — Kyoto University’s Shinya Yamanaka coaxed up to 20% of embryonic fibroblasts to form fully fledged iPS cells. Yamanaka also omitted c-Myc and found he could still reprogram up to 10% of cells with just the three remaining factors. Immobilizing p53 also improved Yamanaka’s reprogramming success with a safer, plasmid-based technique and allowed his team to transform mouse T-lymphocytes — terminally differentiated cells that have proven difficult to reprogram. 3 Going one factor further, Juan Carlos Izpisúa Belmonte of the Salk Institute for Biological Studies in La Jolla, California, and at the Center of Regenerative Medicine in Barcelona, Spain, successfully obtained iPS cells with only two transgenes — Oct4 and Sox2 — when p53 levels were reduced.4
In addition to demonstrating enhanced efficiencies, Konrad Hochedlinger of the Massachusetts General Hospital in Boston showed that handicapping p53 speeds up the reprogramming process. Hochedlinger inactivated either p53 or Ink4a/Arf, a locus that encodes two tumour suppressors that interact with p53, and found that iPS cells developed in only 3 or 4 days — around half the time normally needed5. Finally, two papers authored by Manuel Serrano and Maria Blasco of the Spanish National Cancer Research Centre in Madrid showed that eliminating the anti-tumor pathway improved the reprogramming of cells from older organisms,6 as well as cells with heavy DNA damage or truncated telomeres.7 Blocking the p53 pathway also improved human cell reprogramming, several of the research teams reported.
“We’re starting to unveil the black box of reprogramming,” says Izpisúa Belmonte. His data showed that the p53 pathway normally switches on after the reprogramming factors, which induce DNA damage, are introduced. The p53-associated genes, when intact, then might serve as gatekeepers to eliminate all but the healthiest cells bound for pluripotency. “Only those that are pristine with no DNA damage at all are those that will be able to undergo reprogramming,” says Serrano. In this way, says Blasco, “p53 would be like a very important quality control for getting damage-free iPS cells.” Thus, she adds, modulating p53 introduces a tradeoff between improving efficiencies and deriving only the best possible stem cells, so it might be unwise to knock down p53 when generating stem cells intended for the clinic. But in some cases, notes Hochedlinger, improved reprogramming rates might be a prerequisite to obtaining any iPS cells at all. “If you think about making patient-specific stem cells, you are often confronted with the situation where you have very little material,” he says. With such small samples, targeting the p53 pathway should allow researchers to obtain stem cells “reproducibly and efficiently,” he says.
Silencing p53 should also help build cellular models of many human diseases that have so far proven refractory to reprogramming. For example, Daley’s lab has attempted to derive patient-specific iPS cells for around 50 different diseases, but with only an 80-90% success rate. “That still leaves 10 to 20% unexplained,” Daley says. “By abrogating p53, I think maybe we can get up to 95%.”
The p53 link between pluripotency and cancer could challenge the widely held view that only a subset of specialized cells can trigger tumourigensis, says Izpisúa Belmonte. If most adult cells can be made into iPS cells in the absence of p53, then almost any p53-lacking cells should also be an eligible candidate for cancer initiation — a prediction that runs counter to the cancer stem cell hypothesis. “This set of papers hints that maybe that could be the case,” Izpisúa Belmonte says.
Jacob Hanna of the Whitehead Institute in Cambridge, Massachusetts, notes that although the five new papers offer clues as to why the p53 pathway hinders reprogramming, they don’t shed much new light on how exactly the cells that get past the p53 roadblock go on to become pluripotent. “It’s beneficial to be able to more easily derive iPS cells,” he says, “but we still don’t why we are getting these iPS cells, basically.”
1. Kanatsu-Shinohara, M. et al. Generation of Pluripotent Stem Cells from Neonatal Mouse Testis. Cell 119, 1001-1012 (2004).
2. Zhao, Y. et al. Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell 3, 475-479 (2008).
3. Hong, H. et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature advance online publication doi:10.1038/nature08235 (2009).
4. Kawamura, T. et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature advance online publication doi:10.1038/nature08311 (2009).
5. Utikal, J. et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature advance online publication doi:10.1038/nature08285 (2009).
6. Li, H. et al. The Ink4a/Arf locus is a barrier for iPS cell reprogramming. Nature advance online publication doi:10.1038/nature08290 (2009).
7. Marión, R. M. et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature advance online publication doi:10.1038/nature08287 (2009).