Chromosomal translocation t(11;14) and p53 deletion induced by the CRISPR/Cas9 system in normal B cell-derived iPS cells

Yusuke Azami,Karnan Sivasundaram,Misaki Sugai-Takahashi,Megumi Sasatani,Moe Muramatsu,Tomonari Shigemura,Takayuki Ikezoe,Masafumi Onodera,Ichiro Hanamura,Kenji Kamiya,Naohiro Tsuyama,Yu Abe,Akinobu Ota,Akira Sakai,Ken-Ichi Kudo,Shigehira Saji,Yuko Hashimoto

doi:10.1038/s41598-021-84628-5

Abstract

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain potential to transform by reprograming, and then chromosomal aberrations cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we have already established normal B cell-derived induced pluripotent stem cells (BiPSCs). Here we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eμ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5′-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14). These BiPSCs differentiated into hematopoietic progenitor cells (HPCs). However, unlike cord blood, those HPCs did not differentiated into B lymphocytes by co-culture with BM stromal cell. Therefore, further ingenuity is required to differentiate those BiPSCs-derived HPCs into B lymphocytes.

Highlights

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis
Based on the experiments of transplantation of bone marrow (BM) samples from MM patients into immunodeficient mice, so-called myeloma stem cells have been inferred to be present in C D19−/CD38++/CD138+ or C D138− plasma cell p opulations1,2; the results may indicate the presence of plasma cell populations with cell proliferation ability rather than the cellular origin of myeloma cells
The CCND1 cleavage site on chr 11 was upstream of CCND1 where off-target effects are fewer and setting a protospacer adjacent motif (PAM) site is easy, based on a study showing no hot spots at the cleavage site in the analysis of MM patients8

Summary

Introduction

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. We analyzed the features of cTr t(11;14) between the functional allele or the non-functional allele of IgH and CCND1 and the ability to differentiate into blood cells. We attempted to induce reciprocal cTr t(11;14) in two normal B lymphocyte-derived iPS cell lines (BiPSC13, MIB2-6) using the above method.

Results

Conclusion