title: Reprogramming IgH isotype-switched B cells to functional-grade induced pluripotent stem cells.
abstract: Induced pluripotent stem cells (iPSCs) can be formed from somatic cells by a defined set of genetic factors; however, aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster often hinders their developmental potency and ability to contribute to high-grade chimerism in mice. Here, we describe an approach that allows splenic B cells activated to undergo Ig heavy-chain (IgH) class-switch recombination (CSR) to be reprogrammed into iPSCs that contribute to high-grade chimerism in mice. Treatment of naïve splenic B cells in culture with anti-CD40 plus IL-4 induces IgH CSR from IgM to IgG1 and IgE. CSR leads to irreversible IgH locus deletions wherein the IgM-producing Cμ exons are permanently excised from the B-cell genome. We find that anti-CD40 plus IL-4-activated B cells produce iPSCs that are uniformly hypermethylated in the imprinted Dlk1-Dio3 gene cluster and fail to produce chimerism in mice. However, treatment of activated B cells with the methyltransferase inhibitor 5-aza-2'-deoxycytidine before and at early stages of reprogramming attenuates hypermethylation of the Dlk1-Dio3 locus in resultant iPSCs and enables them to form high-grade chimerism in mice. These conditions allowed us to produce chimeric mice in which all mature B cells were derived entirely from IgG1-expressing B-cell-derived iPSCs. We conclude that culture conditions of activated B cells before and at early stages of reprogramming influence the developmental potency of resultant iPSCs.
authors.affiliation: Program in Cellular and Molecular Medicine and Immune Disease Institute, Boston Children's Hospital, Boston, MA 02115, USA. wesemann@idi.harvard.edu
authors.forename: Duane R
authors.initials: DR
authors.lastname: Wesemann
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Portuguese
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Magee
authors.forename: Michael P
authors.initials: MP
authors.lastname: Gallagher
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Zhou
authors.forename: Rohit A
authors.initials: RA
authors.lastname: Panchakshari
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Alt
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology*; D002469:Cell Separation; D003584:Cytological Techniques; D004268:DNA-Binding Proteins / Q000235:genetics; D005434:Flow Cytometry; D005821:Genetic Techniques; D016678:Genome; D017578:Immunoglobulin Class Switching; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D005810:Multigene Family; D039904:Pluripotent Stem Cells / Q000166:cytology*
chemical_list: D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; C499308:Rag2 protein, mouse