Triggering a pathway designed to sense viral infection can help boost generation of induced pluripotent stem cells (iPSCs), suggesting that the viruses used by many reprogramming methods influence the fate of the cell. The new research, published today (October 25) in Cell, suggests that targeting the pathway without using viruses could avoid the risk that the viruses’ genetic material will integrate into the genome and cause the cell to become cancerous—a common concern for iPSC therapies.
“This is something everyone else missed before,” said Gioacchino Natoli, an experimental oncologist at the European Institute of Oncology in Italy who was not involved in the study. An innate immune response to the viral vector is clearly needed for reprogramming, and “stimulating this leads to reprogrammed cells without exogenous DNA being integrated,” he added.
Six years ago, Shinya Yamanaka identified four key transcription factors, that, when transduced into cells using a viral vector, caused cells to de-differentiate into a stem-cell-like state, capable of generating a multiplicity of human tissues. He shared this year’s Nobel Prize in Medicine for the achievement, which has already made significant contributions to biomedical research. But there was a major hurdle on the path to clinical use: the strategy relies on a retrovirus that can integrate into host genomes, sometimes activating an oncogene that could render the cell cancerous. Injecting such cells as therapies thus raised concerns about the risk of patients developing tumors.
Hoping to devise a non-integrative carrier for Yamanka’s reprogramming factors, John Cooke, who studies vascular regeneration at Stanford University and colleagues created cell-permeant peptides (CPPs)—the transcription factors attached to an 11-amino–acid-long protein tag that allows them to pass through the cell membrane. But using CPPs to reprogram fibroblasts turned out to be a hundred times less efficient than using retroviral vectors. Compared to virally-transduced cells, CPP-programmed cells also expressed critical pluripotency target genes several days later and at much lower levels.
It occurred to Cooke’s postdocs Jieun Lee and Nazish Sayed that the viral vectors themselves might be sending an important signal to the fibroblasts the researchers were trying to reprogram. So they added a GFP-expressing viral particle during their CPP reprogramming protocol—and found that the virus boosted efficiency, even though the CPPs carried the transcription factors.
Sayed and Lee hypothesized that the viral particles were actually stimulating innate immune pathways in the fibroblasts. A key player in the activation of these pathways is the toll-like receptor 3 (TLR3), which is known to be activated by the double-stranded viral RNA (dsRNA) carried by the viruses. Sure enough, knocking down TLR3 reduced the efficiency of a retroviral reprogramming. Furthermore, adding a synthetic mimic of dsRNA during CPP reprogramming had the reverse effect, boosting the number of human fibroblasts that completed the transition to iPSCs.
The researchers were even able to nail down the mechanism. It turns out that TLR3 stimulation leads to epigenetic changes that allow easier transcription factor binding to their target genes.
“In fibroblasts, much of the genome is in a closed conformation, so transcription factors have trouble accessing promoters,” explained Cooke. “But activation of innate immunity [by TLR3] puts chromatin in an open configuration so transcription proteins work.” It may also be possible to target the chromatin directly, he noted.
It’s not the first time epigenetic marks have been fingered as key players in cellular reprogramming, and it’s probably not the last. “It will be interesting to use genome-wide epigenetic studies to elucidate the epigenetic similarity between embryonic stem cells (the gold standard of pluripotency) and reprogrammed cells in the presence and absence of active TLR3 stimulation,” Kitai Kim, a cancer and stem cell biologist at Memorial Sloan-Kettering Cancer Center, wrote in an email.
Additionally, the work may have implications for the origin of cancer. “iPSC reprogramming and tumorigenicity share noticeable levels of the similarity,” said Kim, who did not participate in the research. Cooke and his colleagues agree and are planning to study how inflammation may help create an open chromatin state that enables new differentiation pathways. “The state of chronic inflammation is often associated with cancer,” Cooke noted, speculating that inflammation may push chromatin toward a plastic state that enables malignant transformation.
Jieun Lee et al., “Activation of innate immunity is required for efficient nuclear reprogramming,” Cell, 151: 547-558, 2012.