Stem cells: Inducing pluripotency

The progression from pluripotent to somatic cells typically occurs through a process called differentiation, where pluripotent cells undergo specific genetic and epigenetic changes that restrict their potential and lead them to develop into specialized somatic cells with distinct functions and characteristics. However, recent research indicates that genetic factors can induce pluripotency in somatic cells.

To explore this, researchers developed an assay system in which the induction of the pluripotent state could be detected as resistance to the antibiotic G418. They inserted a βgeo cassette (a fusion of the β-galactosidase and neomycin resistance genes) into the mouse Fbx15 gene, which is specifically expressed in mouse embryonic stem (ES) cells. ES cells homozygous for the βgeo knockin construct (Fbx15${}^{\beta geo/\beta geo}$‍) were resistant to extremely high concentrations of G418 (up to 12 mg/ml), whereas somatic cells derived from Fbx15${}^{\beta geo/\beta geo}$‍ mice were sensitive to a normal concentration of G418 (0.3 mg/ml).

Researchers introduced 24 candidate genes into mouse embryonic fibroblasts (MEFs) from Fbx15${}^{\beta geo/\beta geo}$‍ embryos by retroviral transduction. To determine which of the 24 candidates were critical, they examined the effect of withdrawal of individual factors from the pool of transduced candidate genes on the formation of G418-resistant colonies. Results are shown in Figure 1.

Figure 1 Colony numbers at day 10 and 16 post transduction

Researchers identified the 10 factors whose individual withdrawal from the bulk transduction pool resulted in no colony formation 10 days after transduction and fewer colonies 16 days after transduction in Experiment 1. They then examined the formation of colonies after withdrawal of individual factors from this 10-factor pool transduced into MEFs. They also compared results to MEFs transduced with all 24 of the original genes. Results are shown in Figure 2.

Figure 2 Colony numbers at day 16 post transduction

Researchers analyzed the role of the Oct3/4, Klf4, Sox2, and c-Myc genes, which correspond to factors 14, 20, 15, and 22, respectively. Figure 3 shows the number of G418-resistant colonies at day 16 in cells transduced with these four factors versus all 10 factors analyzed in Experiment 2, as well as the effects of removing one or two of these factors from the pool of four.

Figure 3 Colony numbers at day 16 post transduction