by Monya Baker
Small molecules replace pluripotency gene and Wnt boosts self-renewal and reprogramming
A trio of recent papers shows that differentiated cells can be persuaded more easily into pluripotency or self-renewal by either adding small molecules or stimulating a signalling pathway found in most differentiating cells.
Following on from previous work in neural progenitor cells, researchers led by Sheng Ding of the Scripps Research Institute in La Jolla, California, were able to reprogram mouse embryonic fibroblasts (cultured skin cells) using only two of the standard four pluripotency genes.
By adding a small-molecule inhibitor of histone methyltransferase, BIX-01294, along with the calcium-channel agonist BayK8644, Ding and his colleagues found that they needed only to insert the genes for Klf4 and Oct4 to create induced pluripotent stem (iPS) cells that passed a stringent test for pluripotency — when mixed with mouse embryos, the iPS cells could contribute to sperm formation in the resulting mice. At around 0.2% efficiency, reprogramming rates are similar to those found using the standard four factors.
The researchers identified this combination through a series of phenotypic screens of around 2,000 small molecules. They added the molecules to fibroblast cultures, then looked for the formation of colonies typical of embryonic stem (ES) cells and for expression of the ES cell marker alkaline phosphatase. BIX-01294 had been previously recognized to boost reprogramming, presumably by affecting epigenetic regulation of gene expression. In fact, it is also able to generate IPS cells even without BayK8644, though not as effectively. The screen also found a DNA methyltransferase inhibitor called RG108 that enhanced reprogramming in combination with BIX, but because this mechanism of action is precedented, the researchers chose not to pursue it. However, the researchers describe the identification of the booster activity of BayK8644 as "intriguing"; it is known to induce intracellular signalling and the release of calcium ions. How exactly the two molecules might interact to make the presence of the transcription factor Sox2 unnecessary for reprogramming is a matter for further study.
Cell signalling is also involved in two other papers showing exogenous boosting of rates of reprogramming or self-renewal. A team led by Roel Nusse and Irving Weissman at Stanford University in California found that adding the signal protein Wnt to culture media can boost clonal outgrowth in populations of neural stem cells and that inhibiting Wnt signalling stalls attempts to clone or expand cell cultures2.
Another team led by Maria Pia Cosma at the Telethon Institute of Genetics and Medicine in Naples, Italy, found that periodically activating Wnt signalling boosts the ability of ES cells to reprogram genomes from specialized somatic cells, including neural stem cells and thymocytes3. The team fused the somatic cells with ES cells, and then increased Wnt signalling either by adding Wnt directly or by inhibiting the enzyme glycogen synthase kinase 3, which is inactivated in the course of normal Wnt signalling.
The fused somatic nuclei were demethylated in patterns resembling those found in ES cells, and also began expressing known pluripotency genes. Intriguingly, reprogramming rates corresponded to the accumulation of a specific threshold of beta-catenin, the intracellular mediator of Wnt signaling. Furthermore, the researchers only observed this effect when Wnt signalling was boosted in newly fused cells or in ES cells, not in differentiated cells. Thus, the researchers conclude that Wnt signalling somehow activates the reprogramming capacity of pluripotent nuclei.
Source: Nature