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

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.

www.nature.com/scientificreports/ (CRISPR)/Cas9 system 7 . Furthermore, we generated BiPSC13 with t(11;14) with a deletion in exon 5 of p53 because deletion of p53 is involved in the progression of MM 4,5 ( Figure S2). Subsequently, 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.

Results
Construction of IgH/cyclin D1-specific CRISPR/Cas9. 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 patients 8 . The IgH cleavage site on chr 14 was targeted at a site with fewer off-target effects, between Eμ and Iμ in the class switch region of IgH, and where setting a PAM site is easy 9 . We then designed a CRISPR/Cas9 vector in which these were arranged in tandem to obtain artificial induction of t (11;14) by cutting CCND1 upstream on chr 11 and between Eµ and Iµ regions of IgH on chr 14. We designed two efficient gRNA sequences to be expressed in one CRISPR/Cas9 vector 7 . The functioning of this system was confirmed by induction of cTr t (11;14) in 293 T cells 7 . 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. In both BiPSC13 and MIB2-6, IgH has a complete VDJ rearrangement (VH3-FR1: V 3-9 D 4-23 J 2 in BiPSC13, VH4-FR1: V 4-39 D 3-22 J 6 in MIB2-6), no class switch recombination (CSR) of IgH (Supplemental information, Figure S3), and no somatic hypermutations (SHM) in the VDJ region compared to germline (Supplemental information, Figure S4A and S4C). Two pairs of IgH on chr 14 and CCND1 on chr 11 of BiPSC13 and MIB2-6 are shown in Fig. 1A,D. In BiPSC13, the upper and lower alleles of chr 14 show the non-functional allele that stopped at DJ rearrangement (D 2-21 J 2 ) and the functional allele that completed VDJ rearrangement (V 3-9 D 4-23 J 2 ) of IgH, respectively ( Figure  S4A and S4B). In MIB2-6, the upper and lower alleles of chr 14 show the non-functional allele in which DJ rearrangement was incomplete (D 5-18 J 4 ), and the functional allele in which VDJ rearrangement was complte (V 4-39 D 3-22 J 6 ) for IgH, respectively ( Figure S4C and S4D). The DJ joining of the non-functional allele of MIB2-6 was incomplete because DH4, DH6, and DH7 were deleted between D 5-18 and J 4 , but an intron of 1000 bases or more remained ( Figure S4D).
The features of these cTrs were confirmed with PCR using translocation-specific primers (Fig. 1). The PCR product in lane 1 shows the presence of the VDJ-rearranged functional allele of IgH, and the PCR product in lane 2 shows the non-functional allele that stopped at DJ rearrangement of IgH. Therefore, the PCR product in lane 3 shows the translocation between the functional allele of IgH and CCND1, and the PCR product in lane 4 shows the translocation between the non-functional allele of IgH and CCND1 (Fig. 1G,H). Among these cTrs, when the VDJ side of the IgH functional allele translocated with CCND1 upstream, der(11)t(11;14) was formed. Simultaneously, the CDS side of CCND1 translocated with the constant region of IgH, and der(14)t(11;14) was formed (AZ and BC) (Fig. 1B,E,G-I). On the other hand, when the DJ side of the non-functional allele of IgH translocated with CCND1 upstream, der(11)t(11;14) was formed. Simultaneously, the CDS side of CCND1 translocated with the constant region of IgH, and der(14)t (11;14) was formed (AX and BG) (Fig. 1C,F,G-I). Therefore, we established cell lines carrying reciprocal cTr t (11;14) between CCND1 and either an allele in which VDJ rearrangement of IgH was completed or an allele in which VDJ rearrangement was not completed (stopped at DJ joining) in BiPSC13 t(11;14)(AZ and AX)and MIB2-6 t(11;14) (BC and BG), respectively. We also confirmed the reciprocal cTr t(11;14) using IGH-CCND1 FISH (Fig. 1J,K).
Subsequently, we analyzed the nucleotide sequences near the translocation sites of AZ and AX, and BC and BG (Fig. 1L,M). Thirteen-base deletion of the CCND1 gRNA region and three-base deletion of the IgH gRNA region in der(11)t(11;14) of AX, and three-base addition at the translocation junction in der(14)t(11;14) of AX were confirmed (Fig. 1L). The cleavage site was 140 bases downstream from the PAM region of CCND1 gRNA in der(11)t(11;14) of BC, and 11-base deletion in the CCND1 gRNA region was confirmed (Fig. 1M). One-base deletion in the CCND1 gRNA and 30-base deletion in the Eμ direction from the IgH gRNA in der(11)t(11;14) of BG, and 2-base deletion in the IgH gRNA region in der(14)t(11;14) of BG were confirmed (Fig. 1M).
Effects of CRISPR/Cas9 on alleles of IgH not used in the translocation with CCND1 and on alleles of CCND1 not used in the translocation with IgH were also analyzed (Supplemental information, Figure S5 and S6). Deletions from 11 to 333 bases were found around the cleavage sites of Cas9 in those BiPSCs.
Knockout of TP53 with the CRISPR/Cas9 system. Because deletion of chr 17p, including p53 deletion, is involved in the progression of MM, genome editing was performed on p53 in BiPSC13 t(11;14) (AX) using the CRISPR/Cas9 system. The expression of TP53 was knocked out by deleting 83 bp of exon 5 of p53 (G#28: Fig. 2). Subsequently, we established G#28 and AX with AID expression regulated by the doxycycline-controlled (Tet-off) system (G#28-AID and AX-AID) 6 (Supplemental information, Figure S7).

Differentiation of BiPSCs into blood cells.
Initially, the induction of differentiation into HPC was performed by co-culturing BiPSC13, MIB2-6 with AGM-S3 10 (Supplemenatal information, Figure S8). However, the differentiation efficiency was not stable, and AGM-S3 contamination was present even after purification using CD34 microbeads. We then used the STEMdiff Hematopoietic Kit to induce differentiation of BiPSCs www.nature.com/scientificreports/ www.nature.com/scientificreports/ into HPCs (Fig. 3). Based on a model of hematopoietic differentiation from human embryonic stem cells 11 , the expression of CD43, CD45, and CD38 was examined in CD34 + cells. Except for AX (Fig. 3C), two cell populations were observed (R1 and R2), and most CD34 + cells were CD43 + /CD45 + /CD38 +/− in both populations (Fig. 3). The number of colonies formed in the colony assay is shown in Fig. 3I. Most colonies were a mix of macrophages and granulocytes, and some were mixed with erythrocytes ( Fig. 3A-D,G,H). BC differentiated into CD34 + cells, but colony formation was not observed (Fig. 3F). On the other hand, differentiation into blood cells was observed even in the case of p53 deletion and induction of AID expression in the colony assay in AX (Fig. 3G,H). Therefore, B cell-derived iPS cells were able to differentiate into HPCs even in the presence of cTr t (11;14) or p53 was deletion.

Differentiation of BiPSCs-derived CD34 + cells and cord blood (CB) into B lymphocyte.
We investigated whether HPCs differentiated from iPS cells derived from normal B lymphocytes can differentiate into B lymphocyte again by co-culture with MS-5, with reference to a study by MacLean 12 . First, we confirmed that CB would differentiate into B lymphocytes by co-culture with MS-5, with reference to studies by Nishihara 13 and Hirose 14 . CB proliferated rapidly after initiating the co-culture, and phenotype analysis at day 10 revealed that CD34 + cells were mainly present in the R2 region (Fig. 4A). The main cell population in both the R1 and R2 regions was CD34 − /CD38 +/− /CD43 + /CD45 + , followed by CD34 + /CD38 − /CD43 + /CD45 + (Fig. 4A). Four weeks www.nature.com/scientificreports/ after initiating the co-culture, the CD34 + cells had disappeared and the main cell populations were CD34 − / CD38 +/− /CD43 + /CD45 + , and CD19 + /CD10 − cells were observed in R2 region (Fig. 4B). The number of CD19 + / CD10 − cells further increased 6 weeks after initiation of the co-culture (Fig. 4B). Next, we investigated whether two types of BiPSCs (BiPSC13 and MIB2-6) would differentiate into B lymphocytes by co-culture with MS-5. The cells were differentiated into CD34 + cells using a STEMdiff Hematopoietic kit, and purified CD34 + cells were subsequently cultured on MS-5. Phenotype analysis was performed 3 weeks after initiation of the co-culture, with reference to a previous study 12 . Based on studies reporting the appearance of CD19 + cells from the fourth week of co-culture of CB with MS-5 13,14 , we performed phenotype analyses 4 or 5 weeks after initiation of the co-culture. Analysis of BiPSC13-derived cells showed a non-specific cell population, even after staining with an isotype control using FITC or without staining ( Figure S9). Therefore, the cell population observed by the double staining of CD34/CD38 and CD19/CD10 was likely to be due to autofluorescence. In the co-culture of BiPSC13 or MIB2-6-derived CD34 + cells with MS-5, the CD34 + cells remained mainly in the R1 region and their phenotype were CD34 + /CD38 − /CD43 − /CD45 − (Fig. 4C,D). A population of CD34 − /CD38 − /CD43 + /CD45 + cells was also observed (Fig. 4C,D). No CD19 + cells were observed. In the coculture of G#28-AID-derived CD34 + cells with MS-5, CD34 + cells were still found in both the R1 and R2 regions and their phenotype was CD34 + /CD38 − /CD43 − /CD45 − (Fig. 4E).
Therefore, the critical difference between these three types of BiPSCs-derived HPCs and CB was that the former CD34 + cells remained and did not differentiate into CD38 + cells even after 4 weeks of co-culture with MS-5.

Discussion
We hypothesized that the abnormal cells from which myeloma cells originate may be mature B lymphocytes with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become tumor cells during the process of redifferentiation into B lymphocytes. Here we tried to generate cells to test this hypothesis. First, we established iPS cells from normal B lymphocytes (BiPSCs) that can induce AID expression 6 . We then generated BiPSCs carrying the most frequent reciprocal cTr t (11;14) in MM (BiPSC13 t(11;14) and MIB2-6 t(11;14)), and also generated BiPSC13 t(11;14) with p53 deletion, which is involved in the progression of MM. This means "Strike (chromosomal or genetic changes) while the iron (B lymphocytes) is hot".  www.nature.com/scientificreports/ Studies have reported the possibility of generating reprogrammed tumor cells or precancerous lesions by forcibly expressing the Yamanaka factors in colonic epithelium or pancreatic acinar cells that already have gene mutations 15,16 . Interestingly, in vivo reprograming is possible in the presence of inflammation and senescence instead of Yamanaka factors, and depolarization of the original cell is considered to be critical 17 . We believe that normal B lymphocytes could be transformed into iPS-like cells in the environment of BM or lymph nodes in which Yamanaka factors are induced due to chronic inflammation. The original cells of BiPSCs were normal cells, unlike the cells mentioned above 15,16 , and genomic changes were induced after transformation into iPS cells.
The onset of MM is often caused by a reciprocal cTr between chr 14 with IgH and chr 11 with CCND1 or chr 4 with FGFR3 and MMSET 4,5 . In follicular lymphoma (FL) and mantle cell lymphoma (MCL), immature B lymphocytes in BM are considered the origin of tumor cells; the reciprocal cTr between IgH and oncogenes such as BCL-2 on chr 18 or CCND1 on chr 11 during VDJ rearrangement is responsible for tumorigenesis of B lymphocytes 18 . On the other hand, in MM, mature B lymphocytes, so-called plasma cells, or antibody-producing cells, are considered tumor cells because IgH reciprocally translocate with other genes during CSR 19,20 , and SHM is recognized in the VDJ region of IgH [21][22][23] . Considering the presence of M-protein, a functional allele of IgH is present in MM. Therefore, cTr occurs between another non-functional allele of IgH in which VDJ rearrangement has not been completed, and other chromosomes in MM 3 . The features of cTr in AX and BG exactly mimic that of MM.
When VDJ rearrangement of one allele of IgH is complete, VDJ rearrangement of another allele of IgH is not complete as a non-functional allele due to allelic exclusion 24,25 . In FL and MCL, cTr occurs during VDJ rearrangement in immature B lymphocytes as mentioned above. However, considering that B-cell receptors are expressed on the cell surface, the allele that initiates VDJ rearrangement earlier would be a non-functional allele due to cTr, and in turn, another allele that should have been non-functional, would undergo VDJ rearrangement to create a functional allele that expresses B-cell receptors. Although cTr during VDJ rearrangement in MM was also reported 26 , many cTrs occur during CSR 4,5 .  www.nature.com/scientificreports/ Even in immature B lymphocytes, cTr t(14;18) or cTr t (11;14) alone is believed to not be able to transform these B lymphocytes, as suggested by studies revealing these chromosomal aberrations in normal individuals [27][28][29] . Similarly, mature B lymphocytes would be unlikely to transform into tumor cells by cTr. Myeloma cells could be derived from reprogramed mature B lymphocytes with the following features: the CD19 antigen on the cell surface and its transcription factor, Pax5, are deleted in myeloma cells unlike in other B cell lymphomas 30 . Furthermore, myeloma cells expressing CD33 31 , which is a cell surface marker of granulocytes, or producing amylase 32,33 or ammonia 34 have been reported.
Myeloma cells are derived from mature B lymphocytes because of SHM in the VDJ region of IgH ( Figure  S10). Because neither SHM in the VDJ region nor CSR of IgH occurred in BiPSC13 or MIB2-6, those BiPSCs are considered to be derived from pre-germinal center (GC) B lymphocytes rather than mature B lymphocytes. However, they can induce AID expression with the tet-off system 6 , and we confirmed that those BiPSCs and BiPSCs with t (11;14) can differentiate into HPCs in this study. If they further redifferentiate into B lymphocytes, SHM in the VDJ region and CSR of the functional allele of IgH could be induced during the redifferentiation process by activation of endogenous AID. Furthermore, activation of enhancers involved in IgH expression during the process of redifferentiation into B lymphocytes would lead to overexpression of CCND1 induced by cTr t (11;14), which is observed in MM with t (11;14). Expression of AID also would be expected to induce SHMs on various genes other than IgH, which would lead to the branching pathway theory based on a Darwinian selection perspective 35 , a model for the progression of MM. In addition, if IgH expression is impossible due to a deletion in the constant region instead of CSR in the functional allele, this would be considered a mechanism of development of Bence-Jones-type MM; only the light chain of the M-protein is detected in the urine (and serum), because myeloma cells in these cases do not produce the IgH chain.
Unfortunately, no CD19 + cells were observed in co-culture of BiPSCs-derived CD34 + cells with MS-5. The difference between these BiPSCs-derived HPCs and CB was that the former CD34 + cells remained and CD34 − /CD43 + /CD45 + cells did not express CD38 even after 4 weeks of co-culture with MS-5. Especially, compared with the parent cell (BiPSC13), G#28-AID did not differentiate into CD34 − /CD43 + /CD45 + cells with remaining of CD34 + cells even 6 weeks after the initiation of co-culture with MS-5. A deletion of p53 might be involved in this feature. Given that CD38 is involved in lymphocyte activation 36,37 , the ability to express CD38 in differentiation from HPC could be important for differentiation into B lymphocytes. Considering the results of the colony assay, a differentiation potential into blood cells of BiPSCs-derived HPCs can be expected, so other factors could be required for co-culture with MS5 in order to differentiate them into B lymphocytes in vitro. Moreover, important questions are whether these BiPSCs with cTr or p53 deletion are capable of forming MM or B lymphoid tumor cells, and whether AID induction induces additional genetic aberrations. We are planning to transplant these BiPSC-derived HPCs into BM of immunodeficienct mice.

Induction of translocation. cTr was induced by infection of BiPSCs with IgH-CCND1 lentiCRISPRv2
lentivirus which targets the human IgH Eµ region and 13 kb upstream of the CCND1 coding sequence (CDS) 7 . Briefly, CRISPRscan (http://www.crisp rscan .org/) 38 estimated the gRNA candidate, 5′-GGA GAA CAT ACC AAG CCC CAC-3′ for IgH (105,861,064 to 105,861,045 of NC_000014.9, chromosome (chr) 14, GRCh38.p12 Primary Assembly) and 5′-GGT GGC GAG GTG GGA CCG CGG-3′ for CCND1 (69,627,757 to 69,627,776 of NC_000011, chr 11, GRCh38.p12 Primary Assembly), which were recombined into the lentiCRISPRv2 BsmBI site. The CCND1 gRNA expression unit of CCND1-lentiCRISPRv2 was cloned into IgH-lentiCRISPRv2 to form the IgH-CCND1 lentiCRISPRv2 dual site targeting vector. The vector was packaged into lentivirus, and BiPSCs cultures were infected with the packaged virus in the presence of polybrene (4 µg/mL) for 1 day. Puromycin selection (0.25 µg/mL) started after 2 days. Ten days later, drug-resistant colonies were picked and mechanically divided into two portions using pipet tips; one half was cultured, and DNA was isolated from the other half using DNAiso kit. Confirmation of cTr was performed by PCR using the IgH/CCND1 translocation-specific primer pair (IgH-Fs for the IgH side and CCND1-86-Rs for the CCND1 side) (Fig. 1, Table S1) with Thunderbird SYBR qPCR Mix and a Light Cycler Nano (Loche Diagnostics, Basel, Switzerland). PCR conditions were 94 °C 120 s, 50 cycles of (94 °C 10 s, 62 °C 10 s, 72 °C 20 s), and 72 °C to 94 °C at 0.1 °C/sec for melting temperature measurement. The presence of Tr-positive clones was assessed based on the melting temperature of PCR-amplified DNA compared with positive control DNA, which was the product amplified from the IgH and CCND1 genomic region with PCR and recombined into a translocation-mimic PCR template (5′-IgH-CCND1-3′) 7 .
Detection of VDJ and DJ rearrangements in BiPSCs. Confirmation of the VDJ recombination profile was performed as described 39  www.nature.com/scientificreports/ primer pairs of VHn-FR1 (n = 1-6) and JH consensus for the functional IgH allele, and DH regions were amplified using primer pairs of DHn (n = 1-7) and JH consensus for the non-functional IgH allele. Primers are listed in Table S1. PCR conditions are 95 °C 120 s, 45 cycles of (95 °C 30 s, 62 °C 15 s, 72 °C 40 s) using TB Green Fast qPCR Mix and LightCycler Nano. PCR products were electrophoresed in an agarose gel, and bands of the corresponding size were purified using FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and sequenced with the same primers for PCR (FASMAC Co. Ltd.). Sequence data were analyzed using the IgBLAST database (https ://www.ncbi.nlm.nih.gov/igbla st/) to identify the subtype of every subdomain. An additional primer, DH5-18-F was used for DNA sequence of the non-functional IgH allele of MIB2-6.
Identification of reciprocal chromosomal translocation t (11;14). VH3-FR1 primer for BiPSC13 or VH4-FR1 primer for MIB2-6 was used in combination with the CCND1-86-F primer to detect reciprocal cTr, which formed der(11)t (11;14), between the functional IgH allele and CCND1. DH2 primer for BiPSC13 or DH5 primer for MIB2-6 was used with the CCND1-86-F to detect reciprocal cTr, which formed der(11)t (11;14) between the non-functional IgH allele and CCND1. The combination of IgH-Fs and CCND1-86-R or CCND1-86-Rs was used to detect another reciprocal cTr forming der(14)t (11;14). The presence of the untranslocated allele of IgH was confirmed with PCR in the combinations of DH2 or VH3-FR1 and IgH-Fs for BiPSC13 and DH5 or VH4-FR1 and IgH-Fs for MIB2-6, respectively. The presence of the untranslocated allele of CCND1 was confirmed with PCR in the combination of CCND1-86-F and CCND1-86-R for both BiPSCs (Fig. 1A-F (AX) (1 × 10 6 cells) using a 4D-Nucleofector instrument (Lonza Japan, Tokyo, Japan). After 3 days, cells expressing GFP were sorted using FACSAria (BD Bioscience, Franklin Lakes, NJ, USA), and single-cell cloning was performed. A single clone was selected, expanded, and used for biological assays. For sequence analysis of TP53 exon 5, the following primer set was used to amplify genomic DNA: 5′-TTG CAG GAG GTG CTT ACA CA and 5′-GAA TTC TGA AGG TCT CGT CGT.
Culture of BiPSCs in stem cell differentiation medium for differentiation into HPCs. Differentiation of BiPSCs into HPC was performed using STEMdiff Hematopoietic Kit (STEMCELL Technologies, Vancouver, Canada). BiPSCs were cultured on Matrigel hESC-Qualified Matrix (CORNING, Corning, NY, USA), incubated with Gentle Cell Dissociation Reagent (STEMCELL Technologies) at room temperature for 4-6 min, removed using a scraper, and then, 50 μm-sized pieces were transferred to a matrigel-coated 12-well plate in Complete StemFit AK02N (100 pieces/well). Differentiation experiments were performed according to the instructions of the kit. BiPSCs with AID were cultured in the presence of 10 ng/mL doxycycline (Takara Bio, Kusatsu, Japan) to prevent AID expression. Concurrently, CD34 + cells were purified using MACS CD34 Micro-Beads (Miltenyi Biotec Inc., Auburn, CA, USA) according to the manufacturer's instructions, and the collected cells were used for phenotype analysis and a colony-forming assay.
Colony-forming assay. Purified CD34 + cells were then used in colony-forming assays using Methocult www.nature.com/scientificreports/ Western blot analysis. The method was described in our previous study 6 . Briefly, cells were lysed in lysis