Research Highlights

Nature Reports Stem Cells
Published online: 14 February 2008 | doi:10.1038/stemcells.2008.36

Reprogramming kinetics

Monya Baker¹

Virally introduced genes must be active for more than a week to induce pluripotency

Multiple labs have now shown that adult cells can be reprogrammed into an embryonic-like state. A suite of virally introduced genes are necessary to kick-start the reprogramming process, but gradually the cell's own endogenous pluripotency genes become active, and the viral genes are silenced. Reporting in Cell Stem Cell, Rudolf Jaenisch and colleagues at the Massachusetts Institute of Technology, in Cambridge, Massachusetts, lay out a time frame for this activation and show that transgene expression is essential for at least 12 days. This insight could help researchers develop more efficient and nonviral approaches to reprogramming.

The researchers first showed that endogenous pluripotency genes must become active in a particular sequence, a finding that is consistent with other studies. For this, they used cell-sorting techniques, plus mouse cell lines engineered so that green fluorescent protein would be produced when one of two endogenous pluripotency genes (Nanog or Oct4) was expressed. The first gene to activate was that encoding alkaline phosphatase, at around three days; the next was the gene for stage-specific embryonic antigen-1 (SSEA-1), at around nine days. Nanog and endogenous Oct4 were detectable by cell-sorting machines at around 16 days. Even 21 days after culture, transgene activity greatly boosted reprogramming rates. Rates were still low, however, at well under 1%.

Next, the researchers explored whether activation of the endogenous genes depended on the activity of the transgenes. Using the four genes originally established for inducing pluripotency (Oct4, Sox2, Klf4 and c-Myc), the researchers created versions of these genes that could only turn on in the presence of the small molecule doxycycline.

Though the cells looked morphologically different as early as three days after infection, they always reverted to fibroblasts if doxycycline was removed from the culture within two weeks of delivery of the transgenes.

The researchers also created versions of the transgenes that could not be silenced and found that although transformed cells took on properties that are characteristic of embryonic stem (ES) cells, they did not differentiate when injected into mice, unlike ES cells or other induced pluripotent stem cells.

Jaenisch and his team are now exploring roles and relationships of different transgenes and trying to figure out why reprogramming cells through this method seems to take so much more time than reprogramming through nuclear transfer. "Many sequential events have to happen. We have to re-establish the core circuitry of pluripotency," says Jaenisch.

Author affiliation

1. Monya Baker is news editor of Nature Reports Stem Cells.