LRF Is Indispensable for Hematopoietic Stem Cell Function Via Blocking Notch1-Mediated T Cell-Instructive Signals in the Bone Marrow Niche.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 81-81 ◽  
Author(s):  
Sung-UK Lee ◽  
Manami Maeda ◽  
Nagisa Sakurai ◽  
Freddy Radtke ◽  
Takahiro Maeda
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Target Gene ◽  
T Cell Development ◽  
Cell Development ◽  
B Cell Development ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System

Abstract Abstract 81 Hematopoietic stem cells (HSC) have the ability to self-renew and give rise to all hematopoietic lineage cells. Understanding signals that regulate the balance between self-renewal and differentiation of HSCs is an important issue in stem cell biology as well as regenerative medicine. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function is currently under debate. We explored the role of the LRF (Leukemia/Lymphoma Related Factor), a Notch repressor (also known as Zbtb7a, pokemon, OCZF and FBI-1) in HSC function, as it plays key roles in embryonic development, oncogenesis, and hematopoiesis. Conditional inactivation of the LRF gene in mouse HSCs (LRFF/FMx1-Cre mice) led to the development of CD4/CD8 DP (double positive) T-cells at the expense of B-cell development in the bone marrow (BM) in a Notch-dependent manner. Absolute numbers of the most primitive HSCs (LT-HSCs), defined as CD150+CD48−Flt3−Vcam-1+IL7Rα−LSK (Lin−Sca1+c-Kit+), were significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150−CD48+Flt3+Vcam-1+/−IL7Rα−LSK) and common lymphoid progenitors (CLPs: Lin−CD150−CD48+Flt3+Vcam-1−IL7Rα+) were barely detectable in LRFF/FMx1-Cre mice one month after pIpC injection. Enhanced T cell development and concomitant loss of B cell development was also seen in LRF−/− fetal liver (FL). Lin−IL7Rα+c-Kit+PIR+ (Paired Immunoglobulin-like receptors) T cell precursors were significantly increased in LRF−/− FL, indicating that Notch-mediated aberrant lymphoid fate determination also takes place during fetal hematopoiesis. To address which Notch gene(s) are targeted by LRF, we studied the HSC/progenitor population of conditional LRF knockout (LRFF/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRFF/FNotch1F/FMx1-Cre). In the absence of Notch1, normal B cell development was restored in LRFF/FMx1-Cre mice. Reduction of LT-HSCs in LRFF/FMx1-Cre resulted from high Notch1 activity, as loss of Notch1 rescued LT-HSC numbers, suggesting that LRF functions to maintain HSCs and normal lymphoid fate by blocking Notch1. HSCs in active cell cycle are sensitive to 5-fluoro-uracil (5-FU) treatment, which causes remaining dormant HSCs to be recruited into the cell cycle to rapidly produce new cells and to quickly re-establish the hematopoietic system. To examine the self-renewal capacity of LRF deficient LT-HSC, LRFF/FMx1-Cre mice were treated with 5-FU after pIpC injection and the recovery of LT-HSC numbers examined. While control LT-HSC numbers recovered to pretreatment levels 3 wk after 5-FU treatment, levels in LRFF/FMx1-Cre mice remained low, accompanied by DP T cell development in the BM. Furthermore, after 5-FU treatment, LT-HSC numbers of LRFF/FNotch1F/FMx1-Cre were compatible to those of control and LRFF/FMx1-Cre mice, indicating that lack of self-renewal capacity in LRF deficient LT-HSCs was due to excessive differentiation toward T cells caused by Notch1. In support of this idea, when mice were given 5-FU weekly as a challenge to assess their HSC function in vivo, the survival percentage in LRFF/FMx1-Cre mice was much lower than in controls (0% versus 50% in 1 month, P <0.0001) and that of LRFF/FNotch1F/FMx1-Cre mice was compatible to controls. Serial bone marrow transplant experiments further demonstrated functional defects of LRF deficient HSCs, as they failed to reconstitute the hematopoietic system in secondary recipients. Microarray analysis and subsequent Gene Set Enrichment Analysis demonstrated upregulation of genes that were enriched in progenitor compartments. Since LRF can act as a transcriptional repressor, mRNA levels of Notch receptors and Notch ligands were examined using the same data set. A Notch target gene Hes1, but not Notch1 itself, was upregulated, and increased levels of Hes1 was also confirmed by real-time q-PCR in FACS-sorted LT-HSCs, as well as in 10.5 d.p.c whole embryos. These data suggest that LRF does not transcriptionally regulate Notch1, as LRF loss led to Notch1 target gene activation at the LT-HSC level without affecting Notch1 mRNA. Our genetic studies clearly indicate that LRF is indispensable for the maintenance of the HSC pool by repressing T cell-instructive signals mediated by Notch1 in the BM niche. Our findings shed new light on the regulatory mechanisms underlying the balance between HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

IL-7 Effects on Thymocyte Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3318-3318
Author(s):  
Nahed El Kassar ◽  
Baishakhi Choudhury ◽  
Francis Flomerfelt ◽  
Philip J. Lucas ◽  
Veena Kapoor ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Notch Signaling ◽  
Stromal Cells ◽  
T Cell Development ◽  
Fold Increase ◽  
Cell Development ◽  
B Cell Development ◽  
Over Expression

Abstract IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

A Hypomorphic Cbfb Allele Reveals a Critical Dosage-Sensitive Function of Core Binding Factors at the Earliest Stages of T Cell Development.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 124-124
Author(s):  
Ivan Maillard ◽  
Laleh Talebian ◽  
Zhe Li ◽  
Yalin Guo ◽  
Daisuke Sugiyama ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dna Binding ◽  
B Cell ◽  
Bone Marrow Cells ◽  
T Cell Development ◽  
Cell Development ◽  
B Cell Development ◽  
Hematopoietic Stem

Abstract The family of core binding factors includes the DNA-binding subunits Runx1-3 and the common non-DNA binding partner CBFβ. Runx1 and CBFβ are essential for the emergence of hematopoietic stem cells during fetal development, but not for stem cell maintenance during later ontogeny. Runx1 is also required for megakaryocyte differentiation, B cell development, and for the DN2 to DN3 transition in thymocyte development. Runx2/CBFβ are critical for normal osteogenesis, and Runx3 for CD4 silencing in CD8+ T cells, but their contribution to other steps of hematopoietic development is unknown. To examine the collective role of core binding factors in hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss). CBFβ protein levels were reduced by approximately 2–3 fold in fetuses homozygous for the Cbfbrss allele (Cbfbrss/rss), and 3–4 fold in fetuses carrying one hypomorphic and one knockout allele (Cbfbrss/−). Cbfbrss/rss and Cbfbrss/− fetuses had normal erythroid and B cell development, and relatively mild abnormalities in megakaryocyte and granulocyte differentiation. In contrast, T cell development was very sensitive to an incremental reduction of CBFβ levels: mature thymocytes were decreased in Cbfbrss/rss fetuses, and virtually absent in Cbfbrss/−fetuses. We next assessed the development of Cbfbrss/rss and Cbfbrss/− fetal liver progenitors after transplantation to irradiated adult recipients, in competition with wild-type (wt) bone marrow cells. Wt, Cbfbrss/rss and Cbfbrss/− fetal progenitors replenished the erythroid, myeloid and B cell compartments equally well. The overall development of Cbfbrss/rss T cells was preserved, although CD4 expression was derepressed in double negative thymocytes. In Cbfbrss/− chimeras, mature thymocytes were entirely derived from competitor cells. Furthermore, the developmental block in Cbfbrss/− progenitors was present at the earliest stages of T cell development within the DN1 (ETP) and DN2 subsets. Our data define a critical CBFβ threshold for normal T cell development, and they situate an essential role of core binding factors during the earliest stages of T cell development. In addition, early thymopoiesis appeared more severely affected by reduced CBFβ dosage than by the lack of Runx1 (Ichikawa et al., Nat Med 2004; Growney et al., Blood 2005), suggesting that Runx2/3 may contribute to core binding factor activity in the T cell lineage.

Ammonium Ions Derived from Spontaneous Decomposition of L-Glutamine Inhibit Lymphoid Differentiation from Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2244-2244
Author(s):  
Gerald J. Spangrude ◽  
Birgitta Johnson ◽  
Scott Cho ◽  
Xiaosong Huang ◽  
L. Jeanne Pierce
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Progenitor Cells ◽  
Stromal Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Ammonium Ions ◽  
Hematopoietic Stem ◽  
Lymphocyte Differentiation

Abstract The ability to study lymphocyte differentiation in culture has been greatly advanced by the availability of the OP9 bone marrow stromal cell line, which was derived from an op/op mouse and thus lacks M-CSF. As a result, the normal default myeloid differentiation from bone marrow-derived stem and progenitor cells does not occur, and lymphocyte differentiation is favored. Introduction of the Notch ligand Delta-like 1 into OP9 cells results in promotion of T cell development and parallel suppression of B cell development. While the OP9-DL1 model of T cell development works quite well when fetal liver-derived progenitors are cultured, the success of T cell development from adult bone marrow-derived progenitors has been more difficult to reproduce. We have undertaken a systematic analysis of variables that can prevent efficient T cell development in OP9-DL1 cultures, and have found that one limiting factor that impacts the efficiency of differentiation of both T and B cell lineages is the accumulation of ammonium ions as a result of the spontaneous decomposition of l-glutamine. L-glutamine, which is present at 2 to 4 mM in standard tissue culture media, is unstable and will spontaneously degrade to form ammonium ions and pyroglutamic acid at a rate of 1%/day at 4°C and at a 10-fold higher rate at 37°C. To evaluate the effects of the two major products of l-glutamine decomposition on lymphoid differentiation, we added each product to differentiation cultures at 3 mM in the presence of a stable source of l-glutamine (l-alanyl-l-glutamine). Cultures were established in 1 ml containing 4×104 stromal cells (OP9 for B cell differentiation, OP9-DL1 for T cell differentiation), 1×103 bone marrow-derived lymphoid progenitors enriched by phenotype (c-kit+LinnegSca-1+Thy-1.1neg), and 5 ng/ml Flt3L plus 5 ng/ml IL-7. Every 3 to 4 days, cultures were harvested and passaged onto fresh stromal cell monolayers; lymphoid cells were counted and evaluated for surface antigen expression at each passage. While addition of pyroglutamic acid had no inhibitory effect on lymphocyte growth or differentiation, addition of ammonium chloride slowed growth and prevented differentiation of both T and B lymphocytes. Growth of the stromal cell monolayers was not affected by ammonium chloride at the concentrations utilized in these studies. We conclude that freshly-prepared culture medium, preferably containing a stabilized form of l-glutamine, is a critical aspect contributing to the success of lymphocyte differentiation cultures established from adult bone marrow cells. We also found that decreasing IL-7 concentrations to 1 ng/ml resulted in more rapid differentiation of T cells and a more balanced representation of CD4 and CD8 single positive cells. Our studies help define optimal conditions for differentiation of bone marrow-derived lymphoid progenitor cells into T and B lineages in vitro, and provide evidence that hematopoietic differentiation displays variable degrees of sensitivity to ammonium ions derived from decomposition of l-glutamine. These results will help define optimal conditions for expansion and differentiation of hematopoietic stem and progenitor cells in vitro.

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.

T and B cell development in pituitary deficient insulin-like growth factor-II transgenic dwarf mice

1997 ◽  
Vol 155 (1) ◽  
pp. 165-170 ◽  
Author(s):  
R Kooijman ◽  
SC van Buul-Offers ◽  
LE Scholtens ◽  
RG Reijnen-Gresnigt ◽  
BJ Zegers
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
T Cell Development ◽  
Cell Development ◽  
B Cell Development ◽  
Igf I ◽  
Dwarf Mice

Treatment of mice with IGF-I stimulates T and B cell development. We showed that overexpression of IGF-II in transgenic FVB/N mice only stimulated T cell development. In the present study, we further addressed the in vivo effects of IGF-II in the absence of IGF-I to get more insight into the potential abilities of IGF-II to influence T and B cell development. To this end, we studied lymphocyte development in IGF-II transgenic Snell dwarf mice that are prolactin, GH and thyroid-stimulating hormone deficient and as a consequence show low serum IGF-I levels. We showed that T cell development was stimulated to the same extent as in IGF-II transgenic FVB/N mice. Furthermore, IGF-II increased the number of nucleated bone marrow cells and the number of immature B cells without having an effect on the number of mature B cells in spleen and bone marrow. Our data show that IGF-II has preferential effects on T cell development compared with B development, and that these preferential effects also occur in the absence of measurable IGF-I levels.

scholarly journals Thymus-autonomous T cell development in the absence of progenitor import

2012 ◽  
Vol 209 (8) ◽  
pp. 1409-1417 ◽  
Author(s):  
Vera C. Martins ◽  
Eliana Ruggiero ◽  
Susan M. Schlenner ◽  
Vikas Madan ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Short Life Span ◽  
Receptor Repertoire ◽  
T Cell Progenitors ◽  
Transplantation Experiments

Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell–deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2−/−γc−/−KitW/Wv mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2−/−γc−/−KitW/Wv hosts, γc-mediated signals alone played a key role in the competition between thymus-resident and bone marrow–derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

Notch Repression by LRF Is Necessary for the Maintenance of Adult Hematopoietic Stem Cell Pool.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2633-2633
Author(s):  
Sung-UK Lee ◽  
Min Li ◽  
Manami Maeda ◽  
Nagisa Sakurai ◽  
Yuichi Ishikawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 2633 Among the different stem cells, hematopoietic stem cells (HSCs) are one of the best studied and characterized stem cells. To maintain life-long hematopoiesis in the bone marrow (BM), signals governing the balance between self-renewal and differentiation are tightly regulated in HSC compartment. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function in the BM is currently under debate. LRF (Leukemia/Lymphoma Related Factor, also known as Zbtb7a/pokemon) is a transcription factor that acts as a proto-oncogene and plays a key role in lymphoid and erythroid development. Previously we reported that the pool of LT-HSCs, CD150+CD48−Flt3−Vcam-1+/&minus;IL7Rα−LSK (Lin−Sca-1+c-Kit+), was significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150−CD48+Flt3+Vcam-1+/&minus;IL7Rα−LSK) and common lymphoid progenitors (CLPs: Lin−CD150−CD48+Flt3+Vcam-1−IL7Rα+) were barely detectable in LRF deficient mice. This was due to excessive differentiation of HSC into aberrant CD4/CD8 DP (double positive) T cell development in the BM caused by high Notch activity, implicating LRF role on HSC maintenance. Both gene expression profile (GSEA and DAVID analysis) and Q-PCR results indicated that LRF deficient LT-HSCs had loss of stem cell signature; but gain of T cell signature and up-regulated Notch-target gene, Hes-1, without affecting mRNA expression of Notch (1-4) or related (DLL1, DLL4, Jagged-1) genes. To determine LRF function in HSCs, we performed in vivo and in vitro experiments: 1) 5-FU (5-fluorouracil, the chemotherapy agent) treated LRF deficient mice were not able to compensate for their loss of LT-HSCs; 2) multi-lineage defects were shown in second recipient mice transplanted with 1 million of LRF deficient bone marrow cells in serial bone marrow transplantation assays, suggesting that LRF deficient LT-HSCs had defect in self-renewal and 3) LRF deficient FL-HSCs (CD150+CD48−LSK cells) were cultured on OP9 cells expressing delta-like ligand (DLL1, DLL4 and Jagged1), and enhanced T cell differentiation was only observed when they were co-cultured with delta-expressing OP9 cells. Among the Notch family, these phenotypes were Notch1-dependent. In fact, Notch1flox/floxLRFflox/floxMx1-Cre+ mice demonstrated normal LT-HSC numbers and restored B cell development, and prolonged survival over LRFflox/floxMx1-Cre+ mice in sequential 5-FU treatment in vivo. To explore which Notch-ligand(s) in BM niche is responsible for aberrant T-cell development in LRF deficient mice as well, we treated wild-type and LRFflox/floxMx1-Cre+ with anti-DLL4 antibody twice per week for 3 weeks. DLL4 blockage in LRF deficient mice rescued B cell development and prevented the development of aberrant DP T-cell development in LRF deficient mice. To further elucidate the relationship between LRF and Notch in adult HSC function, we analyzed Notch protein expression levels in HSCs and performed in-depth analysis of HSC/progenitor (HSC, LMPP and CLP) compartments in wild-type and LRF knockout (KO). Interestingly, Notch1 proteins were differentially expressed in LT-HSCs and ~50 % of them were positive for Notch1, while Notch2 was abundantly expressed in LT-HSCs. Notch1 expressing LT-HSCs were in more active cell-cycle (S phase) and absent in LRF conditional knockout mice. It is most likely that Notch1 expressing LT-HSCs were continually differentiating toward T cells in the absence of LRF, as CD4+CD8+ T cells were evident in the BM 10 months after pIpC injection. Taken together, our data strongly indicate that LRF is indispensable for hematopoietic homeostasis by preventing the lymphoid-primed HSCs from Notch/Delta-mediated T-instructive signal in the BM niche. Currently we're investigating the functional significances of Notch1 expressing LT-HSCs in detail. Our studies help us to better understand the underlying mechanism for HSC fate decision (self-renewal v.s. differentiation) in stem cell biology and its therapeutic approach in regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

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.

T and B lymphocyte abnormalities in bone marrow biopsies of common variable immunodeficiency

Blood ◽  
2011 ◽  
Vol 118 (2) ◽  
pp. 309-318 ◽  
Author(s):  
Manuella L. Gomes Ochtrop ◽  
Sigune Goldacker ◽  
Annette M. May ◽  
Marta Rizzi ◽  
Ruth Draeger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Common Variable Immunodeficiency ◽  
Plasma Cells ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Biopsies ◽  
Cell Counts ◽  
Common Variable

Abstract In common variable immunodeficiency (CVID) defects in early stages of B-cell development, bone marrow (BM) plasma cells and T lymphocytes have not been studied systematically. Here we report the first morphologic and flow cytometric study of B- and T-cell populations in CVID BM biopsies and aspirates. Whereas the hematopoietic compartment showed no major lineage abnormalities, analysis of the lymphoid compartment exhibited major pathologic alterations. In 94% of the patients, BM plasma cells were either absent or significantly reduced and correlated with serum immunoglobulin G levels. Biopsies from CVID patients had significantly more diffuse and nodular CD3+ T lymphocyte infiltrates than biopsies from controls. These infiltrates correlated with autoimmune cytopenia but not with other clinical symptoms or with disease duration and peripheral B-cell counts. Nodular T-cell infiltrates correlated significantly with circulating CD4+CD45R0+ memory T cells, elevated soluble IL2-receptor and neopterin serum levels indicating an activated T-cell compartment in most patients. Nine of 25 patients had a partial block in B-cell development at the pre-B-I to pre-B-II stage. Because the developmental block correlates with lower transitional and mature B-cell counts in the periphery, we propose that these patients might form a new subgroup of CVID patients.

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.

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