iPSC Learning

The cells used in this experiment were embryonic fibroblasts belonging to mice. These cells were used to test the 24 potential transcription factors hypothesized to have the potential to turn non-stem cells into ones with characteristics relating to pluripotency.

 

In order to create a pluripotent cell, retroviruses were used to transfer the specific transcription factors to near the Fbx15 gene in the mouse fibroblast chromosomes. Along with the transcription factor, a gene for antibiotic resistance to G418 was included with the retrovirus. Thus, determining whether a cell could be embryonic-like or not depended on whether the infected cells demonstrated antibiotic resistance when exposed to G418.

 

When all 24 transcription factors were added to determine if any of them were viable, a significant amount of colonies demonstrated antibiotic resistance. Thus, the problem remained on narrowing down which of the 24 factors could be identified to contribute to these cells (iPS-MEF24). In order to determine the necessary factors, various lesser combinations were also tested for antibiotic resistance. As a result, the list of 24 factors was able to be narrowed down to a list of 10, which resulted in iPS-MEF10 cells. These 10 were able to be further narrowed down to a smaller subsection of factors, which you will have the opportunity to explore and determine in the game surrounding Yamanaka’s Experiment.

 

Researchers were able to further confirm that the iPS cells were embryonic or stem cell-like from a series of additional tests on the properties. When the infected cells were injected into the mice, teratomas (tumors with cells from all three germ lines - cartilage, muscle, neural, etc.) would form, which is the same response as if an embryonic stem cell would have been injected. Given the similarities in responses, researchers concluded that the iPS-MEF cells were similar to embryonic stem cells and were pluripotent in the mice.