Nuclear reprogramming demonstrates the potential for reversability in cellular differentiation. Reprogramming introduces a small number of transcription factors into somatic cells, such as skin cells, transforming the skin cells into induced pluripotent stem cells (iPS), While a core set of factors necessary for reprogramming has been identified the process is extremely inefficient (less than .1% of cells exposed to factors are transformed). The impetus to improve the efficiency of reprogramming is high. Approaches in regenerative medicine, such as replacing organs and tissues lost to injury and disease, face the obstacle of donor cell rejection by the host. Nuclear reprogramming can create iPS cells that are genetically identical to that of the donor, thus allowing a patient to be the donor for an iPS cell line that may serve as starting material for replacement tissue and organs which will not be rejected by the patient. A key component to increasing the efficiency of reprogramming is a more comprehensive understanding of pluripotency. This would help to define more extensive genetic networks and additional players that could be introduced or engineered to increase reprogramming efficiency.
Much of the information gained from studying pluripotency has been done so using genomic approaches; such as DNA microarrays or "chips", and variations on chip experiments such as chIP-chip, chromatin immunoprecipitation followed by chip analysis, and more recently bio-chIP and bio-chIP-chip which is in vivo biotinylation mediated chIP coupled with target mapping (chip). The significant findings achieved using these approaches reflect the shifting landscape of how biological qustions are being asked-on a global rather than gene by gene level. Innovative and improved methods are enabling us to understand the extensive gene and protein networks involved in pluripotency which will, in turn, inform reprogramming approaches.
Recently, much attention has been focused on a small suite of transcription factors including the "reprogramming factors" (Oct4, Sox2, Klf4, and c-Myc) and others, such as Nanog, found to be required to maintain cells in a pluripotent state (Stadtfeld et al., 2008). In a recent paper by Stu Orkin's lab at the Harvard Stem Cell Institute and Dana Farber Cancer Center (Kim et al., 2008), they explored the molecular basis of these requirements using a powerful bio ChIP (in vivo-biotinylated chIP) and bio chIP-chIP approach, which gets around some of the limitations of many antibodies in traditional chIP experiments.
Orkin's group identified target promoters of nine of the reprogramming factors on a global scale in embryonic stem cells. They were able to examine their promoters and distinguish two classes of target genes based on the requirements to activate or repress them suggesting differential regulation of these targets related to the factors occupying the promoter. While other groups have used chip based approaches to probe the downstream targets of Oct4, Sox2,and Nanog in mouse and human embryonic stem cells, the studies by Orkin and colleagues expand the set analyzed to nine transcription factors, examined on a global scale and provide a background with which to ask more detailed questions about the reprogramming activities of these factors.
Another recent study, also taking a whole genome approach toward understanding the genetic networks involved in pluripotency, Bing Lim and colleagues were interested in defining the mechanism by which the Wnt pathway, ubiquitous in many developmental processes, plays a role in maintaining pluripotency. The researchers coupled chromatin immunpprecipitation and DNA chip analysis (ChIP-on-chip) in order to identify previously unknown targets of Tcf3, a downstream effector of the Wnt pathway known to be involved in pluripotency. Their experiments revealed that, along with a rang of other targets previously implicated in developmental processes, Tcf3. Interestingly, they found that Tcf3 binds and represses Oct4 one of the core reprogramming factors which affects Oct4 as well Nanog levels, another factor involved in pluripotency. The genomic approaches taken allowed researchers to examine multiple genes at once, revealing a complex genetic network that would not have so clearly emerged using a more piecemeal approach.
Whole genome studies have come into their own at an ideal time to serve as a tool to better our understanding of pluripotency, as well as differentiation. A greater knowledge of the two states will ultimately help us understand the means for more efficient and targeted flux between the two.
- Kim, J., Chu, J., Shen, X., Wang, J., Orkin, S.H. (2008) An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132, 1049-61.
- Stadtfeld, M., Maherali, N., Breault, D.T., Hochedlinger, K. (2008) Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell. 2, 230-40.
- Tam, W.L., Lim, C.Y., Han, J., Zhang, J., Ang, Y.S., Ng, H.H., Yang H, Lim, B. (2008) Tcf3 Regulates Embryonic Stem Cell Pluripotency and Self-Renewal by the Transcriptional Control of Multiple Lineage Pathways. Stem Cells. May 8. [Epub ahead of print]