Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells

B cell derived induced pluripotent stem cells (BiPSCs) were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) and C/EBPα using a Sendai virus vector. Here, using a different method, we established BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes. The critical points of our method are pre-stimulation of B cells with IL-21 and CD40-ligand (CD40L), followed by consecutive transfection of highly concentrated Yamanaka factors using a retroviral vector. Following each transfection the cells were centrifuged onto a retronectin coated plate and the activated by IL-4, IL-2, and CD40L. Furthermore, we established BiPSCs (BiPSC-A) in which activation-induced cytidine deaminase (AID) could be induced using the doxycycline-controlled. Both the parental BiPSC and BiPSC-A showed the capability of differentiating into hematopoietic progenitor cells (HPCs) based on confirmation of CD34 expression and colony-formation from CD34-positive cells. The findings that BiPSC-A can differentiate into HPCs suggest that there is a possibility that induction of AID expression would result in chromosomal translocations in the process of differentiation from BiPSCs, and therefore that these BiPSCs could be useful in elucidating the tumor origin of abnormal B cells in myelomagenesis.

of the original T cell. Similarly, if B cell derived iPSC (BiPSC) could be established from mature B cells or plasma cells and then be subsequently redifferentiated into mature B cells or plasma cells, it should be possible to make mature B cells that are specific for an antigen or make plasma cells that are producing monoclonal antibodies. In a mouse system, chimeric mice were produced from iPSC that were established from mouse embryonic fibroblasts (MEFs). Subsequently, BiPSCs that had a B cell receptor (BCR) that was identical to that of B cells isolated from the chimeric mice were established by reactivation of Yamanaka factors together with either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα) or specific knockdown of the B cell transcription factor Pax5 12 . On the other hand, Wada et al. established iPSCs from MEFs and mouse splenic B cells, and both iPSCs differentiated into a T-cell lineage. However, these cells were relatively resistant to B-cell lineage differentiation and it was speculated that this resistance was due to a defect in Pax5 expression in the differentiated cells 13 . Recently, Stefano et al. reported that the overexpression of Tet2 induced by the transfection of C/EBPα enhances Yamanaka factor-induced B-cell reprograming 14 . Subsequently, BiPSCs were established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors and C/EBPα using a Sendai virus vector 15 .
In the present study we were able to establish BiPSCs from normal B cells purified from lymph nodes by using a different method. Here, normal B cells were pre-activated with IL-21 and CD40-ligand (CD40L) and were then consecutively transfected on a retronectin coated plate with highly concentrated Yamanaka factors using a retroviral vector and centrifugation.
Activation-induced cytidine deaminase (AID) is an enzyme that initiates somatic hypermutation (SHM) and class-switch recombination (CSR) 16 . Not only immunoglobulin heavy chain (IgH) gene, but also gene mutations in B cells induced by AID, are supported as a cause of lymphomagenesis [17][18][19][20][21][22] . Recently, model mice with diffuse large B-cell lymphoma (DLBCL) were reported, in which DNA repair abnormalities after mutations or cleavages of DNA induced by AID-generated uracil-guanine (U-G) mismatch regulate malignant transformation, was reported 23 . Since BiPSCs established from this mouse have IgH gene rearrangement, we subsequently established BiPSCs in which AID expression could be induced by using a doxycycline-controlled system (tet-off system), in order to determine whether those BiPSCs would produce B cells with chromosomal translocation, which is thought to be one origin of B cell malignancies.

Results
Generation of iPS cells from CD19 + cells of human lymph nodes. Normal B cells were purified from the LNs of patients who underwent mediastinum surgical treatment. Cell surface analysis of the LN lymphocytes before the CD19 + lymphocytes purification indicated that about 70% of the lymphocytes were T cells and about 20% were B cells (Fig. 1a). In terms of expression of CD27, which is a memory B cell marker, there were two populations within B cells, a CD27 + and a CD27 − population (Fig. 1a). Over 99% pure B cells were obtained by purification using the CD19-microbeads (Fig. 1b), and subsequently, the Yamanaka factors were transfected into these B cells using a retroviral vector. The processes of cell culture and virus transfection are shown in Fig. 2a. The critical points of these processes are that, after pre-stimulation of the B cells with IL-21 and CD40L, three successive transfections and stimulations were performed, in each of which, after the transfection of highly concentrated Yamanaka factors into the B cells, the cells were centrifuged for 2 h and were then stimulated with IL-4, IL-2, and CD40L for 22 h. Subsequently, the infected B cells were cultured with IL-4, IL-2, and CD40L for 1 day and were then cultured on MEFs with IL-4 and IL-2 for 2 days, followed by continuous culture on MEFs with bEGF and iPSC medium until the occurrence of colony formation. Forty-eight colonies were picked up from 3 × 10 6 of the purified B cells and IgH gene rearrangements were confirmed in 17 of these colonies. Teratoma formation was examined in 6 of these 17 clones, and was confirmed in 4 of the clones. Based on these data, we established 4 BiPSCs from normal B cells in LN, in which 3 BiPSCs were confirmed to have a normal karyotype and to be negative for EBV infection (data not shown). Although all colonies were not confirmed to be derived from B cells, BiPSC induction efficiency from normal B cells in LN was 0.0016%. A representative BiPSC, that we called BiPSC13, was analyzed in the following studies. The results of PCR showing the IgH gene rearrangement, the colony formation, the teratoma formation, and the karyotype of BiPSC13 are shown in Fig. 2.

Differentiation of BiPSCs into hematopoietic progenitors and colony-forming assay. In order
to confirm the capacity of these two BiPSCs to differentiate into HSCs, we cocultured BiPSC13 and MIB2-6 with C3H10T1/2 cells. On day 14 of culture, the cells were collected and the emergence of HSCs was analyzed using flow cytometry (Fig. 4a). A population of CD34 + /CD38 − cells was detected and sorted. These cells, which were obtained from both BiPSC13 and MIB2-6 cultures, were morphologically similar to HSC cells (Fig. 4b).
However, these cells were negative for CD43 and CD45. Since the cells were negative for CD43, which is a marker of HSCs, we next performed a colony-forming assay to confirm that these CD34-positive cells had the capacity to undergo differentiation. Although the typical erythroid colony-forming unit was not detected, colony formations with mixed macrophages and granulocytes were observed (2-3/0.6-3.0 × 10 4 of CD34-positive cells) and macrophages, granulocytes, and erythroblasts were confirmed in the cells picked-up from those colonies (Fig. 4c). The phenotype of CD34 + /CD38 − /CD43 − /CD45 − is similar to that of hematoendothelial cells as Vodyanik proposed previously 24 .
Establishment of BiPSCs with inducible AID expression, using the doxycycline-controlled (Tet-off) system. The above data confirmed that the two different BiPSCs tested were capable of differentiating into hematopoietic stem cells. We therefore expected that it would be possible to induce chromosomal translocations in B cells if we could establish BiPSCs with induced AID expression and differentiate them into HSCs. Furthermore, we expected that we could differentiate these BiPSCs into B cells in which double stranded breaks (DSBs) of DNA would be induced by AID expression. We established two BiPSCs for each BiPSC13 and MIB2-6 in which AID expression could be induced by doxycycline-controlled (Tet-off) system (BiPSC13-AID (#1 and #2) and MIB2-6-AID (#16 and #17) termed BiPSCs-AID; Fig. 5). Induced expression of AID mRNA peaked at 72 h after the removal of doxycycline and disappeared at 24 h after doxycycline addition (Supplementary Figure 4) and immunofluorescence staining of AID was detected at 2 or 3 days after the removal of doxycycline (Supplementary Figure 5). These BiPSCs-AID cells expressed Oct3/4 and Nanog and after the induction of AID expression (Fig. 5c,f). AID expression had no effect on cell proliferation (Supplementary Figure 6). We then treated two BiPSCs-AID (BiPSC13#1 and MIB2-6#17) as described above to differentiate them into HSCs and removed doxycycline 10 days before sorting of the CD34-positive cells. Both BiPSC13#1 with induced AID expression and BiPSC13#1 without induced AID expression were capable of differentiating into HSCs (Fig. 6). Furthermore, there was no difference between BiPSC13#1 with induced AID expression in the course of differentiation into HSCs and BiPSC13#1 with constitutive AID expression. AID expression in the sorted CD34-positive cells was confirmed by immunostaining (Fig. 6b). Furthermore, we detected macrophages and granulocytes within the CD34-positive population in a colony-formation assay (3-5/1.3-4.0 × 10 4 of CD34-positive cells) (Fig. 6c). Although the colony-formation assay was not performed due to a small number of CD34-positive cells, both MIB2-6#17 with induced AID expression and MIB2-6#17 without induced AID expression were also capable of differentiating into HSCs (Supplementary Figure 7). Therefore, we succeeded in establishing a BiPSC in which it was possible to induce AID expression using a doxycycline-controlled system, and resulting AID expression did not affect the differentiation of the BiPSC into HSCs.

Discussion
Initially, we tried to establish iPSCs from B cells purified from lymph nodes using a retroviral vector for the transfection of C/EBPα followed by transfection of Yamanaka factors; however no colony formation indicating the generation of iPSCs was observed. It is possible that, compared with the method of Bueno et al. who used a Sendai virus vector 15 , the transfection efficiency of genes using the retroviral vector of our study might be lower. Ultimately, we succeeded in the establishment of BiPSCs from B cells using Inaki's method (unpublished method; supplemental Materials and Methods). The original B cells obtained from the lymph node biopsy were CD20 + / CD27 +/− (Fig. 1) by cell surface antigen analysis after. Therefore, it is not clear whether the original B cells from which the BiPSCs were derived were memory B cells or not. Noteworthy points in our method were the activation of B cells with IL-2, IL-4, IL-21, and CD40L, and subsequently, centrifugation of B cells for 2 h onto a retronectin coated plate during the transfection of Yamanaka factors. Furthermore, these procedure were repeated three times. The reprogramming efficiency (iPSC clones/ infected cells 15 ) of our method appears to be close to the efficiency reported by Bueno.
The purpose of establishing BiPSCs from normal B cells was to find or make an abnormal B cell that could be considered as an origin of B lymphoid tumors. The BiPSCs of the present study originated from B cells in lymph nodes or peripheral blood and have IgH gene rearrangement. Therefore, these BiPSCs were thought to be established from germinal center (GC) or post-GC B cells. Furthermore, taking into consideration the fact that these BiPSCs have a teratoma-forming ability, if a chromosome brake were to occur either in the allele with IgH gene rearrangement (productive allele) or in another nonproductive allele of the IgH gene, and then a chromosome reciprocal translocation with another chromosome with an oncogene were to happen, the reactivation of BiPSCs to differentiate into B cells might be a cause of B cell neoplasms in vivo (Fig. 7).
The chromosomal translocation t(14;18) in follicular lymphoma (FL) and t (11;14) in mantle cell lymphoma (MCL) are thought to occur at the time of VDJ rearrangement of the IgH gene in the process of B cell differentiation. Therefore, the origin of these lymphoma cells is a B cell in the bone marrow (BM) or a B cell in pre-GC. On the other hand, a myeloma cell is thought to originate from a GC or a post-GC B cell and has a productive (functional) allele with IgH gene rearrangement and class switch recombination (CSR), which produces the M-protein, and another nonproductive (nonfunctional) allele of chromosome 14. Usually, the latter allele is thought to reciprocally translocate with other chromosomes in the myeloma cell. Therefore, we hypothesize that the origin of a myeloma cell would be a reprogrammed mature B cell, in which reciprocal chromosome translocation would occur by a double stranded break (DSB) of DNA induced by AID activation (Fig. 7). Indeed, the CD19 antigen on the cell surface and its transcriptional factor, Pax5, are deleted in myeloma cells unlike in other B cell lymphomas 25 . Furthermore, myeloma cells expressing CD33 26 , which is a cell surface marker of granulocytes, or producing amylase 27,28 or ammonia 29 have been reported. The two BiPSCs of our study did not express Pax5 and were negative for CD19 (Fig. 3, Supplementary Figure 3). Those features suggest that myelomagenesis might result from transformation from a mature B cell accompanied by reprogramming of the mature B cell.
Here we reported two BiPSCs that were established from B cells of lymph nodes and peripheral blood, in which AID expression could be induced using the tet-off system. Furthermore, these BiPSCs were able to differentiate into hematopoietic stem cells. Generally, AID is an enzyme that initiates SHM and CSR in B cells. However, we did not detect an increase in DIC formation, which is evidence of DSB of DNA, in the BiPSCs. The DIC analysis was performed at 10 days based on the result that the expression of AID mRNA and protein already peaked at this time after the removal of doxycycline in the tet-off system ( Supplementary Figures 4 and 5). Furthermore, there was no significant difference in the number of DIC formed in the BiPSCs with and without constitutive AID expression (data not shown). On the other hand, only 5% of the sorted CD34-positive cells in the experiment of BiPSCs differentiation into HPCs were AID positive cells. Therefore, the capability of inducing AID expression in the tet-off system might differ between BiPSCs and CD34-positive cells.
If we could differentiate these BiPSCs into mature B cells in vitro or in a mouse, we could prove the relationship between reprogramming and oncogenesis in mature B cells.

Materials and Methods
Ethics Statement. The samples and the medical records used in this study were approved by the Ethics   (Day5-6). The next day, the infected B cells were transferred onto the MEF feeder cells and were cultured in new B cell culture medium containing IL-4 (10 ng/ml) and IL-2 (100 U/ml). After One of the alleles of the IgH gene of these B cells is a productive (functional) allele that had performed VDJ rearrangement as well as class switch recombination and produces the so-called M protein. The other allele, however, is a nonproductive (nonfunctional) allele (c), in which case binding to oncogenes, etc. (b) due to reciprocal translocation with another chromosome following cleavage between the J and C segments due to AID expression results in myelomagenesis.
Scientific RepoRts | 7: 1659 | DOI:10.1038/s41598-017-01627-1 two days (Day6-8), the medium was replaced with human iPSC medium (DMEM/F12 (SIGMA) supplemented with 20% Knocout ™ Serum Replacement (Thermo Fisher Scientific, Yokohama, Japan), L-glutamine (4 mM) (Thermo Fisher Scientific), MEN Non-Essential Amino Acids Solution (1 mM) (Thermo Fisher Scientific), β-mercaptoethanol (0.1 mM) (Sigma), 1% antibiotic-antimycotic (Thermo Fisher Scientific), and basic fibroblast growth factor (10 ng/ml) (bFGF, PeproTech). The human iPSC medium was changed every other day for 6 days (Day8-14). Additionally, sodium butyrate (0.5 mM) (Sigma) was added into the medium until colonies appeared. After culture of the infected B cells for 6 days (Day8-14), the medium was changed to MEF culture medium containing bFGF (10 ng/ml) until colonies were picked up. Established BiPSCs were maintained on a 6-well plate coated with iMatrix-511 (Nippi, Tokyo, Japan) instead of feeder cells in Complete StemFit ® AK02N (BiPSC medium) (ReproCELL, Yokohama, Japan). The human iPSC culture medium was changed every day. DNA extraction. Genomic DNA was isolated with the DNAiso kit (Takara, Kusatsu, Japan) according to the manufacturer's instructions. Briefly, the cell pellet was suspended in DNAiso reagent. After removal of the cell debris by centrifugation, the DNA was precipitated by adding ethanol and was recovered by centrifugation at 4,000 × g. Establishment of BiPSCs with AID expression regulated by the doxycycline-controlled (Tet-off) system. AID cDNA was amplified from pcDNA3.1AID (a kind gift of Dr. Yakushijin, Ehime University School of Medicine) 32 with KOD plus DNA polymerase using the primers: hAID-BamHI, 5′-TGGGATCCgccaccatggac-3′ and hAID-EcoRI, 5′-CAGAATTCtcaaagtcccaaagtacg-3′. Amplified DNA was digested with BamHI and EcoRI, and cloned into the BamHI/EcoRI sites of pRetroX Tight Pur vector (Clontech) to form pRetroX Tight AID Pur. The DNA sequence of the cloned AID fragment was confirmed. Next, pRetroX Tight AID Pur was transfected into gp293 packaging cells with pMD2.G and the culture supernatant was recovered 3 days later. The supernatant containing packaged retrovirus pseudotyped with VSV-G was added to tTA2 s expressing BiPSC13 and MIB2-6 cells. Puromycin resistant cell clones were picked and cultured in the presence of doxycycline (0.2 mg/ml) to prevent AID expression.

Detection of monoclonal immunoglobulin heavy (IgH
Immunofluorescence staining. BiPSCs were washed with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature (RT). After washing with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min at RT, washed 3 times with PBS, and blocked in PBS-10% goat serum for 30 min. Antibodies (diluted with 1.5% goat sera) against Oct4 (Abcam, Tokyo, Japan), SSEA3 (Abcam), SSEA4 (Abcam), Nanog  Western blot analysis. Cells were lysed in lysis buffer (0.5% NP-40, 1% TritonX-100, 150 mM NaCl, and 1 mM EDTA in 20 mM Tris, pH 7.5) in the presence of a protease (Nakarai Tesque, Kyoto, Japan) and a PhosSTOP phosphatase (Roche Diagnosis, Tokyo, Japan) inhibitor cocktail. After 30 min on ice, the lysates were centrifuged at 14,000 rpm for 30 min at 4 °C and the supernatants were collected. Protein samples (70 µg aliquots) were separated in a Mini-PROTEAN TGX precast Gel (BIO-RAD, Hercules, CA, USA) and transferred to a nitrocellulose membrane (BIO-RAD). The membrane was immunoblotted with the indicated antibodies, and the signal was visualized using enhanced chemiluminescence (ECL) Western blotting detection reagents (BIO-RAD) and detected with the ChemDoc XRS+ Imaging System (BIO-RAD). The primary antibodies used included anti-AICDA (Abcam, Tokyo, Japan) and anti-GAPDH (Abcam) and the second antibody was goat anti-Rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA).