scholarly journals Establishment of Induced Pluripotent Stem Cells from Lymph Node B Cells and Induction of Aid Expression in Their Differentiation into Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5049-5049
Author(s):  
Fumihiko Kawamura ◽  
Inaki Makoto ◽  
Atsushi Katafuchi ◽  
Yu Abe ◽  
Naohiro Tsuyama ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
B Cell ◽  
Gene Rearrangement ◽  
Cell Receptor ◽  
Chromosome 14 ◽  
Hematopoietic Stem ◽  
Yamanaka Factors ◽  
Igh Gene Rearrangement ◽  
Induced Pluripotent

Abstract 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.

Conditional Knockout of Sox4 Markedly Affects B Lymphopoiesis in Adult Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2524-2524
Author(s):  
Baohua Sun ◽  
Saradhi Mallampati ◽  
Yun Gong ◽  
Donghai Wang ◽  
M. James You ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Conditional Knockout ◽  
B Lymphopoiesis ◽  
Cell Stage ◽  
Hematopoietic Stem ◽  
Adult Mice

Abstract Abstract 2524 Poster Board II-501 B cells constitute an integral part of the immune system. The development of mature B cells from hematopoietic stem cells is a complex process that is regulated in a hierarchical order by various proteins, particularly transcription factors. Sox4 is a SRY-related HMG box containing transcription factor and is known to be involved in B cell development. However, the role of Sox4 in various stages of B cell development has not been systematically investigated. In this study we used a conditional knockout mouse strain and studied the effect of Sox4 deletion in B lymphopoiesis in adult mice. We crossed the Sox4-floxed mice with different Cre mouse strains that were expected to delete the floxed Sox4 gene at different B cell developmental stages. These Cre strains included Vav-iCre (expressed in hematopoietic stem cell stage, starting from early embryos), MX1-Cre (expression in hematopoietic stem cells, induced by pIpC injection in adults), MB1-Cre (expressed in B cells, starting from early progenitor cells in embryos), and CD21-Cre (expressed in mature B cells). We demonstrated that deletion of Sox4 caused an arrest of B lymphopoiesis at the transition from pre-pro-B cell (fraction A) stage to pro-B cell stage (fraction B): fraction A cells are slightly reduced in number whereas fraction B and later stage cells are nearly absent. The pre-pro-B cells from the Sox4 knockout mice retain a population of AA4.1+ cells, which are considered to be developed into B cells. Deletion of Sox4 in early embryonic stage (Vav-iCre) or in adults (Mx1-Cre) results in a similar phenotype on B lymphopoiesis, except that peritoneal B1 cells appear to be affected with Vav-iCre, but not with Mx1-Cre. MB1-Cre gave rise to similar results as did Vav-iCre, but the arrest was not as dramatic as with Vav-iCre. CD21-Cre produced no significant difference in B cell phenotype. These data suggested that Sox4 is required for early B cell development at the transition from pre-pro-B cells to pro-B cells and is not required for mature B cells. We are currently investigating the transcription program of this transcription factor in B cell development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Distinct pathways regulated by menin and by MLL1 in hematopoietic stem cells and developing B cells

Blood ◽  
2013 ◽  
Vol 122 (12) ◽  
pp. 2039-2046 ◽  
Author(s):  
Bin E. Li ◽  
Tao Gan ◽  
Matthew Meyerson ◽  
Terence H. Rabbitts ◽  
Patricia Ernst
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
B Cells ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Hematopoietic Stem Cell ◽  
B Cell Differentiation ◽  
Hematopoietic Stem ◽  
Key Points

Key Points MLL1 does not require interaction with menin to maintain hematopoietic stem cell homeostasis. Menin and MLL1 are both critical during B-cell differentiation, but largely through distinct pathways.

scholarly journals Silencing of B Cell Receptor Signals in Human Naive B Cells

2002 ◽  
Vol 196 (10) ◽  
pp. 1291-1305 ◽  
Author(s):  
Niklas Feldhahn ◽  
Ines Schwering ◽  
Sanggyu Lee ◽  
Maria Wartenberg ◽  
Florian Klein ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Large Group ◽  
Bcr Signaling

To identify changes in the regulation of B cell receptor (BCR) signals during the development of human B cells, we generated genome-wide gene expression profiles using the serial analysis of gene expression (SAGE) technique for CD34+ hematopoietic stem cells (HSCs), pre-B cells, naive, germinal center (GC), and memory B cells. Comparing these SAGE profiles, genes encoding positive regulators of BCR signaling were expressed at consistently lower levels in naive B cells than in all other B cell subsets. Conversely, a large group of inhibitory signaling molecules, mostly belonging to the immunoglobulin superfamily (IgSF), were specifically or predominantly expressed in naive B cells. The quantitative differences observed by SAGE were corroborated by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) and flow cytometry. In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B cells at least partly results from interleukin (IL)-4 receptor signaling. Conversely, activation or impairment of the inhibitory IgSF receptor LIRB1 affected BCR-dependent Ca2+ mobilization only in naive but not memory B cells. Thus, LIRB1 and IL-4 may represent components of two nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity maturation. In memory B cells, facilitated responsiveness upon reencounter of the immunizing antigen may result from amplification of BCR signals at virtually all levels of signal transduction.

scholarly journals Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer

Blood ◽  
2004 ◽  
Vol 104 (5) ◽  
pp. 1281-1290 ◽  
Author(s):  
Phyllis W. Yu ◽  
Ruby S. Tabuchi ◽  
Roberta M. Kato ◽  
Alexander Astrakhan ◽  
Stephanie Humblet-Baron ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
Gene Transfer ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Serum Immunoglobulin ◽  
Hematopoietic Stem ◽  
B Lineage ◽  
And Function

Abstract X-linked agammaglobulinemia (XLA) is a human immunodeficiency caused by mutations in Bruton tyrosine kinase (Btk) and characterized by an arrest in early B-cell development, near absence of serum immunoglobulin, and recurrent bacterial infections. Using Btk- and Tec-deficient mice (BtkTec–/–) as a model for XLA, we determined if Btk gene therapy could correct this disorder. Bone marrow (BM) from 5-fluorouracil (5FU)–treated BtkTec–/– mice was transduced with a retroviral vector expressing human Btk and transplanted into BtkTec–/– recipients. Mice engrafted with transduced hematopoietic cells exhibited rescue of both primary and peripheral B-lineage development, recovery of peritoneal B1 B cells, and correction of serum immunoglobulin M (IgM) and IgG3 levels. Gene transfer also restored T-independent type II immune responses, and B-cell antigen receptor (BCR) proliferative responses. B-cell progenitors derived from Btk-transduced stem cells exhibited higher levels of Btk expression than non-B cells; and marking studies demonstrated a selective advantage for Btk-transduced B-lineage cells. BM derived from primary recipients also rescued Btk-dependent function in secondary hosts that had received a transplant. Together, these data demonstrate that gene transfer into hematopoietic stem cells can reconstitute Btk-dependent B-cell development and function in vivo, and strongly support the feasibility of pursuing Btk gene transfer for XLA.

scholarly journals Attempt to Prove the Existence of Abnormal B Lymphocyte As Myeloma-Initiating Cells from B Cell-Derived Induced Pluripotent Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1896-1896
Author(s):  
Aki Yanagi ◽  
Naohiro Tsuyama ◽  
Yukari Yanai ◽  
Yu Abe ◽  
Misaki Sugai ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Research Funding ◽  
Chromosomal Translocation ◽  
Company Limited ◽  
Cd34 Cells ◽  
Induced Pluripotent

Abstract 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.

scholarly journals Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1590-1600 ◽  
Author(s):  
Hiromi Iwasaki ◽  
Chamorro Somoza ◽  
Hirokazu Shigematsu ◽  
Estelle A. Duprez ◽  
Junko Iwasaki-Arai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
B Cells ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Cell Maturation ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
B Cell Maturation ◽  
Lymphoid Progenitors

Abstract The PU.1 transcription factor is a key regulator of hematopoietic development, but its role at each hematopoietic stage remains unclear. In particular, the expression of PU.1 in hematopoietic stem cells (HSCs) could simply represent “priming” of genes related to downstream myelolymphoid lineages. By using a conditional PU.1 knock-out model, we here show that HSCs express PU.1, and its constitutive expression is necessary for maintenance of the HSC pool in the bone marrow. Bone marrow HSCs disrupted with PU.1 in situ could not maintain hematopoiesis and were outcompeted by normal HSCs. PU.1-deficient HSCs also failed to generate the earliest myeloid and lymphoid progenitors. PU.1 disruption in granulocyte/monocyte-committed progenitors blocked their maturation but not proliferation, resulting in myeloblast colony formation. PU.1 disruption in common lymphoid progenitors, however, did not prevent their B-cell maturation. In vivo disruption of PU.1 in mature B cells by the CD19-Cre locus did not affect B-cell maturation, and PU.1-deficient mature B cells displayed normal proliferation in response to mitogenic signals including the cross-linking of surface immunoglobulin M (IgM). Thus, PU.1 plays indispensable and distinct roles in hematopoietic development through supporting HSC self-renewal as well as commitment and maturation of myeloid and lymphoid lineages.

Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells

Blood ◽  
2007 ◽  
Vol 109 (9) ◽  
pp. 3706-3712 ◽  
Author(s):  
Jiang Zhu ◽  
Russell Garrett ◽  
Younghun Jung ◽  
Yi Zhang ◽  
Nacksung Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Developmental Process ◽  
Bursa Of Fabricius ◽  
B Lymphopoiesis ◽  
Hematopoietic Stem ◽  
Cell Commitment

Abstract Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin−Sca-1+Rag-2− BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Δ-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.

PBSCT Similar to BMT Allows Active B-Lymphopoiesis Unless Severe GVHD Occurs.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5351-5351
Author(s):  
Julia Winkler ◽  
Armin Biller ◽  
Natalie Schub ◽  
Wolf Rösler ◽  
Kerstin Eckart-Schäfer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
B Cells ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
First Year ◽  
B Lymphopoiesis ◽  
Hematopoietic Stem ◽  
Precursor B Cells ◽  
B Cell Precursors

Abstract Long-term engraftment after stem cell transplantation is dependent on pluripotent hematopoietic stem cells capable to multi-lineage reconstitution. The engraftment and prolonged repopulation of B lymphoid progenitor cells is dependent on pluripotent hematopoietic stem cells. We have recently shown that a high number of B-cell progenitors is detectable in the bone marrow from patients both after allogeneic PBSCT and after allogeneic BMT. The percentage of CD19/CD10+ pro-B/pre-B cells showed a high variability and ranged from 0 to 98% of all B-lymphocytes. Interestingly, no difference in numbers of precursor B-lymphocytes between BM recipients and PBSC recipients was found. In micro-satellite analysis, both in PBSC and in BM recipients the B cell precursors were derived exclusively from the donor. To identify predictors of B lymphopoiesis in the bone marrow after stem cells transplantation, a stepwise, multiple regression analysis was performed. The stem cell source, BM or PBSC, the occurrence of GVHD, the conditioning with or without TBI and whether ATG was given, were entered as categorical variables. Time point of BM sampling after transplantation, age of the donor and the recipient, the quantity of stem cell transplanted and the T cell content of the BM sample served as continuous variables. Time elapsed after transplantation (p=0.002) and severe GVHD (aGVHD grade III/IV or extensive cGVHD) (p=0.036) could be identified as parameters with an independent influence on the percentage of B cell precursors. The highest percentage of precursor B cells was found during the first year after transplantation, unless severe GvHD was present, leading to neglible precursor B-cells. This hyperactive B lymphopoiesis in the first year after transplantation contrasts to low circulating B cell counts. Finally, it could be formally demonstrated, that G-CSF mobilized PBSC contain hematopoietic stem cells capable of long-term reconstitution of the B cell compartment.

The Dual Bromodomain Protein Brd2 Controls Primary B Cell Mitogenesis and Cell Cycle in Mice Reconstituted with Lentivirus-Transduced HSCs

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2465-2465
Author(s):  
Wanda P. Blanton ◽  
Fangnian Wang ◽  
Hongsheng Liu ◽  
Paul Romesser ◽  
Douglas Faller ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Brdu Incorporation ◽  
Cell Compartment ◽  
Hematopoietic Stem

Abstract Transcriptional control of cellular proliferation and differentiation is critically important in hematopoiesis; specifically, the role of chromatin-dependent regulatory processes in this context is poorly understood. The human BRD2 proto-oncogene encodes a double bromodomain protein that binds to acetylated histone H4 in chromatin and is located within the MHC class II locus, suggesting Brd2 plays a role in immunity. However, BRD2 shares no sequence similarity with other MHC genes, nor is Brd2 involved in antigen processing, but rather it plays a role in mitogenic signal transduction. We have previously found that whole-body knockout of Brd2 is lethal to mice. However, when Brd2 was expressed constitutively in the B cells of transgenic mice, Brd2 binds E2F proteins, histone acetylases and Swi/Snf complexes, and co-activates cyclin A leading to B cell lymphoma and leukemia. Importantly, elevated levels of Brd2 have been reported in primary malignant B cells from human and mouse. We therefore hypothesize that Brd2 multiprotein complexes, working through chromatin modification, are crucial in the control of the cell cycle and in the mitogen responsiveness and proliferation of the B cell compartment. To study the effects of Brd2 in B cell development and proliferation, we performed bone marrow transplants of hematopoietic stem cells in a chimeric mouse model. Hematopoietic stem cells were sorted from CD45.1 donor mice with the characteristic ‘side population’ profile by flow cytometry and transduced with lentivirus containing vectors for Brd2 overexpression, shRNA knockdown, or control vectors. Recipient CD45.2 mice were lethally irradiated and a functional immune system was successfully reconstituted with donor cells and CD45.2 competitor BM cells. Mice were immunophenotyped and functional B cell mitogenic capacity was examined by BrdU incorporation into LPS-stimulated B cells. We found that in the spleen, Brd2 expression dramatically expands the CD45.1 (but not CD45.2) B cell compartment at the expense of T cells and renders B cells mitogenically hypersensitive. Compared with control, there was an increase in BrdU incorporation at 24 and 48 hours (29.8% v. 43.5% at T=24 h; 56.9% v. 66.7% at T=48 h). Preliminary results also suggest that B cell development was skewed in the bone marrow and periphery towards B1a phenotype. Moreover, downregulation of Brd2 via shRNA blocked cyclin A transcription and completely arrested B cell development and proliferation. Taken together, these data suggest that Brd2, through epigenetic regulation of the cell cycle, plays an important role in B-lymphopoiesis, proliferation, and stimulation.

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