scholarly journals Preeclampsia is Associated with Sex-Specific Transcriptional and Proteomic Changes in Fetal Erythroid Cells

2019 ◽  
Vol 20 (8) ◽  
pp. 2038 ◽  
Author(s):  
Zahra Masoumi ◽  
Gregory E. Maes ◽  
Koen Herten ◽  
Álvaro Cortés-Calabuig ◽  
Abdul Ghani Alattar ◽  
...  
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Surface Adhesion ◽  
Hematopoietic Stem ◽  
Ampk Signaling ◽  
Placental Dysfunction ◽  
Transcriptional Changes ◽  
Amp Activated Protein Kinase

Preeclampsia (PE) has been associated with placental dysfunction, resulting in fetal hypoxia, accelerated erythropoiesis, and increased erythroblast count in the umbilical cord blood (UCB). Although the detailed effects remain unknown, placental dysfunction can also cause inflammation, nutritional, and oxidative stress in the fetus that can affect erythropoiesis. Here, we compared the expression of surface adhesion molecules and the erythroid differentiation capacity of UCB hematopoietic stem/progenitor cells (HSPCs), UCB erythroid profiles along with the transcriptome and proteome of these cells between male and female fetuses from PE and normotensive pregnancies. While no significant differences were observed in UCB HSPC migration/homing and in vitro erythroid colony differentiation, the UCB HSPC transcriptome and the proteomic profile of the in vitro differentiated erythroid cells differed between PE vs. normotensive samples. Accordingly, despite the absence of significant differences in the UCB erythroid populations in male or female fetuses from PE or normotensive pregnancies, transcriptional changes were observed during erythropoiesis, particularly affecting male fetuses. Pathway analysis suggested deregulation in the mammalian target of rapamycin complex 1/AMP-activated protein kinase (mTORC1/AMPK) signaling pathways controlling cell cycle, differentiation, and protein synthesis. These results associate PE with transcriptional and proteomic changes in fetal HSPCs and erythroid cells that may underlie the higher erythroblast count in the UCB in PE.

SCL/TAL1 Regulates Erythropoietin Receptor Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1195-1195
Author(s):  
Heather M. Rogers ◽  
Xiaobing Yu ◽  
Constance Tom Noguchi
Keyword(s):  
Erythroid Differentiation ◽  
Erythropoietin Receptor ◽  
Receptor Expression ◽  
Binding Motif ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Mild Anemia ◽  
Adult Mice ◽  
High Level

Abstract The basic-helix-loop-helix transcription factor SCL/TAL1, is required for erythropoiesis during development, and conditional deletion in adult hematopoiesis results in hematopoietic stem cells with a competitive repopulation disadvantage and defective erythropoiesis in vitro. However, adult mice with a conditional SCL/TAL1 deletion survive with mild anemia, suggesting defective erythroid proliferation and indicating that SCL/TAL1 is important, but not essential in mature red blood cell production. We find that during erythroid differentiation of primary human hematopoietic CD34+ cells, SCL/TAL1 expression peaks at day 8–10 following erythropoietin (EPO) stimulation, concomitant with peak expression of GATA-1 and EKLF. Treatment with SCL/TAL1 antisense oligonucleotides during erythroid differentiation markedly decreases erythroid differentiation as indicated by decreased expression of GATA-1 and both b- and g-globin expression, along with the absence of the characteristic decrease in GATA-2. Microarray analysis of erythroid cells overexpressing SCL/TAL1 indicate increased gene expression for b- and g-globin, and other genes related to erythropoiesis including EPO receptor (EPO-R), and these results are confirmed in stable cell lines with increasing SCL/TAL1 expression. Examination of EPO-R transcription regulation indicates that E-boxes in the 5′ UTR can bind SCL/TAL1 in vitro and, in addition to the GATA-1 binding motif, provide transcription activity in reporter gene assays. These data indicate that in addition to the importance of SCL/TAL1 DNA binding for proliferation of BFU-E and expression of glycophorin A and protein 4.2, SCL/TAL1 is also necessary for high level expression of EPO-R. Reduction in EPO-R expression likely contributes to the anemia associated with the conditional adult deletion of SCL/TAL1 and to the proliferative defect of erythroid cells observed in vitro. Early expression of SCL/TAL1 in hematopoietic cells may activate expression of EPO-R prior to EPO stimulation of erythropoiesis and induction of GATA-1.

scholarly journals Isolation of erythropoietin-sensitive cells from Friend virus-infected marrow cultures: characteristics of the erythropoietin response

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 751-758 ◽  
Author(s):  
M Bondurant ◽  
M Koury ◽  
SB Krantz ◽  
T Blevins ◽  
DT Duncan
Keyword(s):  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Precursor Cells ◽  
Erythroid Cells ◽  
Friend Virus ◽  
Isolated Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Erythroid Precursor ◽  
Short Time

Abstract Murine erythroid precursor cells, stimulated to proliferate in vitro in the absence of added erythropoietin (EP) by the anemia strain of Friend virus (FVA), will subsequently respond to EP by complete erythrocyte differentiation. If not exposed to EP, the erythroid cells divide for about 120 hr in culture, and they maintain the potential for full differentiation in response to EP added at any time during the period from 72 to 120 hr. Between 96 and 120 hr of culture without added EP, the EP-sensitive erythroid precursor cells that have formed discrete erythroid bursts can be isolated in relatively large numbers from such cultures by plucking with a Pasteur pipette. The addition of EP initiates the final stages of erythroid differentiation, including heme synthesis in 70%-80% of these isolated cells. With respect to homogeneity of the precursor cells, quantity of EP-responsive cells obtainable, and uniformity of EP responsiveness, this system is uniquely favorable for biochemical studies of the late differentiation effects of EP. The overall changes in gene expression accompanying EP- induced terminal differentiation were examined by two-dimensional gel electrophoresis of proteins labeled for a short time with radioactive amino acids. Several new proteins are synthesized in these erythroid cells during terminal differentiation, but the number is a very small percentage of the total number of proteins being made. Thus, in this system, the effect of EP is to initiate expression of a small group of genes, including those for globins, spectrin, and other proteins involved in the final stages of erythroid differentiation.

scholarly journals Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Gordon G. L. Wong ◽  
Gabriela Krivdova ◽  
Olga I. Gan ◽  
Jessica L. McLeod ◽  
John E. Dick ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Erythroid Precursor ◽  
Lineage Differentiation ◽  
Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

Metformin Protects Auditory Hair Cells from Gentamicin-Induced Toxicity in vitro

2015 ◽  
Vol 20 (6) ◽  
pp. 360-369 ◽  
Author(s):  
Andrea Glutz ◽  
Katharina Leitmeyer ◽  
Cristian Setz ◽  
Yves Brand ◽  
Daniel Bodmer
Keyword(s):  
Hair Cells ◽  
Apoptotic Cell ◽  
Mammalian Target Of Rapamycin ◽  
Spiral Ganglion ◽  
Oxygen Species ◽  
Antidiabetic Drug ◽  
Auditory Hair Cells ◽  
Amp Activated Protein Kinase ◽  
Toxicity In Vitro

Metformin is a commonly used antidiabetic drug. It has been shown that this drug activates the AMP-activated protein kinase, which inhibits downstream the mammalian target of rapamycin. In addition, several studies indicate that metformin reduces intracellular reactive oxygen species. Our data, using an in vitro rat model, indicate that metformin is able to protect auditory hair cells (HCs) from gentamicin-induced apoptotic cell death. Moreover, metformin has no toxic effect on spiral ganglion neuronal survival or outgrowth in vitro. These results suggest a protective effect of metformin on auditory HC survival in gentamicin-induced HC loss in vitro.

scholarly journals AMP-activated protein kinase pathway and bone metabolism

2011 ◽  
Vol 212 (3) ◽  
pp. 277-290 ◽  
Author(s):  
J Jeyabalan ◽  
M Shah ◽  
B Viollet ◽  
C Chenu
Keyword(s):  
Protein Kinase ◽  
Energy Homeostasis ◽  
Bone Cells ◽  
Peripheral Tissues ◽  
Ampk Signaling ◽  
Obesity And Diabetes ◽  
Regulate Food Intake ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

There is increasing evidence that osteoporosis, similarly to obesity and diabetes, could be another disorder of energy metabolism. AMP-activated protein kinase (AMPK) has emerged over the last decade as a key sensing mechanism in the regulation of cellular energy homeostasis and is an essential mediator of the central and peripheral effects of many hormones on the metabolism of appetite, fat and glucose. Novel work demonstrates that the AMPK signaling pathway also plays a role in bone physiology. Activation of AMPK promotes bone formationin vitroand the deletion of α or β subunit of AMPK decreases bone mass in mice. Furthermore, AMPK activity in bone cells is regulated by the same hormones that regulate food intake and energy expenditure through AMPK activation in the brain and peripheral tissues. AMPK is also activated by antidiabetic drugs such as metformin and thiazolidinediones (TZDs), which also impact on skeletal metabolism. Interestingly, TZDs have detrimental skeletal side effects, causing bone loss and increasing the risk of fractures, although the role of AMPK mediation is still unclear. These data are presented in this review that also discusses the potential roles of AMPK in bone as well as the possibility for AMPK to be a future therapeutic target for intervention in osteoporosis.

b1 Integrin Deficiency in Both Erythroid Cells and Their Microenvironment Does Not Affect Basal Erythropoiesis but Critically Impairs Survival and Erythroid Response to Phenylhydrazine-Induced Stress in Adult Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3567-3567
Author(s):  
Tatiana Ulyanova ◽  
Gregory V. Priestley ◽  
Yi Jiang ◽  
Stephen Padilla ◽  
Thalia Papayannopoulou
Keyword(s):  
Critical Role ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
In Vivo Experiments ◽  
Adult Mice ◽  
Erythroid Precursors ◽  
And Control ◽  
Deficient Mice

Abstract Previous experiments in vitro have emphasized the important role of a5b1 integrin/fibronectin interactions in terminal stages of erythroid differentiation (JCB1987, 105:3105), whereas in vivo experiments with genetically deficient mice (JI2000, 165:4667) and recent in vitro ones emphasized the important contribution of a4b1 integrin in the expansion of fetal erythroid progenitors (JCB2007, 177:871) or for optimal responses post stress in adult animals (MCB2003, 23:9349). However, no abnormalities in erythropoiesis were reported in a model of conditional ablation of b1 integrins post-transplantation (Blood2006, 108:1857). Therefore, it has not been clear to what extent each of the two major b1 integrins (a4b1 and a5b1) alone or in combination is critical for expansion and/or terminal erythroid differentiation of adult cells at homeostasis and/or after stress. We have made detailed and parallel observations comparing erythropoiesis in two genetic models with conditional ablation of b1 or a4 integrins at homeostasis and after phenylhydrazine (PHZ)-mediated stress. Basal erythropoiesis in b1-, a4-deficient and control mice as assessed by hematocrit levels and total nucleated erythroid cells (Ter119+) in BM and spleen was similar. Furthermore, both b1 and a4-deficient mice showed an increase in circulating progenitors (1275±230 CFC/ml PB, 2446±256 CFC/ml PB, respectively) over controls (338±113 CFC/ml PB). However, post PHZ-induced hemolytic stress there was a dramatic difference in outcomes of b1-deficient, but modest differences in a4-deficient mice compared to controls. Survival of b1-deficient mice by day 6 post PHZ was 33% compared to 100% in a4-deficient and control groups. In b1-deficient animals, no significant increase in spleen cellularity (153±26×106 and194±64×106 cells/spleen at day 0 and 6 post PHZ, respectively) was detected and the expansion of total erythroid precursors (CD71hi,Ter119+) in the spleen was minimal (from 2.08×106 to 10.8×106 cells/spleen at day 6). In contrast, in a4-deficient and control mice by the same time spleen cellularity increased respectively by 3 and 8 fold, and erythroid precursors expanded by 400 and 2,500 fold. Of interest, BM response to PHZ was not significantly different among all groups. To test whether the splenic response was cell-autonomous or environmentally controlled we compared PHZ response in wild type recipients reconstituted with b1-ablated (Cre+b1D/D) or with control (Cre-b1f/f) BM cells. Recipients of b1-ablated cells had an impaired response compared to recipients of control cells, which was somewhat intermediate to that seen in non-transplanted b1-deficient animals; by day 6 post PHZ, spleen cellularity was 300±24×106 cells/spleen and erythroid precursors expanded by 130 fold in recipients of b1-ablated BM cells compared to 859±159×106 cells/spleen and 900 fold precursor increase in control recipients. These data suggest that both erythroid and their environmental cells were responsible for the reduced survival and poor spleen response in b1-deficient mice. The target environmental cells (fibroblasts, endothelial cells, macrophages) and/or matrix involved will be the focus of future studies. It is of interest that in contrast to splenic response, the increased release of progenitors from BM seen in animals reconstituted with b1D/D cells was as high as that seen in non-transplanted b1- deficient animals and with the same qualitative characteristics, suggesting this alteration in biodistribution of progenitors is cell autonomous. Taken together, our data suggest that a combined expression of b1 integrins in erythroid and cells in their microenvironment is critical for survival and optimal splenic response to a PHZ-induced stress in adult mice; release of progenitors seen at homeostasis in both b1 and a4 models is cell autonomous with a preferential erythroid progenitor release from BM seen only in b1-deficient but not in a4-deficient mice; in contrast to results with fetal liver cells showing a critical role of a4b1 but not a5b1 integrin for proliferative expansion of erythroid cells, in adults a5b1 expression in erythroid and environmental cells in the spleen assumes a more critical role. Our data expand the current knowledge on the distinct dependency of a4b1 vs a5b1 integrins in basal vs stress erythropoiesis and bridge previously divergent information from in vitro and in vivo experiments.

TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4829-4829
Author(s):  
David C Dorn ◽  
Wei He ◽  
Joan Massague ◽  
Malcolm A.S. Moore
Keyword(s):  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Transforming Growth Factor Β ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

L-Leucine Improves the Anemia of the 5q- Syndrome Via Activation of the mTOR (mammalian target of rapamycin) Pathway

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 970-970
Author(s):  
Maria Virgilio ◽  
Elspeth Payne ◽  
Anupama Narla ◽  
Hong Sun ◽  
Barry H. Paw ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Mammalian Target Of Rapamycin ◽  
Macrocytic Anemia ◽  
Flow Cytometric Analysis ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Target Of Rapamycin ◽  
Translational Efficiency ◽  
Erythroid Cells ◽  
Hematopoietic Stem

Abstract Abstract 970 The “5q- syndrome” is a subtype of myelodysplastic syndrome (MDS) characterized by a profound macrocytic anemia that is thought to arise from the heterozygous loss of the RPS14 gene on chromosome 5q. The 5q- syndrome shares many characteristics with another ribosomopathy, Diamond Blackfan anemia (DBA), wherein half the patients have a heterozygous loss of a ribosomal protein gene, with RPS19 being the most frequently mutated. Both RPS19 and RPS14 are components of the 40S small ribosomal subunit. Heterozygous loss of both genes is thought to contribute to the pathophysiology of these ribosomopathies by impairing ribosome function and thereby decreasing mRNA translational efficiency. L-Leucine, a branched-chain amino acid has been shown to be a potent stimulator of mRNA translation. In a case report, administration of L-Leucine to a DBA patient resulted in complete recovery of the anemia and improvement in appetite and growth parameters. We therefore hypothesized that L-Leucine could improve the anemia associated with heterozygous loss of RPS14. To test this hypotheisis, we used zebrafish as an in vivo model for del(5q) MDS. Using antisense morpholinos, we knocked down Rps14 expression to haploinsufficient levels and observed anemia in the resulting morphants. Treatment of these morphants with L-Leucine, but not D-Leuicne, resulted in a dramatic increase in hemoglobinization as well as an increase in total erythroid cells. This observation was further validated in vitro using human CD34+ hematopoietic progenitor cells infected with RPS14 shRNA. Flow cytometric analysis demonstrated that treatment with L-Leucine increased the total number of erythroid cells (glycophorin-A/CD71 expressing cells) compared to untreated cells. In previous studies, L-leucine has been shown to upregulate mRNA translation by activating the mTOR (mammalian target of rapamycin) pathway and its downstream targets S6Kinase (S6K1) and 4E-Binding proteins (4E-BPs). We demonstrated increased levels of phospho-S6K1 as a result of L-Leucine treatment in the rps14 zebrafish morphant embryos. This increased phosphorylation of S6K1 was inhibited by rapamycin, suggesting specificity of mTOR activation in bringing about the L-Leucine effect. In summary, we have successfully demonstrated, in both a zebrafish model and in human primary hematopoietic stem cells, that L-Leucine alleviates the anemia associated with del(5q) MDS. This effect is likely mediated by activation of the mTOR pathway resulting in increased mRNA translation and an improvement in the anemia associated with loss of RPS14. Our studies support the further evaluation of L-Leucine as a potential therapeutic agent in the treatment of the 5q- syndrome. Disclosures: No relevant conflicts of interest to declare.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4107-4107
Author(s):  
Susan Hilgendorf ◽  
Hendrik Folkerts ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Erythroid Differentiation ◽  
Lentiviral Vectors ◽  
Apoptotic Response ◽  
Double Positive ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p<0.01) and, to some extent, granulocytic and macrophage colony formation as well (p<0.09, p<0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p<0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

scholarly journals In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Jiafei Xi ◽  
Yanhua Li ◽  
Ruoyong Wang ◽  
Yunfang Wang ◽  
Xue Nan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Red Blood Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

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