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Embryonic stem cells derived from mammalian Blastocytes can grow indefinitely to maintain the pluripotency and the ability to differentiate into different germ layers. Therefore, human embryonic stem cells can be used to treat different diseases like Parkinson’s disease, diabetes, and spinal cord injury, etc. However, due to ethical difficulties and tissue rejection problems, a new strategy needs to be developed.
To cope up with these problems, researchers have tried to reprogram the somatic cells into the pluripotent cells by transferring the somatic cell nuclear content into oocytes or by fusing with embryonic stem cells containing factors that can induce the totipotency.
Transcription factors like Oct3/4, Sox2 and Nanog have been found to maintain the pluripotency of the early embryos and embryonic stem cells while some genes that upon upregulation leads to the tumor formation, such as Stat3, Klf4, β-catenin, E-Ras, etc. are also found to be associated with the long-term maintenance of the embryonic stem cells.
In the present study, researchers have examined whether these factors can induce pluripotency in somatic cells or not based on a hypothesis that these factors also play a vital role in the embryonic stem cell identity. The researchers evaluated 24 candidate genes using an assay that can detect the induction of pluripotency as a resistance to the G418 (geneticin).
Researchers inserted a fusion of β-galactosidase and neomycin resistance gene (βgeo construct) into the mouse Fbx15 gene through homologous recombination. Later on, it was found that embryonic stem cells with the βgeo construct (Fbx15βgeo/βgeo) were resistant to geneticin while somatic cells derived from Fbx15 βgeo/βgeo were sensitive to the geneticin indicating that partial activation of the Fbx15 leads to the development of resistance to the geneticin.
Researchers performed the same experiment using mouse embryonic fibroblasts in which they introduced the same number of candidate genes (24) from Fbx15βgeo/βgeo by retroviral transduction. However, in this case, they did not observe geneticin resistance in any of the colonies indicating that none of these candidate genes are able to activate the Fbx15 locus in the embryonic fibroblasts.
Among the geneticin-resistant colonies, upon further cultivation in the growth media, researchers were able to get some clones having morphological structures as well as proliferation properties similar to that of the embryonic stem cells. Later on, these cells were designated as iPS-MEF24 that mean pluripotent stem cells induced from Mouse Embryonic Fibroblasts by 24 factors.
Reverse Transcription PCR analysis of these clones revealed the presence of embryonic stem cell markers like Oct3/4, Nanong, E-Ras, etc. while Bisulfite genomic sequencing reveals that the promoters of the Fbx15 were demethylated and Oct3/4 promoters were in the methylated state in these cells.
Researchers examined the pluripotency of the induced pluripotent stem cells by teratoma formation in which they obtained tumors with different iPS-MEF clones by injecting these clones into the subcutaneous of the nude mice. By examining the histology of the iPS-MEF clones, researchers found that these clones were differentiated into all three germ layers.
However, some tumors of some iPS-MEF clones were composed of only undifferentiated cells, indicating that factors like Oct3/4, c-Myc and Klf4 can induce the expression of some of the embryonic stem cell marker genes are not able to induce the pluripotency.
In the next step, researchers introduced four selected factors into the tail-tip fibroblasts of male Fbx15βgeo/βgeo mice of 7-week old and a 12-week old female Fbx15βgeo/βgeo mouse where they obtained three colonies resistant to the geneticin from which they established the induced pluripotent stem cells. Induced pluripotent stem cells obtained from these mice were transplanted into the nude mice where they produced tumors with all three germ layers.
In conclusion, Oct3/4, Sox2, and Nanog are the core transcription factors that maintain the pluripotency while Oct3/4 and Sox2 are essential in the formation of induced pluripotent stem cells.
Article doi: 10.1016/j.cell.2006.07.024