Novel technique for manipulation cell fate
The demonstration that simply by introducing four selected proteins it is possible to change mammalian somatic cells from one phenotype to another is providing important new opportunities in medicine. However, this approach has several limitations. In contrast to other methods of changing cell fate such as cloning and cell fusion it is very slow, very inefficient and it is necessary to have identified the key transcription factors. In both cloning and fusion the nucleus is exposed to the cytoplasm of the recipient cell and it is this that changes nuclear function. With the hope of creating the same effect, extracts from cells of the desired cell type have been introduced into candidate cells. This induced some changes in cell function, but did not change cells from one phenotype to another. The aim of this project was to improve methods for the introduction of extract when human skin cells were exposed to extracts of mouse pluripotent stem cells. During early studies, it was noticed that external materials could enter cells spontaneously at a specific stage of cell cycle, metaphase. When cells in metaphase were exposed to cell extracts pluripotent cell-like colonies were formed. These cells expressed markers of pluripotency such as SSEA4 and Tra-1-60 and could form embryoid bodies that would further differentiate to all three germ layers. Unfortunately this protocol was found to be unrepeatable. In subsequent studies the temperature of exposure to extract was raised from 37oC to 40oC with the aim of promoting the fluidity of the cell membrane and so enhancing uptake of extract. A new treatment regime was introduced to increase the proportion of cells exposed to extract while in metaphase. A new protease inhibitor was introduced in order to promote persistence of the extract within the cells. Also, a new TLR3 agonist was introduced to enhance chromosome modification. After these modifications were made pluripotent cell-like colonies formed within 14 days of treatment with extract and these colonies were positive for alkaline phosphatase live staining. Further research is required to complete the development of a routine procedure.