Establishment of Induced Pluripotent Stem Cells from Lymph Node B Cells and Induction of Aid Expression in Their Differentiation into Hematopoietic Stem Cells

Induced pluripotent stem cells (iPSCs) have been established from a variety of somatic cells not only for regenerative medicine but also for studies of the pathogenesis of inherited genetic diseases or neoplasms. In iPSCs established from T cells, the rearrangement of the T cell receptor (TCR) of the established T cell-derived iPSCs (TiPSCs) took over that of the original T cells. If B cell-derived iPSCs (BiPSCs) could be similarly established from mature B cells or plasma cells, the rearrangement of the B cell receptor (BCR) of the BiPSCs would take over that of the original B cell. BiPSCs were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) together with ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα) using a Sendai virus vector (Bueno C, et al. Leukemia 2016).

We were able to also establish BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes using a method different from the above. The main points of our method are consecutive transfection of a high concentration of Yamanaka factors into B cells, which were pre-stimulated with IL-21 and CD40L, using a retrovirus vector, and centrifugations of the cells after their activation by IL-4, IL-2, and CD40-ligand (CD40L) on a retronectin coated plate. We further established doxycycline-controlled (Tet-off system) activation-induced cytidine deaminase (AID)-induced BiPSCs (BiPSCs-A). AID is an enzyme that initiates somatic hypermutation (SHM) and class-switch recombination (CSR) in B cells. We hypothesized that the origin of a myeloma cell is a reprogrammed mature B cell, in which reciprocal chromosome translocation occurs by double stranded breakage (DSB) of DNA induced by AID activation in the nonproductive (nonfunctional) allele of chromosome 14. First, we did not detect an increase in dicentric chromosome (DIC) formation, which is evidence of DSB of DNA, in the BiPSCs-A.

We next analyzed the ability of these BiPSCs to differentiate into hematopoietic stem cells (HSCs). Both the parental BiPSCs and BiPSCs-A were capable of differentiating into HSCs as judged by confirmation of CD34 expression and colony-formation of macrophages, granulocytes, and erythrocytes from CD34-positive cells. However, these cells were negative for CD38, CD43, and CD45; we therefore think that these CD34⁺/CD38^-/CD43^-/CD45^- cells might be hematoendothelial cells as Maxim proposed previously (Vodyanik MA, et al. Blood 2006). Furthermore, both BiPSCs with induced AID expression and BiPSCs without induced AID expression were capable of differentiating into HSCs. Based on the findings regarding the differentiation of BiPSCs-A into HSCs and their retention of the IgH gene rearrangement, there is a possibility that the induction of AID expression might induce chromosomal translocations in the process of differentiation of these BiPSCs into HSCs and further into B cells in experiments using mouse. Thus these BiPSCs might be useful in elucidating the tumor origin of abnormal B cells in B cell tumor formation. These cells might be especially useful in understanding multiple myeloma, which is thought to originate from germinal center (GC) or post-GC B cells and has a productive (functional) allele with IgH gene rearrangement that produces M-protein, and another nonproductive (nonfunctional) allele of chromosome 14.