Most of our somatic cells are highly differentiated and they are not able to divide and regenerate our body. Some specialised cells are able to divide, but huge step in human medicine would be discovery of genes which can turn somatic cells to a pluripotent state.
Discovery of a master gene, which can program somatic cells to a pluripotent state, would lead to great progress for all of modern human regenerative medicine; it would obviate the need for human cloning, with all its ethical or moral implications. Pluripotent stem cell is able to give rise to differentiated derivatives of all three germ layers. Cells of the inner cell mass and embryonic stem cells are pluripotent.
Several years ago was demonstrated that pluripotent embryonic stem cells could be generated from both embryonic and adult mouse fibroblasts by retroviral conversion of four genes: Oct4, Sox2, Klf4 and c-Myc. The induced pluripotent stem cells, displayed morphology and growth properties typical of embryonic stem cells. Global gene-expression profiling of induced pluripotent stem cells discovered that these cells cluster more closely with embryonic stem cells than with fibroblasts. The different analysis showed genes which were expressed at higher levels in embryonic stem cells than in induced pluripotent stem cells. Oct4 was found partially methylated, or incompletely reprogrammed in induced pluripotent stem cells. When the pluripotent potential of the cells was tested, it turned out that, even though induced pluripotent stem cells were prepared for multi-lineage differentiation capability in vitro, in vivo they could contribute to fetal but not to adult mouse development. This was probably due to the fact that expression of all four factors was driven by constitutively active promoters, which are not able to mediate transgene down-regulation throughout differentiation.
Induced pluripotent stem cell isolation from drug selected fibroblast which carries a neomycin gene inserted within the Oct4 locus resulted instead in the generation of induced pluripotent stem cells with gene expression, chromatin, and DNA methylation characteristics. Oct4 in induced pluripotent stem cells displayed increased developmental potency, which was identical to the one showed by embryonic stem cells. Expression analysis of transduced and endogenous Oct4, Sox2, Klf4 and Myc genes showed that retroviral transgenes are silenced during the induction of Oct4 induced pluripotent stem cells. This result indicates that exogenous Oct4, Sox2, Klf4, and Myc are essential for the induction of embryonic stem- like characteristics, however that the activity of the endogenous genes must be engaged in order to achieve pluripotency and full differentiation potential. But, induced pluripotent stem cells derived animals developed tumors, probably due to the reactivation of the some transgenes, like Myc transgene. Successful reprogramming of fibroblasts was shown to be accomplished without c Myc, but evidence that induced pluripotent stem cells derived by Oct4, Sox2, and Klf4 transduction will not induce tumor formation in adult mice is still missing.
Induced pluripotent stem cells can be isolated from non-transgenic fibroblasts by using exclusively morphological criteria for the recognition of embryonic stem- like colonies after viral transduction. This methodology provides a rate of reprogramming efficiency higher than observed using drug selection. Fetal and adult fibroblasts are not the only differentiated somatic cell type that can give rise to induced pluripotent stem cells. Non-terminally differentiated cells were shown to be reprogrammed to a pluripotent state by inducible expression of the four factors, while mature cell reprogramming needed furtheter genetic manipulation.
The scientists came to the brilliant conclusion. When the process of mouse induced pluripotent stem cells derivation was monitored at totally different time-points for pluripotency marker gene expression, alkaline phosphatase first, SSEA1, and at last Nanog and Oct4 were consistently found to be switched on in a sequential temporal order. This knowledge would point towards the existence of an iter of gene reprogramming which is defined and gradual, rather than chaotic. Further confirmation of this hypothesis was given by a comprehensive integrative genomic characterization of cells during induced pluripotent stem cells generation. Cells were transduced with inducible viral vectors encoding for the four factors and sampled at day 4, 8, 12, and 16 of initial induction for expression profiling.
The therapeutic potential of induced pluripotent cells was tested in a sickle cell anemia mouse model. Autologous induced pluripotent stem cells were generated from skin cells of a diseased animal, targeted for correction of the endogenous human sickle hemoglobin gene and induced to differentiate into hematopoietic progenitors. The efficient treatment of sickle cell anemia obtained by transplantation of these host specific cells into the diseased mouse provides a proof of principle for the clinical achievements that combined gene and cell therapy can lead to. Significantly, induced pluripotent cells have also been derived from human fetal, neonatal, and adult fibroblasts by ectopic expression of the same four-factor cocktail that yielded mouse induced pluripotent stem cells. Unfortunately, genetic manipulation of cells brings inevitable drawbacks: ectopic expression of tumor suppressor genes causes tumorigenicity, and random insertion of the viral sequences within the genome may produce unwanted mutagenesis events. Nonetheless, an essential step forward in coupled gene repair plus cell therapy has been made. We have learned that the fundamental transcriptional network governing pluripotency in humans and mice is conserved, regardless of the differences between the two species for growth factor requirements. As of these days, the complete image eventually appears in its amazing completeness, thanks to scientists who apply the missing piece of the puzzle and obtained induced pluripotent stem cells by the ‘simple’ protein transduction of the four factors. Some ethical issues, such as failure rate, problems during later development, and abnormal gene expression patterns may still represent the problem, but with further advances in medicine and technology will solve these issues as well as ethical and moral.