Btg1-Deficiency Promotes ETV6-RUNX1-Mediated Leukemic Transformation By Upregulation of BCL6

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5193-5193
Author(s):  
Esther Tijchon ◽  
Liesbeth van Emst ◽  
Dorette van Ingen Schenau ◽  
Laurens T van der Meer ◽  
Simone de Rijk ◽  
...  
Keyword(s):  
B Cell ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Housekeeping Gene ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Capacity ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Targeted Deletion ◽  
Effector Pathways

Abstract Translocation t(12;21) (p13;q22), giving rise to the ETV6-RUNX1 fusion gene, is the most common genetic abnormality in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The ETV6-RUNX1 translocation arises in utero, but its expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Deletions affecting the transcriptional coregulator BTG1 are commonly observed in BCP-ALL (9%), but are significantly enriched in ETV6-RUNX1-positive leukemia (25%). The BTG1 protein displays no intrinsic enzymatic activity but may act by recruiting effector molecules like protein arginine methyltransferase 1 (PRMT1) to specific transcription factors. Here, we show that ETV6-RUNX1 interacts both with BTG1 and PRMT1, and this interaction is lost in c-Kit+Ter-119-Btg1-/- fetal liver (FL) derived hematopoietic progenitors (HPCs). Moreover, targeted deletion of Btg1 enhanced the proliferative capacity of ETV6-RUNX1 in FL-HPCs as measured by enhanced colony-forming and serial replating capacity (Figure 1). The combined loss of Btg1 function and ETV6-RUNX1 expression correlated with strong upregulation of the proto-oncogene Bcl6 and downregulation of BCL6 target genes, such as p19Arf and Tp53 (Figure 2). Similarly, ectopic expression of BCL6 promoted both proliferation and replating capacity of FL-derived progenitor cells in the presence of SCF, FLT3L and IL-7. This phenotype correlated with a fivefold suppression of p19Arf and a twofold suppression of Tp53 expression. Inhibition of BCL6 in a variety of human BCP-ALL cell lines by the peptide inhibitor RI-BPI resulted in decreased proliferation and induction of apoptosis as measured by Annexin-V staining. These included the ETV6-RUNX1-positive cell lines UOC-B6, AT-2 and REH, the BCR-ABL1-positive cell line SD1, as well as Nalm6. Together our results point to a novel role for BCL6 in promoting cell proliferation of primitive progenitor B cells and suggest that targeted inhibition of BCL6 may be effective in the treatment of various BCP-ALL subtypes. Figure 1. Btg1-deficiency enhances the proliferative capacity of early FL-HPCs expressing ETV6-RUNX1. FL-derived hematopoietic progenitor cells (FL-HPCs) (cKit+Ter119-) were isolated from wild-type and Btg1-/- embryos at day 13.5dpc and transduced with control and ETV6-RUNX1 virus. Control and ETV6-RUNX1 transduced FL-HPCs (1x104 cells) were added 48 hours after transduction in B cell specific methylcellulose in the presence of FLT-3L, IL-7 and SCF. Serial replating was performed under identical conditions. Mean colony counts (and SEM) were determined (>30 cells/colony) after 6-10 days of culture. Data is a representative of 2 independent experiments. *, P< 0.05, **, P< 0.01. Figure 1. Btg1-deficiency enhances the proliferative capacity of early FL-HPCs expressing ETV6-RUNX1. FL-derived hematopoietic progenitor cells (FL-HPCs) (cKit+Ter119-) were isolated from wild-type and Btg1-/- embryos at day 13.5dpc and transduced with control and ETV6-RUNX1 virus. Control and ETV6-RUNX1 transduced FL-HPCs (1x104 cells) were added 48 hours after transduction in B cell specific methylcellulose in the presence of FLT-3L, IL-7 and SCF. Serial replating was performed under identical conditions. Mean colony counts (and SEM) were determined (>30 cells/colony) after 6-10 days of culture. Data is a representative of 2 independent experiments. *, P< 0.05, **, P< 0.01. Figure 2. Targeted deletion of Btg1 cooperates with ETV6-RUNX1 in regulating critical effector pathways implicated in leukemia. Relative expression levels of Bcl6, Tp53 and p19arf in empty control (Ctrl) and ETV6-RUNX1 transduced wild-type and Btg1-deficient fetal liver-derived hematopoietic progenitor cells by real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments. *, P< 0.05, **, P< 0.01, ***, P< 0.001. Figure 2. Targeted deletion of Btg1 cooperates with ETV6-RUNX1 in regulating critical effector pathways implicated in leukemia. Relative expression levels of Bcl6, Tp53 and p19arf in empty control (Ctrl) and ETV6-RUNX1 transduced wild-type and Btg1-deficient fetal liver-derived hematopoietic progenitor cells by real-time PCR and normalized to the expression of the housekeeping gene TATA box binding protein (TBP). Data represent the mean and SEM of three independent experiments. *, P< 0.05, **, P< 0.01, ***, P< 0.001. Disclosures No relevant conflicts of interest to declare.

scholarly journals Wilms' Tumor Gene (WT1) Competes With Differentiation-Inducing Signal in Hematopoietic Progenitor Cells

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2969-2976 ◽  
Author(s):  
Kazushi Inoue ◽  
Hiroya Tamaki ◽  
Hiroyasu Ogawa ◽  
Yoshihiro Oka ◽  
Toshihiro Soma ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Tumor Suppressor ◽  
Progenitor Cells ◽  
Wilms Tumor ◽  
Suppressor Gene ◽  
Hematopoietic Progenitor Cells ◽  
Wt1 Gene ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Oncogenic Function

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3–dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

scholarly journals EBF Deficient Hematopoietic Progenitor Cells Potentialy Differentiated into Immature B Cell: EBF1 Dispensable for B Cell Linage Commitment from Pro-B to Pre-B Stage

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5127-5127
Author(s):  
Bidisha Chanda ◽  
Tomokatsu Ikawa ◽  
Kazuki Okuyama ◽  
Katsuto Hozumi ◽  
Kiyoshi Ando ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
B Cell Differentiation ◽  
Tgf Beta ◽  
Hematopoietic Progenitor ◽  
Vdj Recombination ◽  
Igh Locus ◽  
Non Coding Rna

Abstract Introduction: Canonical notion demonstrates that Cell fate is determined by transcriptional factor. Accordingly, the lineage specific transcriptional factors have been investigated for various kinds of cells. Especially, in the hematopoietic system, the extensive research for lineage specific transcriptional factors had elucidated the transcriptional factors which regulated lineage commitment. B cell commitment and development requires the activities of multiple transcription factors, including the early B cell factor (EBF), PAX5, and E2A. These transcription factors regulate B cell development in a stage specific manner. The hematopoietic progenitor cells which are deficient for any of them, cannot commit B cells. Among them EBF1 is presumed to be more potent. Rescue early pro B cell to induce the expression of several key proteins including RAG that enable gene rearrangement to occur by opening of IgH locus. We found that the B cell developmental arrest caused by EBF1 deficiency can be rescued by a single non coding RNA. These B cells which are deficient EBF1 but showed the expression of CD19, B cell lineage specific surface marker and VDJ recombination, molecular markers of B cell commitment in vitro B cell differentiation system cocultured with Tst4 cells, stromal cell lines. We further investigated the quality and differentiation potential of these B lineage commitment cells in the in vivo mouse model and elucidated the mechanism of this phenomenon. Material and methods: We collected EBF1−/− fetal liver hematopoietic progenitor (Lin−) cells and cultured them on TSt-4 stromal cells after infecting with non coding RNA and control vector in IMDM medium containing cytokines and then injected it into NOG mice. Collect bone marrow (BM), thymus and spleen from those mice. Then comprehensive Gene-Expression analysis, real time PCR for VDJ recombination analysis was performed and checked surface marker by flowcytometry. Result: We analyzed BM and spleen of non coding RNA infected EBF1 KO cells injected mice and found the expression of CD19 in BM as well as in spleen and upregulated of B220 also, comparing with control vector expressed cells. Furthermore, surface IgM expression of CD19 positive cells in the spleen is upregulated compared with the cells in the BM (Figure 1). Several target genes of the non-coding RNAs were identified by use of cDNA array analysis and luciferase reportor assay. Among them, several genes were involved in TGF beta pathway. As TGF beta family and the pathway, has been reported a critical factor which is negatively regulating B lymphopoiesis　(Figure 2). We hypothesized that TGF beta family genes such as Tgfβr3, Acvr2a, are responsible for B cell differentiation for which EBF1 is dispensable .We cultured EBF KO cells for 14 days with and without TGF beta 1,2,3 antibody and Activin A antibody on TST4 cells. We found that increase mean intensity (MFI) of B220 into antibodies positive cocultured cells (Figure 3) to suggest, the suppression of TGF beta pathway is partially responsible for B cell differentiation under EBF1 deficiency. Conclusion: Canonical notion of cell fate determination of B cells defines that EBF1 is an indispensible factor for B lymphopoiesis. However, from our previous and present study it is proved that without EBF1 B cell development can progress to pro B to pre B cell and Immature B cell stage and “VDJ recombination” occur in the absence of EBF. Furthermore, in vivo mouse model, EBF1 deficient hematopoietic progenitor cells differentiated into IgM positive cells. Therefore we can conclude that EBF is dispensable of VDJ recombination, opening of IgH locus, binding of RAG protein and B cell differentiation to the mature stage. One of the mechanisms is possibly due to the stimuli from microenvironment, such as TGF beta family and pathway. Furthermore, the detail mechanism of IgH locus opening, epigenetic changes and chromatin remodeling around the IgH locus in the absence of EBF is under investigation. Disclosures Chanda: Japan Society for the Promotion of Science(JSPS): Research Funding.

Oxidative Stress-Mediated Activation of AKT/mTOR Signaling Pathway Leads to Myeloproliferative Syndrome in FoxO3 Null Mice: A Role for Lnk Adaptor Protein

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 509-509 ◽  
Author(s):  
Safak Yalcin ◽  
Sathish Kumar Mungamuri ◽  
Dragan Marinkovic ◽  
Xin Zhang ◽  
Wei Tong ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Reactive oxygen species (ROS) are toxic byproducts of oxidative metabolism implicated in many debilitating human disorders including hematological malignancies and aging. ROS are also generated by growth factors and cytokine stimulation and play critical functions in normal cellular signaling. However, not much is known of how ROS impact physiological processes in normal and diseased states. We and others have recently shown critical functions for box (O) family of forkhead transcription factors (Fox)O in the regulation of physiological ROS in primitive hematopoietic cells. In particular, FoxO3 has emerged as the principal FoxO whose regulation of ROS is essential for the maintenance of hematopoietic stem cell pool. Although FoxO3’s activity is constitutively repressed by several oncoproteins that play critical roles in myeloproliferative disorders the role of FoxO3 in the regulation of primitive hematopoietic progenitors remains elusive. FoxO’s function is restrained by AKT serine threonine protein kinase. AKT supports growth, survival and proliferation by promoting inhibition of FoxO and activation of the mammalian target of rapamycin (mTOR) and its downstream target p70 S6 Kinase (S6K) through phosphorylation. We demonstrate that loss of FoxO3 leads to a myeloproliferative-like syndrome characterized by leukocytosis, splenomegaly, enhanced generation of primitive progenitors including colony-forming-unit-spleen (CFU-S) in hematopoietic organs and hypersensitivity of hematopoietic progenitor cells to cytokines in FoxO3 null mice. These findings were intriguing since we had not found FoxO3 null hematopoietic stem cells to exhibit enhanced cycling in vivo or to generate excessive hematopoietic progenitors ex vivo (Yalcin et al., JBC, 2008). To investigate the mechanism of enhanced myeloproliferation, we interrogated cytokine-mediated activation of signaling pathways in freshly isolated FoxO3 null versus wild type bone marrow cells enriched for hematopoietic progenitors. To our surprise we found that stimulation with cytokines including IL-3 led to hyperphosphorylation of AKT, mTOR and S6K but not STAT5 proteins in FoxO3 null as compared to wild type cytokine-starved hematopoietic progenitors. In agreement with these results, in vivo administration of the mTOR inhibitor rapamycin resulted in significant reduction of FoxO3 null- but not wild type-derived CFU-Sd12 in lethally irradiated hosts. These unexpected results suggested that AKT/mTOR signaling pathway is specifically overactivated as part of a feedback loop mechanism and mediates enhanced generation of FoxO3 null primitive multipotential hematopoietic progenitors in vivo. We further showed that phosphorylation of AKT/mTOR/S6K is highly sensitive to ROS scavenger N-Acetyl-Cysteine (NAC) in vivo and ex vivo in both wild type and FoxO3 null primitive hematopoietic progenitors indicating that ROS are involved in cytokine signaling in primary hematopoietic progenitor cells. Interestingly, in vivo administration of NAC normalized the number of FoxO3 null-derived CFU-Sd12 in lethally irradiated hosts without any impact on wild type CFU-Sd12 strongly suggesting that ROS mediate specifically enhanced generation of primitive hematopoietic progenitors in FoxO3 null mice. In this context, we were surprised to find similar levels of ROS concentrations in FoxO3 mutant as compared to control hematopoietic progenitors. Thus, we asked whether the increase in FoxO3 null primitive hematopoietic progenitor compartment is due to an increase sensitivity of cytokine signaling to ROS as opposed to increased ROS build up per se in these cells. In search for a mechanism we found the expression of Lnk, a negative regulator of cytokine signaling, to be highly reduced in FoxO3 null primitive hematopoietic progenitor cells. We further demonstrated that retroviral reintroduction of Lnk but not vector control in FoxO3 null primitive bone marrow cells reduced significantly the number of FoxO3 null-derived CFU-Sd12in vivo. Collectively, these results suggest that reduced expression of Lnk hypersensitizes FoxO3-deficient hematopoietic progenitors to ROS generated by cytokine signaling leading to myeloproliferation. These cumulative findings uncover a mechanism by which deregulation of cellular sensitivity to physiological ROS leads to hematopoietic malignancies specifically in disorders in which FoxO play a role.

Stromal Expression of Jagged 1 Promotes Colony Formation by Fetal Hematopoietic Progenitor Cells

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1505-1511 ◽  
Author(s):  
Philip Jones ◽  
Gill May ◽  
Lyn Healy ◽  
John Brown ◽  
Gerald Hoyne ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Culture Conditions ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract The Notch signaling system regulates proliferation and differentiation in many tissues. Notch is a transmembrane receptor activated by ligands expressed on adjacent cells. Hematopoietic stem cells and early progenitors express Notch, making the stromal cells which form cell-cell contacts with progenitor cells candidate ligand-presenting cells in the hematopoietic microenvironment. Therefore, we examined primary stromal cell cultures for expression of Notch ligands. Using reverse transcription-polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting, we demonstrate expression of Jagged 1 in primary stromal cultures. To investigate if the stromal expression of Jagged 1 has functional effects on hematopoietic progenitors, we cultured CD34+, c-kit+ hematopoietic progenitor cells derived from the aorto gonadal mesonephros region of day 11 mouse embryos on the Jagged 1− stromal cell line S17 and on S17 cells engineered to express Jagged 1. The presence of Jagged 1 increased the number of colonies formed in subsequent methylcellulose culture fourfold. Larger increases in colony numbers were observed under the same culture conditions with CD34+, c-kit+ hematopoietic progenitor cells derived from d11 fetal liver. These results obtained in vitro table Jagged 1 as a candidate regulator of stem cell fate in the context of stromal microenvironments in vivo. © 1998 by The American Society of Hematology.

scholarly journals Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1591-1591
Author(s):  
Juliana M. Xavier ◽  
Lauremilia Ricon ◽  
Karla Priscila Vieira ◽  
Longhini Ana Leda ◽  
Carolina Bigarella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 2003-2013 ◽  
Author(s):  
Maria Teresa Mitjavila-Garcia ◽  
Michel Cailleret ◽  
Isabelle Godin ◽  
Maria Manuela Nogueira ◽  
Karine Cohen-Solal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (αIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41– cells from embryoid bodies converted to CD41+ hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (β3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41+, whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34+. Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.

Development of dendritic cells in vitro from murine fetal liver–derived lineage phenotype-negative c-kit+hematopoietic progenitor cells

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 138-146
Author(s):  
Yanyun Zhang ◽  
Yi Zhang ◽  
Yong Wang ◽  
Masafumi Ogata ◽  
Shin-ichi Hashimoto ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Nk Cell ◽  
Fetal Liver ◽  
Critical Role ◽  
Hematopoietic Progenitor Cells ◽  
Mouse Tumor ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
And Function

We describe here that lineage phenotype- negative (Lin)−c-kit+ hematopoietic progenitor cells (HPCs) from day 13 postcoitus (dpc) murine fetal liver (FL) can generate dendritic cell (DC) precursors when cultured in vitro in the presence of PA6 stromal cells plus granulocyte/macrophage colony-stimulating factor (GM-CSF) + stem cell factor (SCF) + Flt3 ligand (Flt3L) for 12 to 14 days, and develop into mature DCs when stimulated with GM-CSF plus mouse tumor necrosis factor  (mTNF) for an additional 3 to 5 days. A transwell culture system showed that the generation of DC precursors depended on the support of PA6 cell-secreted soluble factor(s). The mature DCs derived from 13 dpc FL Lin−c-kit+ HPCs showed characteristic morphology and function of DCs and expressed high levels of Ia, CD86, and CD40 molecules, low levels of DEC205, E-cadherin, and F4/80 molecules, but barely detectable CD11c antigen. Once FL-derived HPCs were cultured without GM-CSF, NK1.1+ cells developed in the presence of PA6 cells + SCF + Flt3L. These NK1.1+ cells could develop into DC precursors at an earlier stage of differentiation by reculturing with PA6 cells + SCF + Flt3L + GM-CSF, but they would be irreversibly committed to NK cell precursors without GM-CSF after 3 days, suggesting that GM-CSF plays a critical role in controlling the transition of DC and NK cell precursors from 13 dpc FL-derived Lin−c-kit+ HPCs. This study represents the first success in generating mature DCs in vitro from murine FL HPCs. (Blood. 2000;95:138-146)

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