Background Functional analysis of gene polymorphisms associated with increased lupus risk cannot be adequately addressed using mouse systems and it is difficult to obtain sufficient relevant immune cells from individuals with these polymorphisms to perform the necessary studies. Furthermore, such studies are complicated by the strong linkage disequilibrium that exists between certain gene polymorphisms as well as by the inherent genetic variability between individual donors unrelated to the gene polymorphisms themselves. To address these issues, we propose that having an abundant supply of isogenic lupus-relevant immune cells in which gene-editing can be done would enable such functional analyses of gene polymorphism function to be performed.
Methods We reprogrammed human peripheral blood mononuclear cells with a lentiviral vector containing the Yamanaka transcription factors Oct4, Klf4, Sox2 and cMyc to obtain induced pluripotent stem (iPS) cells. The iPS cells were treated with a cocktail of growth factors and cytokines to generate definitive mesoderm and subsequently myeloid dendritic cells (mDC), utilizing feeder-free, chemically defined media.
Results Differentiated cells expressed the mDC markers CD11c, CD1c, and Zbtb46. The differentiated cells produced a cytokine profile characteristic of primary human mDCs in response to a panel of Toll-like receptor (TLR) agonists and were also able to effectively activate T cells in a mixed lymphocyte reaction. In contrast, differentiated cells generated from iPS cells with a loss of function mutation in Unc93B, a key component in endosomal TLR signaling, did not respond to endosomal TLR stimulation but responded normally to cell surface TLR activation.
Conclusions The use of mDC generated from iPS cells may provide a way to study polymorphism function in a lupus-relevant immune cell type by direct gene editing in the iPS cells. Gene editing enables the generation of isogenic lines that are genetically identical, except for the polymorphism of interest, and showing differences in the function of mDC derived from these isogenic lines would be the most definitive demonstration that a particular gene polymorphism induces functional effects that could plausibly be relevant to lupus pathogenesis. In addition, as mDC constitute less than 0.5% of peripheral blood mononuclear cells, this methodology may be valuable not only for the study of human genetic variants but also for providing sufficient numbers of mDC for the study of human myeloid dendritic cell function more generally.
Acknowledgements This work was supported by grant AI30199 from NIH/NIAID (to I.R.R.).
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