Two separate research teams have published papers describing the creation of a novel cassette for reprogramming stem cells to achieve induced pluripotency. The research was conducted by Rudolph Jaenisch and colleagues at the Whitehead Institute of the Massachusetts Institute of Technology, and Konrad Hochedlinger and colleagues at Harvard Medical School. Hochedlinger was a member of the Jaenisch laboratory for over five years, completing his Ph.D. and conducting post doc work in epigenetics and stem cell biology.
“Here we used gene targeting to generate a mouse strain with a single copy of an inducible, polycistronic reprogramming cassette, allowing for the induction of pluripotency in various somatic cell types,” wrote Hochedlinger’s team in their paper.
Traditionally, creating induced pluripotent stem cells (iPS cells) requires inserting the reprogramming factors Oct4, Sox2, Klf4, and c-Myc in separate steps. Inserting four reprogramming factors requires the use of multiple retroviruses, which place the factors in random locations in the mouse genome. This can interfere with the function of the mouse’s own genes. The mice, therefore, must be screened from generation to generation for the reprogramming genes.
“Although secondary models have been used to generate transgenic mice carrying all or a subset of reprogramming factors, it remains difficult to maintain all factors in one mouse because the multiple transgenes segregate in each generation,” wrote Jaenisch’s team in their paper.
To expedite the process, the two research teams undertook the same protocol to insert all the reprogramming factors at once. First, all four reprogramming factors were placed on a single piece of DNA—called a cassette. Prior to breeding the mice, the cassette—referred to as 4F2A in the Jaenisch paper and OKSM in the Hochedlinger paper—was inserted at a single locus in the mouse genome, the untranslated region of the collagen type I, alpha 1 gene (Col1a1). By administering all four reprogramming factors at a single locus without the help of retroviruses, the stem cell cassette eliminated the randomized location of the reprogramming factors. Finally, the antibiotic doxycycline was administered to the mice to trigger the cassette to express all four reprogramming factors at once, which caused the somatic cells in the mice to be reprogrammed into iPS cells. Therefore, the stem cell reprogramming cassette bypasses the problems of the traditional reprogramming method.
In their research, the Jaenisch laboratory observed that some donor cell types—including intestinal epithelial cells, mesenchymal stem cells, pro-B cells, and tail-tip fibroblasts—produced iPS cells only in mice with two copies of the transgene. “These cell types require higher amounts of factor expression for reprogramming than other cell types, such as MEFs or hepatocytes,” the team wrote.
They concluded that inserting the reprogramming cassette at Col1a1 has the potential for being widely used to create iPS cells. “We observed no tumors or other adverse effects in the transgenic mice suggesting that the Col1a1 4F2A system can be both safe and practical for extended breeding,” said the team.
However, the Hochedlinger laboratory did find tumor growth in their test subjects. “Notably, some reprogrammable mice developed a single aggressively growing tumor that histologically presented as a largely undifferentiated teratoma,” wrote the team in their paper. They concluded that this did not preclude the Col1a1 reprogramming cassette from being used to reprogram stem cells in the future. “This phenotype is likely due to leaky transgene expression in an undefined cell type,” they wrote, “and raises the possibility that somatic cells can be reprogrammed into pluripotent cells not only in vitro but also in vivo.”
Both the 4F2A and OKSM stem cell cassettes reduce the number of steps in the reprogramming process, and also facilitate the breeding of mice to have the reprogramming factors at the Col1a1 locus. “As these ‘reprogrammable mice’ can be easily bred, they are a useful tool to study the mechanisms underlying cellular reprogramming,” wrote Hochedlinger’s team.
The papers, “A reprogrammable mouse strain from gene-targeted embryonic stem cells,” from the Hochedlinger lab and “Single-gene transgenic mouse strains for reprogramming adult somatic cells,” from the Jaenisch lab, were published online on Dec. 13 online at Nature Methods.