Role of CD95/Fas and its ligand in the regulation of the growth of human CD34++CD38− fetal liver cells

1999 ◽  
Vol 27 (9) ◽  
pp. 1428-1439 ◽  
Author(s):  
A Bárcena
Keyword(s):  
Fetal Liver ◽  
Liver Cells ◽  
Human Cd34 ◽  
Fetal Liver Cells

The Role of K-ras Signaling in Erythropoiesis In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3136-3136
Author(s):  
Jing Zhang ◽  
Yangang Liu ◽  
Caroline Beard ◽  
Rudolf Jaenisch ◽  
Tyler Jacks ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Ras Signaling ◽  
Erythroid Progenitors ◽  
Akt Activation ◽  
Fetal Liver Cells ◽  
Terminal Erythroid Differentiation

Abstract K-ras plays an important role in hematopoiesis. K-ras-deficient mouse embryos die around E12-E13 with severe anemia. In humans, oncogenic mutations in K-ras gene are identified in ~30% of patients with acute myeloid leukemia. We used mouse primary erythroid progenitors as a model system to study the role of K-ras signaling in vivo. Both Epo- and stem cell factor (SCF) - dependent Akt activation are greatly reduced in K-ras-/- fetal liver cells, whereas other cytokine- induced pathways, including Stat5 and p44/p42 MAP kinase, are activated normally. The reduced Akt activation in erythroid progenitors per se leads to delayed erythroid differentiation. Our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo. Overexpression of oncogenic Ras in primary fetal erythroid progenitors led to their continual proliferation and a block in terminal erythroid differentiation. Similarly, we found that primary fetal liver cells expressing oncogenic K-ras from its endogenous locus undergo abnormal proliferation and terminal erythroid differentiation is partially blocked. We are currently investigating the signal transduction pathways activated by this oncogenic K-ras that underlies these cellular phenotypes.

MiR34a Contributes to Defective Erythropoiesis in RPS19 Insufficient Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2404-2404
Author(s):  
Elena Bibikova ◽  
Shuo Lin ◽  
Kathleen M Sakamoto
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Target Genes ◽  
Fetal Liver ◽  
K562 Cells ◽  
Liver Cells ◽  
Molecular Pathways ◽  
Human Cd34 ◽  
Control Shrna ◽  
Fetal Liver Cells

Abstract Abstract 2404 Diamond-Blackfan Anemia (DBA) is characterized by defective erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a defect in the RPS19 gene, which encodes a ribosomal protein on the 40S ribosomal subunit. Previous studies in zebrafish and mice have shown that knockdown of RPS19 is correlated with the upregulation of p53, a tumor suppressor gene that regulates cell cycle and apoptosis. However, the molecular pathways that link ribosome dysfunction and defective erythropoiesis remain unknown. Among the targets of p53 is microRNA34a (miR34a). Overexpression of miR34a in human myeloid leukemia K562 cells inhibits cell proliferation, leads to cell cycle arrest at the G1 stage, and promotes differentiation toward the megakaryocytic lineage in a p53-independent manner. MiR34a downregulates numerous proteins, including c-Myb, which is essential for normal erythropoiesis and has been found to be expressed at lower levels in bone marrow cells from DBA patients. We hypothesize that miR34a induction downstream of p53 activation in RPS19 insufficient normal hematopoietic progenitor cells contributes to defective erythropoiesis observed in DBA patients. To study the effects of RPS19 insufficiency and downstream signaling pathways, we transduced primary CD34+ fetal liver cells with two different shRNA constructs against RPS19. Knockdown was confirmed by Western blot analysis and by qRT-PCR (73%, p< 0.0001 for shRNA1, and 87%, p< 0.0001 for shRNA2, respectively), compared with a scrambled control shRNA. Cells were sorted for a GFP marker 72 hours post-transduction and either collected for downstream analysis or propagated further in methylcellulose. Our results demonstrated that knockdown of RPS19 in primary human CD34+ fetal liver cells leads to an increase in p53 protein by Western blot analysis, and upregulation of miR34a mRNA (3-fold, p<0.01). This upregulation correlates with downregulation of miR34a target genes including c-Myb (p<0.01) and c-Myc (p<0.01). Induction of miR34a and downregulation of c-Myb and c-Myc, occurs through a p53-dependent pathway since the expression of those genes was not altered in p53-null K562 cells transduced with RPS19 shRNA lentivirus. To correlate molecular pathways with the differentiation potential of RPS19 insufficient CD34+ fetal liver cells, methylcellulose colony assays were performed. Total colony formation was reduced by 87% (p<0.01) in cells treated with RPS19 shRNA compared to scrambled control shRNA. Erythroid colony formation in methylcellulose containing IL-3, SCF, GM-CSF, and EPO, decreased by 90% (p<0.01) in shRNA transduced cells compared to control shRNA. FACS analysis for erythroid markers CD71 and Glycophorin A showed a similar pattern, with the CD71+GlyA+ population representing 2.6% of RPS19 shRNA transduced cells, compared to 9.9% in cells transduced with the scrambled control. Preliminary data with lymphoblastoid cell lines (LCLs) from DBA patients with RPS19 insufficiency demonstrate 2-fold upregulation of miR34a. Therefore, we conclude that RPS19 insufficiency in human CD34+ fetal liver cells leads to p53-dependent upregulation of miR34a, down-regulation of miR34a target genes c-Myc and c-Myb, and defects in erythroid differentiation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cited2 is required for normal hematopoiesis in the murine fetal liver

Blood ◽  
2007 ◽  
Vol 110 (8) ◽  
pp. 2889-2898 ◽  
Author(s):  
Yu Chen ◽  
Peter Haviernik ◽  
Kevin D. Bunting ◽  
Yu-Chung Yang
Keyword(s):  
Fetal Liver ◽  
Liver Cells ◽  
Cathepsin G ◽  
Molecular Regulation ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Fetal Liver Cells ◽  
B Lymphoid ◽  
First Time

Abstract Cited2 (cAMP-responsive elementbinding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]–rich tail 2) is a newly identified transcriptional modulator. Knockout of the Cited2 gene results in embryonic lethality with embryos manifesting heart and neural tube defects. Cited2−/− fetal liver displayed significant reduction in the numbers of Lin−c-Kit+Sca-1+ cells, Lin−c-Kit+ cells, and progenitor cells of different lineages. Fetal liver cells from Cited2−/− embryos gave rise to markedly reduced number of colonies in the colony-forming unit assay. Primary and secondary transplantation studies showed significantly compromised reconstitution of T-lymphoid, B-lymphoid, and myeloid lineages in mice that received a transplant of Cited2−/− fetal liver cells. Competitive reconstitution experiments further showed that fetal liver hematopoietic stem cell (HSC) function is severely impaired due to Cited2 deficiency. Microarray analysis showed decreased expression of Wnt5a and a panel of myeloid molecular markers such as PRTN3, MPO, Neutrophil elastase, Cathepsin G, and Eosinophil peroxidase in Cited2−/− fetal livers. Decreased expression of Bmi-1, Notch1, LEF-1, Mcl-1, and GATA2 was also observed in Cited2−/− Lin−c-Kit+ cells. The present study uncovers for the first time a novel role of Cited2 in the maintenance of hematopoietic homeostasis during embryogenesis and thus provides new insights into the molecular regulation of hematopoietic development.

scholarly journals Role of Dexamethasone and Oncostatin M on the Formation of Vacuoles in Human Fetal Liver Cells

2009 ◽  
Vol 32 (2) ◽  
pp. 209-212 ◽  
Author(s):  
Tsuyoshi Teramoto ◽  
Tamihide Matsunaga ◽  
Mie Toba ◽  
Toshiaki Sunazuka ◽  
Satoshi Omura ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Liver Cells ◽  
Oncostatin M ◽  
Human Fetal Liver ◽  
Fetal Liver Cells ◽  
Human Fetal Liver Cells

Physiology: The regulatory role of tri-iodothyronine on the production of α-fetoprotein and albumin by mouse fetal liver cells*

1993 ◽  
Vol 8 (10) ◽  
pp. 1576-1578 ◽  
Author(s):  
Eyal Anteby ◽  
Perach Shpan ◽  
Matat Dushnik ◽  
Adi Zvang ◽  
Tamar Zer ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Liver Cells ◽  
Regulatory Role ◽  
Fetal Liver Cells

Hematopoietic Defects in Cited2 Mutant Fetal Liver.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2272-2272
Author(s):  
Yu Chen ◽  
Yu-Chung Yang
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Cited2 [cAMP-responsive element-binding protein (CBP)/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is a newly identified transcriptional modulator. Knockout of Cited2 gene is embryonic lethal because of heart and neural tube defects. Cited2 binds directly to CBP and p300, which have been shown to be crucial for hematopoietic stem cell self-renewal and proper hematopoietic differentiation, respectively. Cited2 also induces the expression of a polycomb-group gene, Bmi-1, which is essential for self-renewal of adult hematopoietic stem cells. These connections provided rationale to study the potential role of Cited2 in hematopoiesis. Mouse fetal liver is the major hematopoietic organ from day 10 postcoitus until right before birth. The smaller sized Cited2−/− fetal liver and significantly decreased fetal liver cellularity strongly suggest the potential defect in hematopoiesis. In vitro colony formation assay in methycellulose-based medium was used to characterize the hematopoietic progenitors. We found that fetal liver cells from E13.5, 14.5 and E15.5 Cited2−/− embryos gave rise to much less colonies, which reflects the decreased number and proliferative ability of hematopoietic progenitors due to Cited2 deficiency. Immunostaining of lineage-specific cell surface markers followed by flow cytometry was performed to characterize different hematopoietic populations in E14.5 and E15.5 fetal liver of wild type and Cited2−/− embryos. Cited2−/− fetal liver cells displayed a significant reduction in numbers throughout the hematopoietic hierarchy including hematopoietic stem cells (Lin− c-Kit+ Sca-1+), progenitor cells (Lin− c-Kit+), and differentiated cells of different lineages (CD45+, Ter119+, Mac-1+, Gr-1+), thus revealing a multi-level hematopoietic deficiency of Cited2−/− embryos. Long-term reconstitution experiment was then carried out to measure the ability of hematopoietic stem cells from Cited2−/− fetal liver cells to engraft and reconstitute hematopoietic system of congenic recipient mice. Mice transplanted with Cited2−/− fetal liver cells showed reconstitution of T cells whereas a 2-fold decrease in the reconstitution of B cell and myeloid lineages was observed, indicating a compromised ability of Cited2−/− fetal liver hematopoietic stem cells to maintain hematopoiesis. The results suggest an important role of Cited2 in hematopoietic differentiation and a selective function of Cited2 in B lymphoid &myeloid induction. The underlying mechanisms responsible for these defects will be pursued by microarray analysis of gene expression profile of Cited2−/− fetal liver cells, followed by more detailed phenotypic analyses of B and myeloid lineage markers plus in vitro and in vivo functional assays.

scholarly journals Loss of KDM6A/UTX Accelerate the Development of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1004-1004
Author(s):  
Daphne Dupere-Richer ◽  
Jianping Li ◽  
Sayantan Maji ◽  
Crissandra Pipe ◽  
Sharon Norton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Fetal Liver ◽  
Liver Cells ◽  
Wild Type ◽  
Post Transplantation ◽  
Demethylase Activity ◽  
Fetal Liver Cells

Abstract In multiple myeloma (MM), inactivating mutations and deletions affecting the histone demethylase KDM6A locus are found in up to 10% of newly diagnosed patients and associated with poor prognosis. KDM6A (also named UTX, Ubiquitously transcribed Tetratricopeptide repeat, X chromosome) belongs to a family of Jumonji-C (Jmj-C)-containing demethylases that work as a scaffold for a multiprotein complex containing H3K4 specific methyltransferases KMT2D and/or KMT2C (MLL2/3), the histone acetyltransferase CBP/p300 and members of the SWI/SNF chromatin-remodeling complex. In a concerted manner this complex appears to add activation marks on histones and remove methylation of lysine 27 on histone H3 (H3K27me) associated with gene repression. Importantly, all these coregulators are found significantly mutated in MM and their function may converge into a tumor suppressive pathway. Our goal is to understand how KDM6A loss contributes to the development of MM. We modeled the loss of KDM6A in MM cell lines using CRISPR-Cas9 ribonucleotide protein (RNP) technology. Mutant allele frequency over time post electroporation of RNP revealed a growth advantage of KDM6A mutant alleles. By 2 weeks of growth most of the cells in culture harbored KDM6A gene disruption and exhibit elimination of KDM6A protein confirming the tumor suppressive role of KDM6A in MM. We used these isogenic polyclonal edited cell lines with KDM6A wild type or mutated to identify KDM6A binding sites and enhancers affected by the loss of KDM6A. As well, we knock-in an HA tag on endogenous KDM6A to identify DNA regions occupied by KDM6A. To understand the importance of KDM6A demethylase activity in the tumor suppressive effect of KDM6A, we developed stable cell lines with a doxycycline-inducible form of KDM6A wild-type (WT) or lacking demethylase activity (JmjC-dead). We found that about 20% of the genes deregulated by re-expression of WT and jmjC-dead KDM6A overlap suggesting that demethylase activity is not essential for all KDM6A functions in MM. Importantly, we confirmed the tumor suppressive role of KDM6A in a novel mouse model of MM in which KDM6A is deleted specifically in the B cell compartment. Briefly, we isolated CD19cre-/+ (control) or CD19cre-/+ Kdm6afl/fl fetal liver cells and transduced these cells with a constitutively activated form of the IL-6 coreceptor (L-GP130) that activates the JAK/STAT pathway. Mice transplanted with CD19cre-/+ Kdm6afl/fl fetal liver cells developed MM by 7 weeks post transplantation while mice transplanted with CD19cre+/- fetal liver cells did not developed MM by20 weeks. Necroscopy and flow cytometry analysis demonstrated infiltration of CD138+ cells in bone marrow, spleen, liver and kidney of mice that developed MM. In the future we will use this model to explore how loss of KDM6A affects chromatin structure in vivo and how it changes the characteristics of MM. These studies are expected to provide new insights that lead to the development of more effective MM therapies which directly target mechanisms of chromatin structure regulation. Disclosures Licht: Celgene: Research Funding.

Distinct Roles of Integrins α6 and α4 in Fetal Liver Hematopoietic Stem and Progenitor Cell Homing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 353-353
Author(s):  
Hong Qian ◽  
Elisabeth Georges-Labouesse ◽  
Alexander Nyström ◽  
Anna Domogatskaya ◽  
Karl Tryggvason ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Critical Role ◽  
Liver Cells ◽  
Hematopoietic Stem ◽  
Knockout Mouse Model ◽  
Genetic Ablation ◽  
Integrin Α4 ◽  
Fetal Liver Cells

Abstract During development and after transplantation, intravenously injected hematopoietic stem and progenitor cells (HSPCs) selectively transmigrate through the sinusoidal walls into the bone marrow (BM) niches to engraft and reconstitute hematopoiesis. The rate of reconstitution following transplantation varies depending on the source of hematopoietic stem cells (HSCs) (To, et al 1992). However, the molecular pathways that control the homing of HSCs, in particular, of fetal HSCs are still not well understood. In the present study we studied the contribution of α6 and α4 integrins in homing of fetal liver HSPCs into adult BM by using function-blocking antibodies and an integrin α6 knockout mouse model. We found an ubiquitous expression of both integrin α6 and α4 receptors on fetal liver Lin−Sca-1+c-kit+ (LSK) HSPCs. Genetic ablation of integrin α6 resulted in reduced homing of fetal liver progenitors (HPCs) to BM of lethally irradiated adult recipients. In agreement with this, the integrin α6 antibody inhibited homing of fetal liver HPCs into BM and spleen. The role of integrin a6 in homing and engraftment of fetal liver HSCs was studied by a competitive repopulation assay by using integrin α6−/− or α6+/+ fetal liver cells. Absence of α6 integrin in fetal liver cells did not cause any engraftment defect or mobilization hypersensitivity as compared to wild-type cells. In agreement with this, anti-integrin α6 antibody did not either inhibit BM homing of short-term or long-term HSCs. In contrast, homing of fetal liver HSCs and HPCs to BM was virtually abrogated after treatment with integrin α4 antibody. Our results show that the α6 integrin receptors are functional during homing of fetal liver HPCs, but not multilineage repopulating HSC in vivo. Furthermore, we show the critical role of integrin α4 receptor for homing of both fetal liver HPCs and multilineage repopulating HSCs to BM, indicating distinct developmentally regulated functions for integrin α6 and α4 receptors during fetal hematopoiesis

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