Development of a Method to Reprogram Antibody Producing B Cells to Functional-Grade Induced Pluripotent Stem Cells
Because of their ability to generate (and regenerate) any tissue in the body, embryonic stem cells (ESCs) harbor the potential to make an enormous impact on human disease. However, serious ethical issues surrounding the use of human ESCs hamper research in this area. In 2006, Yamanaka and colleagues discovered that enforced expression of a defined set of transcription factors can reprogram any cell in the body to induced pluripotent stem cells (iPSCs), which have the potential to behave very much like ESCs. Because iPSCs can be derived from any cell in an adult body, they are considered to potentially hold the promise of ESCs without the weighty ethical concerns.
Excitement in the iPSC field has been tempered by the realization that not all iPSCs are created equal. Although iPSCs can be readily created with widely available techniques, most iPSCs generated in laboratories are poor quality in that they fall short of true ESC-like developmental potential, such as the ability to form certain tissue types and contribute to chimerism in mice. Poor quality iPSCs have modifications of their DNA not present in regular ESCs. One important DNA modification is in the form of methylation of cytosine residues. Poor quality iPSCs are heavily methylated in a particular stretch of DNA in the mouse chromosome 12 called the Dlk-Dio3 locus.
New work in the lab of Fred Alt has found that the use of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytodine (5aza) can reduce DNA methylation at the Dlk-Dio3 locus and dramatically improve the quality of iPSCs. In PNAS, first author Duane Wesemann and colleagues report using 5aza to treat activated mouse B cells in culture before reprogramming to iPSCs. This approach was designed to remove the DNA methylation marks before reprogramming so that reprogramming may "rearrange the epigenetic furniture" so as to ameliorate potential toxic effects of whole genome demethylation.
B-lymphocytes recognize foreign invaders (e.g. viruses and pathogenic bacteria) and then become activated to make antibodies that specifically recognize and eliminate the foreign invaders in the context of an adaptive immune response. Antibodies are made up of immunglobulin heavy (IgH) and light (IgL) chain subunits. During the immune response, the B cells carry out IgH class switching, a process that changes a non-antigen-binding constant portion of the heavy chain into a form that that is most effective in eliminating the foreign invaders once recognized. Mouse B cells can be activated in cell culture with the stimulatory anti-CD40 and IL-4 molecules to undergo IgH class switching, during which the initially expressed IgM heavy chain is changed to other IgH isotypes such as IgG1 and IgE.
IgH class switching results from a DNA excision process in which the DNA that encodes IgM is permanently removed from the genome and replaced with DNA that encodes another IgH class. The authors found that iPSCs derived from activated B cells that had undergone IgH class switching were all uniformly heavily methylated at the Dlk-Dio3 locus and were poor quality because they could not develop back into B or T cells when assayed by injection into early embryos ("blastocyts") from mice that are unable to generate their own B lymphocytes (an novel technique developed by the Alt lab termed RAG-2 deficient blastocyst complementation or "RDBC"). However, many of the tested iPSCs that were reprogrammed with early 5aza treatment were able to develop into functional B-lymphocytes when assayed by RDBC.
These studies have established a new method to enhance ability to reprogram somatic cells into iPSCs. In addition, this method has allowed the generation of a new approach to study the aspects of IgH class switch recombination important for particular immune responses and implicated immune diseases such as asthma and allergies in which IgE production is a key disease element. In this regard, IgM-expressing B cells may switch to IgE directly or may switch first to IgG1 and then switch to IgE at a later time. The mechanisms regulating this "decision" are not well understood. By making good quality iPSCs from activated B cells that had already switched to IgG1, the authors were able create mouse models in which all of the mature B cells in the mouse expressed IgG1. They then showed that these IgG1+ B cells could indeed switch again to IgE upon stimulation providing a model to study this important aspect of adaptive immune response.
Finally, the authors also note that the approach of reprogramming activated B cells into high-grade iPSCs followed by RDBC may be used as a rapid method to generate mice with particular antibody specificities in the context of a desired IgH isotype.
This study was supported by National Institutes of Health R01 and K08 grants to Dr. Alt and Wesemann, respectively, as well as by American Academy of Allergy, Asthma and Immunology and Burroughs Wellcome Fund career development awards to Dr. Wesemann. Dr. Alt is an Investigator of the Howard Hughes Medical Institute.
Duane R. Wesemann, Andrew J. Portuguese, Jennifer M. Magee, Michael P. Gallagher, Xiaolong Zhou, Rohit A. Panchakshari and Frederick W. Alt. Reprogramming IgH isotype-switched B cells to functional-grade induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2012 Aug. 6. [Epub ahead of print]