Authors Chad Cowan, Doug Melton and Kevin Eggan
Most cells in our tissues have a limited ability to become other cell types in the body and therefore are thought of as being “terminally differentiated.” Animal cloning experiments, such as those that generated the sheep Dolly, have demonstrated that the mammalian egg can relieve these constraints and restore developmental potency to a cell, allowing it to instruct the development of an entire organism. The process by which the egg removes these constraints and reinstates developmental potency has been termed “reprogramming.” We study this process because it is of fundamental interest to us as developmental biologists, and because a thorough understanding of it might allow the direct conversion of adult cells into embryonic stem cells. Such embryonic stem cells might then be used as a source of replacement tissues for those lost in diseases such as Parkinson’s or diabetes.
While the egg is an important tool for the study of nuclear reprogramming, eggs are hard to isolate in large quantities, and it is difficult to use them to discover which genes are involved in this process.These problems are particularly acute if we wish to understand how human nuclei can be reprogrammed, so researchers have searched for alternative ways of inducing developmental potency in somatic cells.
Like the oocyte and early embryo, human embryonic stem (hES) cells have the capacity to differentiate into a variety of cell types. Because of this developmental potential, we wondered if hES cells themselves might also harbor reprogramming activities that could restore developmental potential to adult cells. As hES cells can be propagated indefinitely in vitro and their genome is readily manipulated, hES cells would provide an important alternative to oocytes for source of material for studying reprogramming.
To test whether hES cells could reprogram human somatic cells, we have fused hES cells with human skin cells and investigated the characteristics of the resulting “hybrid” cells. These experiments were carried out in collaboration with Chad Cowan, Jocelyn Atienza and MCB Profesor Douglas Melton, the Thomas Dudley Cabot Professor of the Natural Sciences and Investigator, Howard Hughes Medical Institute, and are reported in the August 26th issue of Science Magazine [Cowan, C.A., Atienza, J., Melton, D.A., Eggan, K. (2005). Nuclear Reprogramming of Somatic Cells After Fusion with Human Embryonic Stem Cells. Science 309: 1369-1373]. We found that the appearance, growth characteristics and developmental potential of these hybrids cells were identical to an hES cell, i.e., these cells showed the sought-after characteristics of hES cells. Importantly, our analysis demonstrated that the transcriptional activity and underlying epigenetic configuration of the adult nucleus had been reprogrammed to an embryonic state. Therefore, our results suggest that components of an hES cell can act to reprogram somatic cells, and that like eggs, hES cells are a powerful reagent for both executing and understanding this process.
In the immediate future, we will continue to characterize the process by which the hES cell reprograms the somatic nucleus and begin to test mechanistic aspects of reprogramming. Is DNA replication required for reprogramming of the somatic nucleus? Is transcription or translation required in the hybrid cell for modifying chromatin structure in the somatic chromosomes or can it be accomplished by pre-assembled complexes already present in the hES cells? With these mechanistic insights in hand, we plan to exploit the genetic and biochemical tractability of our system to identify the factors that exact these changes. For instance, it has been proposed that there are enzymatic activities that can remove 5-methyl-cytosine from DNA, and that these activities are necessary for reprogramming to occur. However, the molecular identity of demethylating activities remains unknown. We would seek to identify such factors and investigate their function in both reprogramming and mammalian development.