scholarly journals Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment.

1994 ◽  
Vol 14 (1) ◽  
pp. 382-390 ◽  
Author(s):  
S Okada ◽  
Z Q Wang ◽  
A E Grigoriadis ◽  
E F Wagner ◽  
T von Rüden
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Lymphoid Cells ◽  
Mutant Mice ◽  
Hematopoietic Stem ◽  
Developmental Potential

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment

1994 ◽  
Vol 14 (1) ◽  
pp. 382-390
Author(s):  
S Okada ◽  
Z Q Wang ◽  
A E Grigoriadis ◽  
E F Wagner ◽  
T von Rüden
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Lymphoid Cells ◽  
Mutant Mice ◽  
Hematopoietic Stem ◽  
Developmental Potential

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.

LRF Regulates Self-Renewal of Hematopoietic Stem Cells by Blocking Notch1-Mediated T Cell Differentiation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 75-75 ◽  
Author(s):  
Sung-UK Lee ◽  
Manami Maeda ◽  
Nagisa Sakurai ◽  
Julie Teruya-Feldstein ◽  
Freddy Radtke ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Knockout Mice ◽  
Cell Development ◽  
B Cell Development ◽  
Hematopoietic Stem

Abstract The proto-oncogene LRF, encoded by the Zbtb7a gene, is a transcriptional repressor that belongs to the POK (POZ/BTB and KrŸppel) protein family. Along with its oncogenic property, recent evidence has shown that POK proteins play distinct roles in hematopoiesis and immune system development. Conditional inactivation of the LRF gene in mouse hematopoietic stem cells (HSCs) results in the development of CD4/8 double positive (DP) T cells in bone marrow (BM) at the expense of B cell development (Maeda et al. Science 2007). While LRF acts as a master regulator of B versus T lymphoid lineage fate decision by suppressing Notch-mediated signals, it is unclear as to which Notch genes LRF targets and whether LRF is required for the maintenance of HSCs per se. To address these questions, we analyzed HSC/progenitor population of conditional LRF knockout mice (LRFF/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRFF/FNotch1F/FMx1-Cre). In the absence of Notch1, LRF deficient HSCs/lymphoid progenitors (LRFF/FNotch1F/FMx1-Cre) could successfully give rise to early B cells (Pro B, Pre B and immature B). There were no abnormal DP-T cells seen in the BM, suggesting that LRF primarily targets Notch1 at the HSC/progenitor stages to maintain normal lymphoid development. However the loss of the LRF gene did not rescue the phenotype of Notch1F/FMx1-Cre mice (Radtke et al. Immunity 1999). Immature B cell development in the thymus was still observed in LRFF/FNotch1F/FMx1-Cre mice, suggesting that LRF acts genetically upstream of Notch1 during the early lymphocyte development. Notably, LRFF/FNotch1F/FMx1-Cre mice still exhibit a block of terminal erythroid differentiation and macrocytic anemia as seen in LRFF/FMx1-Cre mice. Thus, LRF is required for erythropoiesis via Notch-independent mechanisms. To further identify distinct HSC/progenitor compartments, we performed multicolor-FACS analysis utilizing antibodies for SLAM family members (CD41, CD48 and CD150), c-Kit, Sca-1, Flt3, IL7R-α, Vcam-1 and lineage markers (Lin). Remarkably, no Flt3 positive HSC/progenitors were observed in LRFF/FMx1-Cre mice. While IL7R-α+ T cell precursors (IL7Rα+Lin-Sca1+c-Kit+Flt3-), which were previously reported as common lymphoid progenitors (Maeda et al. Science 2007), existed abundantly. Absolute numbers of the long-term HSCs (LT-HSCs), defined as CD150+CD48-Flt3-Vcam-1+IL7Rα-LSK (Lin-Sca1+c-Kit+), were significantly reduced in LRFF/FMx1-Cre mice one month after pIpC injection. At the same time, CD150+CD48high+Flt3-Vcam-1-IL7Rα-LSK cells, which are likely T-committed lymphoid precursors, are increased in LRFF/FMx1-Cre mice. To investigate the presence of a population of quiescent HSC/progenitors, we treated LRFF/FMx1-Cre mice with 5-fluorouracil (5-FU), a S phase-specific cytotoxic chemotherapeutic agent, and examined recovery of HSCs in BM. LT-HSCs in LRFF/FMx1-Cre mice did not repopulate as many as their counterpart one month after 5-FU treatment. Our data indicates that LRF deficient HSCs are unable to maintain its quiescent status and are on the state of cell differentiation toward T cells due to the high Notch activity. In fact, loss of the Notch1 gene partially rescued reduced LT-HSCs numbers seen in LRFF/FMx1-Cre mice.

The Obligate Role of TAK1 in the Survival of Hematopoietic Stem Cells and Progenitors in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 642-642
Author(s):  
Minghui Tang ◽  
Zhenbiao Xia ◽  
Shubin Zhang ◽  
Shanshan Zhang ◽  
Xudong Wei ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
B Cell ◽  
Knockout Mice ◽  
Mutant Mice ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Expression And Activity

Abstract TGFβ1-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of stress and proinflammatory signals. TAK1 can be activated by inflammation-mediating cytokines, including tumor necrosis factor-α (TNF-α and interleukin-1b (IL-1β), as well as by T- and B- cell receptors (TCR/BCR), and Toll-like receptors (TLRs) signals. Activated TAK1 induces the nuclear localization of NF-kB and the activation of JNK/AP1 by stimulating IKKβ and MKK3/MKK6 phosphorylation respectively. TAK1 has been found to play an important role in inflammation, immunity, T- and B-cell activation, and epithelial cell survival. The TAK1−/ − phenotype is lethal in mice at the early embryonic stage. We found higher levels of TAK1 expression and activity in hematopoietic stem cells and progenitors (HSC/Ps), and reduced expression and activity in differentiated mature hematopoietic cells. To study the role of TAK1 in bone marrow hematopoiesis, we generated inducible-TAK1 knockout mice by crossing TAK1loxp mice with Mx1Cre mice, the latter being an interferon-inducible Cre mouse line. After injection of polyI:C to induce the knockout, we found that all the TAK1 knockout mice died within 8 to 10 days after the first polyI:C injection, showing severe hematopoietic and other defects; heterozygotes were phenotypically comparable to wild-type control animals. The TAK1 deletion in these mice resulted in ablation of bone marrow hematopoiesis due to the loss of C-Kit+ HSC/Ps. Annexin-V staining showed a 3-fold increase in apoptosis in the C-Kit+ HSC/Ps from TAK1 mutant mice compared to those from littermate control mice. Almost all of the mutant animals showed intestinal bleeding as well as other hemorrhaging due to the significant reductions in platelet counts. In reciprocal bone marrow transplantation experiments, we found that the TAK1-mutant bone marrow microenvironment was able to support the growth and function of wild-type HSC/Ps, while HSC/Ps from TAK1−/ − mice failed to grow within the wild-type bone marrow microenvironment. These observations suggest that the bone marrow ablation phenotype which develops in TAK1-mutant mice is the result of intrinsic defects in HSC/P’s. We propose that TAK1-mutant HSC/Ps might mediate a survival signal for HSC/Ps stimulated by hematopoietic growth factors and cytokines, such as stem cell factor (SCF). The details of possible mechanisms by which this phenomenon might occur is currently under active investigation by our group.

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.

Dlk-1 Regulates B Cell Development Through Altered Bone Marrow Stromal Microenvironment, Not by Affecting Hematopoitic Stem/Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3465-3465
Author(s):  
Edyta Pawelczyk ◽  
Heba A Degheidy ◽  
Allison L Branchaw ◽  
Kenn Holmbeck ◽  
Steven R Bauer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Methyl Cellulose ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Microenvironment ◽  
Wild Type ◽  
Mineral Density ◽  
Hematopoietic Stem

Abstract Abstract 3465 Introduction: DLK-1(delta-like 1) is a member of the EGF-like homeotic protein family whose expression is known to influence cell fate decisions through cell-cell interactions. It is also known to influence the differentiation of bone marrow stromal cells (BMSC) and hematopoietic stem cells (HSC) in bone marrow. Recently, we reported the essential role of DLK-1 in B cell development, which showed that the absence of DLK-1 led to accumulation of the earliest B cell progenitors (pre-pro B cells or Fraction A (Fr A)) in bone marrow, an altered pattern of B cell development in the spleen, and an altered humoral immune response. The objective of this study was to determine whether alterations in the HSC compartment or the BMSC microenvironment contributed to Fr A accumulation in mdlk1−/− mice. Methods: The mdlk1−/− and wild type bone marrow osteoblast and HSC compartments were analyzed by multicolor flow cytometry and in vitro methyl-cellulose colony forming cell assays. Bone marrow harvested from mdlk1−/− and wild type mice was assessed for BMSCs colony forming efficiency (CFU-F) and cultured. Supernatants from cultured BMSCs were analyzed by protein arrays. Since osteoblasts are an important component of the bone marrow microenvironment, OPN+CD45-TER119-ALP+ osteoblasts were identified in the bone marrow and quantified by flow cytometry. Finally, the femurs of mdlk1−/− and wild type mice were analyzed by micro-computed tomography (uCT) scanning. Results: Using flow cytometry, we observed no statistically significant changes in the HSC and progenitor populations in the absence of DLK-1 in mice at 4 and 16 weeks of age. The results of methyl-cellulose assay confirmed the findings of flow cytometry experiments and showed no statistically significant differences in the number of CFU-G, CFU-GM, and CFU-M of 4 and 16 week old mdlk1−/− mice as compared to wild-type control mice. However, significant alterations in the microenvironment of the mdlkl −/− were observed. CFU-F efficiency of mdlk1−/− bone marrow BMSC isolated from 4 week old mice was significantly decreased when compared to age-matched controls. Furthermore, the uCT scans showed the mineral density of the femoral bone significantly decreased in 4 week old mdlk1−/− mice and the number of osteoblast cells analyzed by flow cytometry was decreased by 10%. The analysis of BMSC supernatants revealed a striking down regulation of factors associated with osteoblast function and differentiation such as osteoactivin, PF-4, Follstatin-like 1, Frizzled-6, IGF-1, M-CSF, DKK-1 and others. Conclusions: Our results indicate that accumulation of the earliest B cell progenitors with DLK-1 ablation is the result of multiple defects in the bone marrow microenvironment including decreased CFU-F, decreased number of osteoblasts, decreased bone mineral density or alterations in factors important for osteoblast function but not from increase in numbers of hematopoietic stem or progenitors cells. Our laboratory is investigating this further. Disclosures: Pawelczyk: Baxter Inc.: currently employed by Baxter Inc. Other.

scholarly journals Age-Associated Characteristics of Murine Hematopoietic Stem Cells

2000 ◽  
Vol 192 (9) ◽  
pp. 1273-1280 ◽  
Author(s):  
Kazuhiro Sudo ◽  
Hideo Ema ◽  
Yohei Morita ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Lymphoid Cells ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Functional Changes

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34−/lowc-Kit+Sca-1+ lineage marker–negative (CD34−KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34−KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34−KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34−KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

scholarly journals In vivo stem cell function of interleukin-3-induced blast cells

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 318-322
Author(s):  
J Tsunoda ◽  
S Okada ◽  
J Suda ◽  
K Nagayoshi ◽  
H Nakauchi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Function ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Interleukin 3 ◽  
Donor Cells ◽  
Blast Cells

The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, we obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. We examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 x 10(3) blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 x 10(3) blast cells, donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.

scholarly journals The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 79-79
Author(s):  
Zev J. Greenberg ◽  
Darlene A. Monlish ◽  
Rachel L. Bartnett ◽  
Jeffrey J. Bednarski ◽  
Laura G. Schuettpelz
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Development ◽  
B Cell Development ◽  
Cellular Migration ◽  
Physical Interaction ◽  
Hematopoietic Stem ◽  
Human Phenotype

The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. 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.

Absence of the Wnt Antagonist Sclerostin Adversely Affects B Cell Development in the Bone Marrow Niche

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 220-220 ◽  
Author(s):  
Corey J Cain ◽  
Randell Rueda ◽  
Bryce T McLelland ◽  
Nicole Collette ◽  
Gabriela Loots ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Wnt Signaling ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Niche ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Osteoblast Activity

Abstract Abstract 220 Hematopoietic cell fate decisions are dependent on their localized microenvironmental niche. In the bone, endosteal osteoblasts have been shown to support hematopoietic stem cells (HSC) self-renewal, as demonstrated by transgenic and knockout mouse models in which osteoblast populations were increased or decreased. In addition, Wnt signaling and the Wnt antagonist Dkk-1 have been implicated in various aspects of hematopoiesis and HSC self-renewal. Sclerostin (Sost) is a secreted protein that is primarily expressed by fully mature osteocytes and acts on osteoblasts as a negative regulator of bone growth, by antagonizing Wnt signaling by its binding to the Wnt co-receptors Lrp4, Lrp5, and/or Lrp6. Here, we investigated the role of Sost on hematopoiesis in the bone marrow niche. Increased osteoblast activity in sclerostin-knockout (Sost−/−) mice results in hypermineralized bones with small bone marrow cavities. As such, Sost−/− mice contain markedly reduced numbers of CD45+hematopoietic cells in the bone marrow. Since hematopoietic stem cell activity is dependent on osteoblast function, we examined whether the hyperactive osteoblast activity in Sost−/− mice influences the numbers of hematopoietic stem cells, lymphoid progenitor cells and myeloid progenitor cells in the bone marrow. Surprisingly, no differences were observed in hematopoietic stem and progenitor cell frequency and cell number. However, we found the bone marrow of Sost−/− mice to be depleted of B cells, and this reduction can be attributed to premature apoptosis beginning at the pre-pro-B cell stage. Examination of Sost expression showed that no hematopoietic cells expressed Sost, however, pre-pro, immature and recirculating B cells expressed Lrp5 and Lrp6. These gene expression patterns suggested that the defect in B cell development in Sost−/− mice is non-cell autonomous and that absence of Sost could affect Wnt signaling in these populations. We observed that the expression of Wnt target genes CCND1 and Lef-1 were not affected by the absence of Sost, but c-Myc was significantly upregulated in recirculating B cells in the bone marrow. We also observed a significant decrease in CXCL12 expression in the bone marrow stroma in Sost−/− mice, consistent with their inability to adequately support B cell development. Taken together, our results indicate that the B cell developmental defects in Sost−/− mice are non-cell autonomous, and we are currently performing reciprocal bone marrow transplantation experiments to further support this hypothesis. Our studies demonstrate a novel role for Sost in the regulation of B cell development in the bone marrow, and demonstrate that distinct Wnt antagonists play specific roles in the regulation of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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