Question

how did yamanak find the magic four transcription factors?

Answer 1

Shinya Yamanaka and his postdoctoral researcher Kazutoshi Takahashi in 2006, programmed mice fibroblast to become induced pluripotent stem cells (iPSCs). These stem cells could be programmed to from different cells in the body. Yamanaka was working at Kyoto University to find new ways to acquire human stem cells as use of human embryos for obtaining stem cell has ethical constraints. Yamanaka’ work was based on John Gordon’s work showing that mature cells from Xenopus (frog) could be reprogrammed. Further, somatic cell nuclear transfer could reprogram nucleus of somatic cells to produce Dolly, the sheep provided critical evidence that adult cells can be reprogrammed.

Twenty four genes were initially selected by Yamanaka and Takahashi on the hypothesis that they can induce pluripotency. These genes were tested one at a time. Retroviruses were used to insert each of the genes into into differentiated mouse embryonic fibroblasts chromosomes. Each gene was individually inserted near the mouse Fbx15 gene expressed during development in embryonic stem cells. This gene inserted has resistance G418 antibiotic gene segment inserted next to it. Resulting cells with retroviruses that were mixed with the host DNA (Retroviral factors), were cultured in media with G418 along with mouse feeder cells. However, none of the cells could show resistance to G418 antibiotics.

In order to overcome this negative result, Yamanka inserted multiple retroviral factors into cells simultaneously. When all 24 factors were added simultaneously into mouse fibroblast cells, initially only 22 cells were obtained that has G418 resistance. These cells had embryonic stem cell-like properties and were called iPS-MEF24.

Next, the researchers removed one retroviral factor from the cocktail of 24 factors at each time and infected the mouse fibroblast with the retroviruses containing the modified cocktail. If no G418 resistant cells were obtained when that factor was removed, then that factor is critical for iPSCs generation. From these experiments, they obtained 10 genes that can generate G418 resistant stem cells from mouse fibroblast (iPS-MEF 10). Further experimentation with these 10 factors, revealed that only four were critical for generating iPSCs. When Oct3/4, KIf4, Sox2, and c-Myc (iPS-MEF4) were inserted in mouse fibroblast using retroviruses, they formed most number of iPSCs. These cells were examined to see whether they induced teratoma formation by injecting them in nude mice lacking Fox1 gene, which is required for immune system generation. The teratomas formed were found to differentiate into the three germ layers- ectoderm, mesoderm, and endoderm. Cells from these tissue type aggregates or embryoid bodies were injected in mouse blastocysts that were put back in pseudopregnant recipients. Cloned progeny could be obtained, demonstrating that these iPS-MEF4 genes could induce pluripotent stem cell properties in mouse fibroblast.

Yamanaka continued these experiments in humans (2007) by injecting the four factors in human fibroblast. They could now generate human iPSC.