ALBERT

Description: Abstract 909 Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into all blood lineages. Lineage commitment can take place at any stage from daughter cells of HSCs to multipotent progenitors. We have previously suggested that lineage commitment takes place at the level of HSCs (Takano et al. 2004). According to our asymmetric model, one HSC gives rise to two daughter cells consisting of one HSC and one lineage-committed progenitor. To verify this model, we performed a series of single-cell transplantation, followed by paired daughter cell experiments. The Ly5 congenic system has been commonly used to distinguish test donor cells from recipient cells and competitor cells. Since the Ly5 antigen is only expressed in leukocytes, platelets and erythrocytes have not been examined for evaluation of multi-lineage reconstitution. To assess lineage contribution more precisely, we have recently generated a transgenic mouse line in which Kusabira Orange (KuO) is expressed in all blood lineages including platelets and erythrocytes. CD150+CD41−CD34−/lowc-Kit+Sca-1+Lin− (CD150+CD41−CD34−KSL) cells, in which HSCs are highly enriched (Kiel et al. 2005, Morita et al. 2010), and CD150−CD41−CD34−KSL cells were individually isolated from KuO transgenic mice (B6-Ly5.1), and were transplanted into lethally irradiated mice (B6-Ly5.2), along with 2×105 bone marrow cells (B6-Ly5.1/Ly5.2). For secondary transplantation, 1×107 bone marrow cells from recipient mice were transplanted to lethally irradiated mice. Peripheral blood cells of primary and secondary recipient mice were periodically analyzed after transplantation. In some mice, full-lineage reconstitution was detected after transplantation with single CD150+CD41−CD34−KSL cells. In others, only common myeloid progenitor-type reconstitution was detected whereas common lymphoid progenitor-type reconstitution was not detected at all. Interestingly, megakaryocyte lineage (Meg)-restricted reconstitution and Meg/erythrocyte lineage-restricted (ME) reconstitution were observed after transplantation with single CD150+CD41−CD34−KSL cells, but not after transplantation with single CD150−CD41−CD34−KSL cells. These results suggest that Meg and ME progenitors are closely related to HSCs in their development. Furthermore, we performed paired daughter cell assays combined with single cell transplantation. Following division of CD150+CD41−CD34−KSL cells in culture, individual two daughter cells were separated by micromanipulation techniques and were transplanted into lethally irradiated mice with 2 × 105 competitor cells. Preliminary data showed in effect that the recipient mice of each single daughter cell derived from one CD150+CD41−CD34−KSL cell exhibited asymmetric differentiation; one with full-lineage reconstitution and the other with Meg-restricted reconstitution. Our findings offer a new perspective of HSC differentiation pathways. Disclosures: No relevant conflicts of interest to declare.

Description: Generation of induced pluripotent stem cells (iPSCs) generally uses fibroblastic cells, but other cell sources may prove useful in both research and clinical settings. Although proof of cellular origin requires genetic-marker identification in both target cells and established iPSCs, somatic cells other than mature lymphocytes mostly lack such markers. Here we show definitive proof of direct reprogramming of murine hematopoietic cells with no rearranged genes. Using iPSC factor transduction, we successfully derived iPSCs from bone marrow progenitor cells obtained from a mouse whose hematopoiesis was reconstituted from a single congenic hematopoietic stem cell. Established clones were demonstrated to be genetically identical to the transplanted single hematopoietic stem cell, thus proving their cellular origin. These hematopoietic cell–derived iPSCs showed typical characteristics of iPSCs, including the ability to contribute to chimerism in mice. These results will prompt further use of hematopoietic cells for iPSC generation while enabling definitive studies to test how cellular sources influence characteristics of descendant iPSCs.

Description: Abstract 1888 Hematopoietic stem cells (HSCs) represent the unique cell population capable of self-renewal and multi-lineage differentiation, thereby lifelong sustainment of the hematopoiesis. HSC transplantation has proven beneficial for various diseases, it is therefore important to elucidate the molecular determinants for successful HSC engraftment. Signaling through the chemokine receptor CXCR4 has been implicated in HSC engraftment by the observation that transplantation of HSCs lacking this molecule results in poor hematopoietic reconstitution. Because this impairment, however, can be attributed to the defects in any of the post-transplantation processes that include bone marrow (BM)-homing, -repopulation, or –retention, it is still unclear whether CXCR4 plays an essential role in HSC self-renewal upon transplantation. To elucidate the role of CXCR4 signaling in HSC self-renewal in conjunction with transplantation, we used a purified CD34neg/low c-Kit+ Sca-1+ Lineage-markerneg population as the defined stem cell source. As a loss-of-function study, CXCR4 was conditionally deleted in HSCs before transplantation. As a gain-of-function study, we generated the HSC populations overexpressing either wild-type (wt)- or C-terminal truncated (δC)-CXCR4 (OE-HSCs), the latter of which is known to exhibit enhancement in the SDF-1 signaling, by gene transfer and subsequent cell sorting. We compared these cells with control HSCs in in vitro assays with regard to the biological characteristics including chemotaxis, proliferation, colony formation, and cobblestone-area (CA) forming ability. To dissect in vivo post-transplantation processes, we investigated hematopoietic repopulation kinetics in the recipient BM at the homing/lodging phase (within 1 wk) and the early repopulation phase (2–3 wks) for the above test HSCs. The self-renewal potential of each HSC population was estimated by competitive repopulation assay. In vitro studies: OE-HSCs with wt- or δC-CXCR4 exhibited enhanced chemotaxis and proliferation in response to SDF1, confirming the gain-of-function effects of these modifications. CA forming ability was greater in OE-HSCs with δC-CXCR4 than control counterparts and absent in CXCR4-KO HSCs, suggesting the critical role of CXCR4-signaling in HSC proliferation in the presence of stromal support. In vivo studies: 1) the homing/lodging phase. Unexpectedly, we did not find significant alteration in the numbers of early progenies detectable in recipient BM 3 days after transplantation of HSCs receiving either loss- or gain-of-function modification to CXCR4, indicating that this signaling is indispensable in HSC homing. 2) the early repopulation phase. Impairment of hematopoietic repopulation in BM became evident for CXCR4-KO HSCs through 2–3 wks. On the other hand, OE-HSCs with CXCR4, more remarkably of ΔC-mutation, showed enhanced BM repopulation kinetics at ∼3 wks post transplantation, suggesting the importance of CXCR4 signaling in HSC amplification at this post-transplantation phase. 3) long-term hematopoiesis. CXCR4-KO-HSCs showed poor hematopoietic reconstitution potentials, consistent with previous observations. Interestingly, impaired peripheral repopulation was also observed with OE-HSCs with wt- or ΔC-CXCR4. Further characterization revealed that the recipients of CXCR4-overexpressing HSCs did retain their progenies, which showed multilineage differentiation, but exhibited impaired release of mature leukocytes from the BM to the peripheral blood. Most importantly, however, test-cell chimerism in the long-term HSC fraction was significantly higher in the mice receiving OE-HSCs with CXCR4, especially of ΔC-type, than those transplanted with control HSCs, indicating that the augmentation of CXCR4 signaling enhanced competitive repopulation ability of HSCs. These modified HSCs demonstrated repopulation abilities also in secondary recipients. We demonstrated that CXCR4 signaling is indispensible for HSC homing and that continuous overexpression of CXCR4 cannot benefit the peripheral reconstitution in contrary to the expectation. More importantly, our studies showed that augmentation of CXCR4 signaling leads to HSC expansion in vivo upon transplantation. We thus conclude that CXCR4 signaling has a role in HSC self-renewal and that its regulation may find the approach that will improve HSC transplantation outcomes. Disclosures: No relevant conflicts of interest to declare.

Description: We previously established normal B cell-derived induced pluripotent stem cells (BiPSCs; BiPSC13 and MIB2-6). BiPSCs are known to 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). Using these BiPSCs, we attempted to prove the existence of abnormal B cells, which are thought to be myeloma-initiating cells, originating from mature B cells transformed by reprogramming. We speculated that BiPSCs could develop into myeloma-initiating cells that undergo chromosomal translocation or gain genetic abnormalities during redifferentiation into mature B cells. First, using the comet assay, we confirmed the DNA-damaging effect of AID in BiPSCs-AID. Secondly, we differentiated BiPSC13-AID into CD34+/CD38-/CD43-/CD45- cells by co-culture with stromal cells (mouse embryo cell line: 10T1/2), and we subsequently transplanted the cells into the bone marrow (BM) of immunodeficient NRG mice. The presence of CD34+ cells was still observed in mouse BM 4 months after transplantation; however, no differentiation into B cells was detected. Next, using the CRISPR/Cas9 system, we attempted to make BiPSCs with chromosomal translocation t(11;14); we succeeded in establishing a 293T cell line with t(11;14), then confirmed t(11;14) in MIB2-6-AID, and the clone is now being established. Furthermore, we established BiPSC13-Pax5, which can be induced by the tet-off system to express Pax5, and we then differentiated BiPSC13-Pax5 into CD34+/Pax5+/CD38-/CD43-/CD45- cells by co-culture with stromal cells (10T1/2). We expect that the HPCs or hematopoietic stem cells (HSCs) derived from BiPSCs will further differentiate into B cells due to the expression of Pax5 in the BM of NRG mouse. We also established BiPSC13-AID-p53-/-, in which p53 was deleted using the CRISPR/Cas9 system, and the cells differentiated into HPCs. Interestingly, we detected some CD43+/CD45+ cells among CD34+/CD38- cells after the co-culture of BiPSC13-AID with aorta-gonad-mesonephros-derived stromal cell (AGM-S3) instead of 10T1/2. Therefore, AGM-S3 may promote the differentiation of BiPSCs into cells that are more similar to HSCs. These CD34+ cells differentiated from BiPSCs will be transplanted into the BM of NRG mouse. Disclosures Hanamura: CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Company, Limited: Research Funding; Fujimoto Pharmaceutical Corporation: Research Funding; Takeda Pharmaceutical Company Limited.: Other: Lecture fee; Bristol-Myers Squibb: Other: Lecture fee, Research Funding; Celgene: Other: Lecture fee.

Description: 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.

Description: Induced pluripotent stem cells (iPSCs) have been established from a variety of somatic cells not only for regenerative medicine but also for studies of the pathogenesis of inherited genetic diseases or neoplasms. In iPSCs established from T cells, the rearrangement of the T cell receptor (TCR) of the established T cell-derived iPSCs (TiPSCs) took over that of the original T cells. If B cell-derived iPSCs (BiPSCs) could be similarly established from mature B cells or plasma cells, the rearrangement of the B cell receptor (BCR) of the BiPSCs would take over that of the original B cell. BiPSCs were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) together with ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα) using a Sendai virus vector (Bueno C, et al. Leukemia 2016). We were able to also establish BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes using a method different from the above. The main points of our method are consecutive transfection of a high concentration of Yamanaka factors into B cells, which were pre-stimulated with IL-21 and CD40L, using a retrovirus vector, and centrifugations of the cells after their activation by IL-4, IL-2, and CD40-ligand (CD40L) on a retronectin coated plate. We further 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. We hypothesized that the origin of a myeloma cell is a reprogrammed mature B cell, in which reciprocal chromosome translocation occurs by double stranded breakage (DSB) of DNA induced by AID activation in the nonproductive (nonfunctional) allele of chromosome 14. First, we did not detect an increase in dicentric chromosome (DIC) formation, which is evidence of DSB of DNA, in the BiPSCs-A. We next analyzed the ability of these BiPSCs to differentiate into hematopoietic stem cells (HSCs). Both the parental BiPSCs and BiPSCs-A were capable of differentiating into HSCs as judged by confirmation of CD34 expression and colony-formation of macrophages, granulocytes, and erythrocytes from CD34-positive cells. However, these cells were negative for CD38, CD43, and CD45; we therefore think that these CD34+/CD38-/CD43-/CD45- cells might be hematoendothelial cells as Maxim proposed previously (Vodyanik MA, et al. Blood 2006). Furthermore, both BiPSCs with induced AID expression and BiPSCs without induced AID expression were capable of differentiating into HSCs. Based on the findings regarding the differentiation of BiPSCs-A into HSCs and their retention of the IgH gene rearrangement, there is a possibility that the induction of AID expression might induce chromosomal translocations in the process of differentiation of these BiPSCs into HSCs and further into B cells in experiments using mouse. Thus these BiPSCs might be useful in elucidating the tumor origin of abnormal B cells in B cell tumor formation. These cells might be especially useful in understanding multiple myeloma, which is thought to originate from germinal center (GC) or post-GC B cells and has a productive (functional) allele with IgH gene rearrangement that produces M-protein, and another nonproductive (nonfunctional) allele of chromosome 14. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2970 Poster Board II-946 Acquired uniparental disomy (aUPD) is a common feature of myeloid neoplasms, especially myelodysplastic syndromes (MDS) / myeloploriferative neoplasms (MPN). aUPDs preferentially affected several chromosomal arms in distinct subsets of patients, and frequently associated with mutated oncogenes and tumour suppressor genes. Among these, the most common aUPDs are those involving 11q, which defined a unique subset of myeloid neoplasms that were clinically characterized by frequent diagnosis of chronic myelomonocytic leukaemia (CMML) with normal karyotypes. Recently, we and other groups reported that 11qUPD are genetically defined by the presence of homozygous mutations of C-CBL. C-CBL proto-oncogene is the cellular homolog of the v-Cbl transforming gene of the Cas NS-1 murine leukemia virus. C-CBL is thought to be involved in the negative modulation of tyrosine kinase signalling, primarily through their E3 ubiquitin ligase activity that is responsible for the down-regulation of activated tyrosine kinases. As expected from the latter function, we demonstrated that wild-type C-CBL has tumour suppressor functions; c-Cbl null mice showed expanded hematopoietic progenitor pools, promoted blastic crisis induced by a bcr/abl transgene, and spontaneous development of late-onset invasive cancers in complete penetrance. On the other hand, mutated C-CBL showed clear oncogenic potential; all tested mutants strongly transformed NIH3T3 fibroblasts, and prolonged replating capacity of hematopoietic progenitors. All reported C-CBL mutations involved the linker-RING finger domains that are central to the E3 ubiquitin ligase activity. We demonstrated that mutated C-CBL not only lost their E3 ubiquitin ligase activity, but also inhibited that of wild-type C-CBL, leading to prolonged activation of a broad spectrum of tyrosine kinases after ligand stimulations in fibroblasts and hematopoietic cells. In accordance with this, c-Cbl−/− hematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines, but unexpectedly, transduction of C-CBL mutants into c-Cbl−/− HSPCs further augmented the sensitivity to a broader spectrum of cytokines, indicating the presence of gain-of-function in mutated C-CBL that is not simply mediated by inhibition of wild-type C-CBL functions. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in the c-Cbl+/+ background or by co-transduction of wild-type C-CBL, which may suggest the pathogenic importance of loss of wild-type c-Cbl alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a novel insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some of myeloid cancer subsets. Currently, further functional studies regarding the molecular mechanism of the gain-of-function are ongoing. Disclosures: Omine: Alexion: Consultancy, Research Funding.

Description: Ten patients with adenosine deaminase deficiency (ADA−) have been enrolled in gene therapy clinical trials since the first patient was treated in September 1990. We describe a Japanese ADA− severe combined immune deficiency (SCID) patient who has received periodic infusions of genetically modified autologous T lymphocytes transduced with the human ADA cDNA containing retroviral vector LASN. The percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% during the 12 months since the fourth infusion. ADA enzyme activity in the patient's circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual and was associated with increased T-lymphocyte counts and improvement of the patient's immune function. The results obtained in this trial are in agreement with previously published observations and support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA−SCID.