B Cell-Derived Induced Pluripotent Stem Cells Are Useful for Determining the Cellular Origin of Abnormal B Cells in Multiple Myeloma

Abstract

Induced pluripotent stem cells (iPSCs) are generated by the genetic reprogramming of somatic cells. In addition to regenerative medicine, these cells have been used in studies on the pathogenesis of inherited genetic diseases or neoplasms. If B cell-derived iPSCs (BiPSCs) could be developed from mature B cells or plasma cells, the genomic rearrangement of the B cell receptor of the BiPSCs would take over that of the original B cell. BiPSCs were recently established from peripheral blood B cells by the simultaneous introduction of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) with CCAAT/enhancer-binding protein-alpha using a Sendai virus vector (Bueno C, et al. Leukemia 2016). In this study, we were also able to generate BiPSCs, albeit using different method. The BiPSCs had immunoglobulin heavy chain (IgH) gene rearrangement but no class-switch in both alleles from normal B cells purified from lymph nodes and peripheral blood; therefore, we postulated that they were most likely established from naive B cells or IgM-expressing mature B cells. The main points of our method are consecutive transfection of a high concentration of Yamanaka factors into B cells using a retrovirus vector, which were pre-stimulated with interleukin (IL)-21 and CD40 ligand (CD40L), combined with consecutive centrifugations of the cells after their activation by IL-4, IL-2, and CD40L on a retronectin coated plate. We also 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. Furthermore, it has been noted recently that AID has similar effects on other than immunoglobulin genes. We hypothesized that myeloma cells are derived from reprogrammed mature B cells, in which reciprocal chromosome translocation occurs by double-strand DNA breaks induced by AID activation in the nonproductive (nonfunctional) allele of chromosome 14 and in another chromosome containing genes such as cyclin D1, Maf. To test our hypothesis, we used the comet assay to confirm that DNA cleavage was induced by AID in the BiPSCs-A. Next, we analyzed the ability of these BiPSCs to differentiate into hematopoietic progenitor cells (HPCs), and found that both parental BiPSCs and BiPSCs-A were capable of differentiating into HSCs, as determined by evaluating CD34 expression and the colony-formation of macrophages, granulocytes, and erythrocytes from CD34-positive cells. However, these cells were negative for CD38, CD43, and CD45 expression, indicating that they may be hematoendothelial cells as Maxim proposed previously (Vodyanik MA et al. Blood 2006). Based on our findings regarding the differentiation of BiPSCs-A into HPCs and their retention of the IgH gene rearrangement, it is possible that the induction of AID expression might induce chromosomal translocations in the process of differentiation of these BiPSCs into HPCs and further into mature or antibody-producing B cells in experiments using mouse. Because the BiPSCs had teratoma-forming ability, if a chromosome break were to occur in either an allele with IgH gene rearrangement (productive allele) or in another nonproductive IgH allele followed by reciprocal translocation with another chromosome and oncogene activation, BiPSCs reactivation and differentiation into B cells might be a cause of multiple myeloma in vivo . Thus, these BiPSCs might be useful for elucidating the origin of abnormal B cells in this disease. DisclosuresNoji:Alexion Pharma GK: Honoraria, Research Funding.