Introduction of Chromosomal Translocation t(11; 14) and a p53 Deletion into Normal B Cell-Derived iPSCs to Elucidate the Cellular Origin of Myeloma Cells
Authors: Yanagi Aki , Tsuyama Naohiro , Sugai Misaki , Abe Yu , Azami Yusuke , Yanai Yukari , Ota Akinobu , Sivasundaram Karnan , Muramatsu Moe , Shigemura Tomonari , Sasatani Megumi , Hashimoto Yuko , Kamiya Kenji , Hanamura Ichiro , Ikezoe Takayuki , Onodera Masafumi , Sakai Akira 
Published: November 2019

The cellular origin of multiple myeloma (MM) has not ben identified. Based on the results of transplantation experiments using bone marrow samples from MM patients in immunodeficient mice, so-called myeloma stem cells have been inferred to be present in CD19-/CD38++/CD138+ or CD138- plasma cells populations; however, it is possible that the results indicated the presence of plasma cell populations with cell proliferation ability rather than the cellular origin of myeloma cells. On the other hand, based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation play a critical role in early tumorigenesis of MM. We hypothesize that the reprograming of mature B cells, in which IgH gene rearrangements have maintained, are the origin of MM. We propose that mature B cells gain potential by reprograming, and then chromosomal aberrations cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To identify myeloma-initiating cells, we established normal B cell-derived induced pluripotent stem cells (BiPSCs; BiPSC13 and MIB2-6). These BiPSCs maintain VDJ rearrangement of the IgH gene, and they can be induced by the tet-off system to express activation-induced cytidine deaminase (AID; BiPSC13-AID and MIB2-6-AID) and differentiate into hematopoietic progenitor cells (HPCs) (Scientific Rep, 2017). Subsequently, we used the CRISPR/Cas9 system (Oncology Letters, 2019) to establish two BiPSCs with chromosomal translocation t(11;14); the cleavage site were located in the IgH Eμ region of the VDJ non-rearranged allele of the IgH gene and the CCND1 5'-upsteam region of the CCND1 gene (BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system (Ota A, J Cell Sci, 2017) in BiPSC13 with t(11;14) and BiPSC13-AID with t(11;14). These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43- HPCs in co-culture with stromal cell, AGM-S3, and their ability to subsequently differentiate into granulocytes, macrophages, and erythroblasts was confirmed by colony-formation assay. Until now, the co-culture of BiPSC13, MIB2-6, and MIB2-6 with t(11;14) with AGM-S3 followed by co-culture with stromal cell, MS-5, showed the appearance of CD34-/CD45+ and CD34-/CD10+ cell population. Also of interest, by RT-PCR, the expression levels of E2F, EBF, GATA3 and CD10 were higher in CD34+ HPCs differentiated by the co-culture of BiPSC13 and MIB2-6 with AGM-S3 when compared to the levels in cord blood-derived CD34+ cells. Overall, the results suggest that these BiPSCs have the ability to differentiate into HPCs, and that they may further differentiate into B cells. If these BiPSCs could be differentiated into mature B cells, they may be useful for the elucidation and study of myeloma-initiating cells derived from mature B cells.

Disclosures
Hanamura: Mundi: Honoraria; Yamada Yohojo: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Nihon Shinyaku: Honoraria, Research Funding; Fukuyu Hospital: Research Funding; Fujimoto: Research Funding; Taiho: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Chugai: Research Funding; Eisai: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Eli Lilly: Research Funding; Asai Clinic: Research Funding; AbbVie: Honoraria; MSD: Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Zenyaku: Research Funding; Sanofi: Research Funding; Shionogi: Honoraria, Research Funding; Kyowa Kirin: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Astellas: Research Funding; Pfizer: Honoraria, Research Funding.