Nuclear reprogramming

The reversal of the differentiated state of a mature cell to one that is typical of the undifferentiated embryonic state is known as nuclear reprogramming (NR). Successful reprogramming may have many applications in regenerative medicine. For example, altering cell fate would be beneficial for producing isogenic replacement cells for the treatment of a variety of life-threatening diseases. It would also reduce the limitations of current cell therapeutic approaches resulting from graft rejection, use of immunosuppressants and the safety and ethical issues raised by the use of cells of embryonic foetal or animal origin.

Several approaches are being examined to generate cells with embryonic stem (ES) cell-like characteristics. Results obtained using somatic cell nuclear transfer demonstrates that NR is possible. Using this technology, pluripotent embryonic stem cells have been derived from blastocyst stage embryos and cloned animals have been produced.

The use of cellular fusion, which requires the fusion of somatic cells with ES, embryonal carcinoma (EC) or embryonal germ (EG) cells, has also been utilised to gain further information regarding NR. It has been shown that when mouse thymic lymphocytes are fused with EG cells, demethylation of several non-imprinted genes in the thymocyte nucleus takes place. These EG-thymocyte heterokaryons display pluripotential cell-like properties. More recently, the retrovirus mediated introduction of four transcription factors was also used to induce ES cell-like properties in mouse fibroblasts. Three different groups generated induced pluripotent stem (iPS) cells. These are similar to ES cells in terms of morphology, proliferation and teratoma formation.

Another method of reprogramming requires the use of cellular extracts. This system consists of incubating purified somatic nuclei or reversibly permeabilised somatic cells in an extract of another differentiated cell type or of undifferentiated EC cells. The rationale behind this methodology is that the extract will provide all the necessary regulatory components that mediate alterations in the gene expression and protein expression profile of the target genome.

In 2005 Taranger et al reported that epithelial 293T (human embryonic kidney epithelial cells line) cells treated with extract of undifferentiated human embryonic carcinoma (NCCIT) cells induces expression of pluripotency genes such as OCT4 and NANOG and causes down-regulation of somatic cell specific genes like lamin A (LMNA). Recently, the same group demonstrated that chromatin relaxation at pluripotency associated loci is central to the reprogramming process.

All reprogramming approaches to date show that chromatin remodelling factors, DNA and histone modifications and alterations in gene expression are involved in the process of NR. However, little is known about how the proteomic profile of a somatic cell/ nuclei changes due to the effects of reprogramming. Our current investigation aimed to characterise the proteomic profile of 293T cells that were treated with cellular extract of NCCIT cells and have undergone reprogramming, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We hypothesised that those proteins that are altered in these reprogrammed 293T cells compared to the original 293T cells but display comparable expression values to that of NCCIT cells are the potential protein candidates that confer the reprogramming phenomena.