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Keywords:

  • activation-induced cytidine deaminase;
  • induced pluripotent stem cells;
  • pluripotency;
  • reprogramming

Abstract

  1. Top of page
  2. Abstract
  3. Heterokaryon-based reprogramming: Fast track toward pluripotency
  4. DNA demethylation: AID in reprogramming
  5. Acknowledgements
  6. References

Current methods of reprogramming differentiated cells into induced pluripotent stem cells remain slow and inefficient. In a recent report published online in Nature, Bhutani et al.1 developed a cell fusion strategy, achieving quick and efficient reprogramming toward pluripotency. Using this assay, they identified an immune system protein called activation-induced cytidine deaminase, or AID, which unexpectedly is actually able to “aid” in reprogramming due to its involvement in DNA demethylation that is required for induction of the two key pluripotency genes, Oct4 and Nanog. More recently, Popp et al.2 also reported online in Nature that AID is important for complete cell reprogramming in mammals. Together, these findings provide new insights into how cells are reprogrammed, identify the specific role of AID in cell fate reversal, and advance the field of regenerative medicine.

Heterokaryon-based reprogramming: Fast track toward pluripotency

  1. Top of page
  2. Abstract
  3. Heterokaryon-based reprogramming: Fast track toward pluripotency
  4. DNA demethylation: AID in reprogramming
  5. Acknowledgements
  6. References

The ability to reprogram ordinary human somatic cells such as skin fibroblasts to become induced pluripotent stem cells (iPSCs), which can then be coaxed to develop into various mature cell types, promises to usher in a new wave of research in regenerative medicine. Using reprogramming methods, researchers can create cells from individual patients and examine them for physiological and genetic abnormalities or use them to test potential therapies. Cell reprogramming via simultaneous overexpression of four “factors” (either the combination of Oct4, Sox2, Klf4, and c-Myc or another combination of Oct4, Sox2, Nanog, and Lin28) has led to generation of iPSCs that are similar to embryonic stem cells (ESCs) 3–5, and has opened a new path to the derivation of stem cell lines with clinical and research potential. The generation of pluripotent cells from a patient's own somatic cells has been the holy grail of regenerative medicine because (i) it provides a much-needed means to obtain cellular models of disease directly from patients as invaluable tools for disease characterization and drug screening, and (ii) it provides an unparalleled potential for cell-based therapies, eliminating the immunological considerations of rejection of non-autologous cells in patients and the ethical concerns associated with the use of human ESCs that are generated by destroying embryos.

Unfortunately, current iPSC technologies are still time consuming (>14 days) and ineffective (<0.01%). To realize the potential of clinical applications of iPSCs, it is crucial to elucidate the molecular events that underlie reprogramming of differentiated somatic cells toward a pluripotent state and to use this knowledge to develop improved methods for inducing pluripotency in somatic cells.

In a report published online in Nature on December 21, 2009, Bhutani et al.1 developed a strategy in which reprogramming was initiated by fusing mouse ESCs with human skin cells to create hybrids called heterokaryons, resulting in astonishingly quick and efficient reprogramming in just 1 day and with extremely high efficiency (∼70%). Key advantages of this heterokaryon-based approach included rapid access to the earliest reprogramming events and a more efficient and synchronous environment through which specific pluripotency mechanisms could be uncovered. Moreover, the researchers showed that reprogramming toward pluripotency in single heterokaryons was initiated without cell division or DNA replication 1. The use of interspecies (between mouse and human) heterokaryons allowed for a distinction of transcripts derived from the two fused cell types, and reprogramming was assessed immediately after fusion without the need for cell division or DNA replication. Thus, an advantage of the heterokaryon technology is that species-specific differences can be utilized to track the unique gene expression profiles of each cell type to facilitate mechanistic investigations 6.

Because all cellular factors, including RNAs and proteins, from both species mingle freely in heterokaryons, mouse ESC factors can be made to dominate and overwhelm the human fibroblast, and initiate reprogramming of the human nucleus rapidly and efficiently, leading to a vast leap from the time-consuming and low-efficiency iPSC generation induced with just the four “reprogramming factors.” This leap provided by this cell fusion technique is consistent with the recent demonstration that iPSC generation by overexpression of reprogramming factors is a continuous random process in which almost all donor cells will eventually give rise to reprogrammed cells, and this stochastic process can be accelerated either (i) by increasing the rate of cell division via p53/p21 inactivation or Lin28 overexpression, or (ii) via Nanog overexpression, independent of the cell division rate 7. In heterokaryons, it is possible that certain RNA species may activate expression of endogenous genes encoding the known reprogramming factors. Although small RNAs have normally been known to silence gene expression, recent studies surprisingly revealed that certain RNAs can also induce potent transcriptional activation of endogenous genes by targeting gene promoters 8, 9. If specific RNAs that target the promoter regions of the pluripotency genes can be identified, they can be used to activate these genes. These RNAs may bring about relatively safe reprogramming and may be used to replace virally driven reprogramming factors in the standard iPSC protocols. In addition to RNAs, the presence of a full set of yet-to-be-characterized proteins in heterokaryons may be necessary for, and directly contribute to, reprogramming toward pluripotency.

Without making random gene insertions into the genomes, the heterokaryon technology may represent a relatively holistic reprogramming approach toward pluripotency. The logic of this approach relies on the action of the entire regulatory components of the fused cells, enabling nuclear reprogramming with “whole systems,” rather than by a small set of master genes. Unlike reprogramming methods dependent on overexpression of a small set of genes, this fusion-based technology permits the simultaneous exposure to all cellular factors and yields a reprogramming system in the cellular environment. In addition, this method acts on endogenous genes, thereby negating problems of gene copy number. Although the hybrid cells have a low probability of ultimate use in human therapy, the heterokaryon technology provides a quick and efficient approach for cell reprogramming and represents a powerful tool for investigating mechanisms of cellular plasticity.

Current techniques for iPSC generation involve introducing viruses and random gene insertions into the target cell, which is dangerous and can lead to tumors, teratomas, and infections. As a result, iPSCs created to date are not suitable for use in the clinic. Novel methods are greatly needed to create iPSCs without the use of viruses and free of unsafe genetic manipulations. Given the fear of the risk of tumorgenicity and other safety concerns associated with the current iPSC reprogramming methods, success in developing “benign” approaches is greatly needed to provide quick, efficient, safe, and useful methods for iPSC generation, and to speed the use of these cells in a wide variety of clinical applications.

DNA demethylation: AID in reprogramming

  1. Top of page
  2. Abstract
  3. Heterokaryon-based reprogramming: Fast track toward pluripotency
  4. DNA demethylation: AID in reprogramming
  5. Acknowledgements
  6. References

This heterokaryon method represents a fast track toward pluripotency and provides a useful tool for identifying key reprogramming mechanisms. Using this method, the researchers demonstrated that activation-induced cytidine deaminase (AID), a protein known for its role in generating antibody diversity, is crucial for the reprogramming process. The identified protein is involved in targeted DNA demethylation, i.e., modifying the DNA by removing its molecular tags, the methyl groups. In particular, AID was shown to be required for demethylation of the promoter regions, which was correlated with the induction of the two well-known pluripotency genes Oct4 and Nanog. The researchers demonstrated that this activity of AID enables reprogramming; they used loss-of-function experiments with siRNA knockdown to validate the role of AID in regulating Oct4 and Nanog gene expression in heterokaryons, and also showed that overexpressing AID in the knockdown cells fully rescued Nanog and partially rescued Oct4 demethylation and expression. These results suggest that demethylation of two key pluripotency genes is an essential part of the reprogramming, and that AID plays a critical role in this process.

It is well established that reprogramming of somatic cell nuclei requires extensive DNA demethylation of pluripotency genes. The status of DNA methylation of these genes mirrors their expression patterns; silent genes are methylated and active genes are unmethylated. Devoid of this process of DNA promoter demethylation, pluripotency genes are silent in differentiated cells. However, little is known about the molecular mechanisms underlying DNA demethylation. The researchers observed demethylation in the absence of cell division and DNA replication, suggesting that it is an active process during reprogramming. To understand the mechanism of demethylation, the researchers focused on AID. Although AID is primarily known as an immune system molecule, it has recently been shown to be present at low levels in mammalian germ cells 10, and has been suggested to play a role in global demethylation in zebrafish embryos 11. Using chromatin immunoprecipitation to target the AID protein and detect its substrate, the researchers confirmed the direct role of AID in demethylation. AID bound to methylated but not unmethylated regions of the genes. The results of this study indicate that removing methyl groups from specific regions of cellular DNA via the action of AID eliminates a major constriction in reprogramming toward pluripotency. However, the exact mechanism of AID's involvement in DNA demethylation is unclear; it is unlikely that AID works alone, and the key components of the DNA demethylation machinery are largely unknown. A recent study indicates that AID initiates a Gadd45-dependent DNA repair pathway 11. Future investigations are warranted to determine the detailed mechanisms of action of AID in reprogramming.

Given the newly identified role of AID in reprogramming, it will be interesting to directly test AID for its ability to enhance and accelerate the derivation of iPSCs, and examine whether overexpression of AID alone can speed up iPSC generation, and whether AID plays a role in existing protocols for enhancing the reprogramming efficiency of iPSC production. iPSC generation can be enhanced by p53 inactivation 12–14 or by several small molecules that can modify cellular gene expression patterns by inhibiting histone deacetylases (HDACs), histone methyltransferases (HMTs), and DNA methyltransferases (DNMTs) 15. Could AID and DNA demethylation be involved in the mechanisms of action of p53 or these small molecules in enhancing reprogramming? Emerging evidence appears to show some cross-talking among p53, histone modifications and DNA methylation. First, although the mechanisms of p53 in the reprogramming pathway are still unclear, p53 is critical in iPSC genomic integrity, and p53 may regulate DNA demethylation in DNA repair pathway that replaces the methylated base with an unmethylated one 12–14. Second, although HDACs are generally believed to act on the chromatin modification machinery to silence target gene expression, the HDAC inhibitor valproic acid, which enhances the efficiency of iPSC generation, has also been shown to bring about active demethylation of methylated DNA 16. Third, both the DNMT inhibitor 5-aza-cytidine and the HMT inhibitor BIX-01294 induce expression of pluripotency genes and impact on DNA demethylation 15. Thus, AID may in part mediate the effects of p53 inactivation and the small-molecule drugs on enhancing epigenetic reprogramming by altering DNA methylation patterns. Currently, reprogramming somatic cells into iPSCs is inefficient, due in part to the epigenetic memory imparted by DNA methylation tags in differentiated cells. The identification of AID and its function in epigenetic signaling pinpoint how DNA demethylation can be driven, and may help to overcome a pivotal barrier in iPSC production. Could AID actually “aid” iPSC generation? Hopefully, the study of AID in reprogramming will truly lead to a fast track to iPSCs in 2010 and beyond.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Heterokaryon-based reprogramming: Fast track toward pluripotency
  4. DNA demethylation: AID in reprogramming
  5. Acknowledgements
  6. References

The author is supported by grants from National Institutes of Health (RO1 NS059043 and RO1 ES015988), National Multiple Sclerosis Society, Feldstein Medical Foundation, and Shriners Hospitals for Children. The author declares no conflicts of interest that relate to this article.

References

  1. Top of page
  2. Abstract
  3. Heterokaryon-based reprogramming: Fast track toward pluripotency
  4. DNA demethylation: AID in reprogramming
  5. Acknowledgements
  6. References