Overexpression of Oncogenic KRAS G12V in Human Stem and Progenitor Cells Enhances Proliferation and Initiates Monocytic Differentiation Via Intrinsic and Extrinsic Pathways.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3975-3975
Author(s):  
Szabolcs Fatrai ◽  
Djoke van Gosliga ◽  
Lina Han ◽  
Simon M. G. J. Daenen ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Signal Transduction Pathways ◽  
Monocytic Differentiation ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells ◽  
P38 Pathway

Abstract Abstract 3975 Poster Board III-911 In human hematopoietic malignancies, Ras mutations are frequently present in monocytic and T-cell leukemias. In this study we investigated KRAS G12V-induced phenotypes in human stem and progenitor cells and identified signal transduction pathways that are involved. Using a retroviral expression system, KRAS G12V was introduced to human CD34+ cord blood (CB) cells and proliferation, differentiation and stem cell/progenitor frequencies were evaluated. Overexpression of constitutively active KRAS G12V induced a strong increase in cell expansion over 5-fold in MS5 bone marrow stromal cocultures as well as in cytokine-driven liquid cultures, which coincided with increased early cobblestone formation and induction of monocytic differentiation. Erythroid progenitors were greatly reduced by introduction of KRAS G12V and Q-PCR analysis revealed that expression of PU.1 was increased in conjunction with reduced GATA1 expression in KRAS G12V cells. Progenitor frequencies were increased 6-fold in KRAS-transduced cells within 1 week after plating on MS5. By week three progenitors were exhausted and KRAS-transduced cells were terminally differentiated into monocytes/macrophages. These results were in line with the strong reduction in LTC-IC frequencies at week 5, indicating that also the stem cell pool was exhausted. Intriguingly, when KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, we observed that the non-transduced cells also displayed a strong growth advantage, coinciding with enhanced early cobblestone formation. Furthermore, the addition of conditioned medium from KRAS G12V-transduced cells grown on MS5 to non-transduced CB cells induced a strong growth advantage and formation of early CAFCs. These observations indicate that, besides intrinsic pathways, secreted factor(s) play an important role in the phenotypes induced by KRAS G12V in human CB CD34+ cells. Current studies include mass-spectroscopy analysis of the secretome of KRAS G12V-transduced CB CD34+ cells to identify the factor(s) that are involved. In order to elucidate signal transduction pathways that mediate KRAS G12V-induced phenotypes, Western-blot analysis was performed. These experiments revealed an increase in phospho-ERK1/2, phospho-p38 and phospho- STAT5 (Y694) levels in KRAS-transduced cells, whereas phospho-JNK was not induced and phospho-C/EBPa (S21) levels were slightly reduced. Induction of STAT5 Y649 phosphorylation by KRAS G12V was confirmed by intracellular phosphoFACS analysis, whereby both in HSCs as well as in more committed MPPs KRAS-induced phosphorylation of STAT5 was observed. KRAS-transduced cells did not show GM-CSF hypersensitivity in any measured cell population upon activation. Inhibition of the ERK/MAPK pathway using the MEK inhibitor U0126 resulted in strongly reduced expansion in MS5 cocultures, whereby both intrinsically induced proliferation as well as proliferation induced via secreted factor(s) were impaired. KRAS G12V-induced monocytic differentiation was not significantly affected by MEK inhibition. While inhibition of the JNK pathway hardly affected proliferation and differentiation of KRAS G12V cells, inhibition of the p38 pathway using SB203580 inhibitor impaired both proliferation and differentiation. When KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, inhibition of p38 predominantly affected the transduced cells but not the non-transduced cells, suggesting that the p38 pathway particularly mediates intrinsic phenotypes imposed by KRAS G12V. In conclusion, we show that overexpression of oncogenic KRAS G12V in human CD34+ cells enhances proliferation and initiates monocytic differentiation via intrinsic and extrinsic pathways. Disclosures: No relevant conflicts of interest to declare.

Multicolor Flow Cytometry Allows Quantitative Analysis of Signal Transduction in Murine and Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5393-5393
Author(s):  
Tamara Riedt ◽  
Claudia Lengerke ◽  
Lothar Kanz ◽  
Viktor Janzen
Keyword(s):  
Signal Transduction ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Intracellular Signaling ◽  
Hematopoietic Stem ◽  
Specific Antibodies ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract The regulation of cell cycle activity, differentiation and self-renewal of stem cells are dependent on accurate processing of intrinsic and extrinsic signals. Traditionally, signaling pathway activation has been detected by immunobloting using phospho-specific antibodies. However, detection of signal transduction in rare cells within heterogeneous populations, such as hematopoietic stem and progenitor cells (HSC) has been difficult to achieve. In a recently reported approach to visualize signaling in selected single c-Kit+ Sca-1+ Lin− (KSL) bone marrow cells, cells were sorted onto glas slides by flow cytometry and signaling was detected by confocal fluorescence microscopy, a very time consuming method that thus restricts the number of cells that can be analysed simultaneously. Moreover it permits only qualitative, but not quantitative signaling evaluation (Yamazaki et al., EMBO J. 2006). Here, we report a new protocol allowing quantitative measurement of signaling activity in large numbers of defined murine and human hematopoietic cells. The cells are stained with established surface markers and then phospho-specific antibodies are used to detect the levels of active intracellular signaling molecules. Signals are quantified by flow cytometry fluorescence measurement. Importantly, the protocol developed in our laboratory enables preservation of surface marker staining identifying the cells of interest inspite the fixation and permeabilization procedures necessary for intracellular signaling detection. This applies also for antigens previously reported to be particularly vulnerable to standard fixation and permeabilization approaches (e.g. the murine stem cell markers c-Kit and Sca1). Thus, our protocol provides an easy and reliable method for quantifying the activation degree of several intracellular signaling pathways on single cell level in defined hematopoietic (stem) cells within the heterogeous bone marrow (BM) compartment. Using cytokines known to exert a biological effect on HSCs, we have examined the susceptibility of KSL murine BM cells and human BM CD34+ cells to cytokine-induced signaling. We have performed extensive dosage titration and time course analysis for multiple cytokines (SCF, TPO, Flt-3, IL-3, IL-6, Ang-1, SDF-1α, TGF-β, and BMP-4) and signaling pathways (ERK, Akt, p38MAPK, Jak-Stat, TGF-β/BMP-Smad) in murine KSL BM cells. The activation intensity and the duration of signal activity as measured by the expression of corresponding phosphorylated proteins were cytokine specific. The obtained results can be used as a platform to explore signaling alterations in distinct compartments of the hematopoietic system, and may provide mechanistical insights for observed bone marrow defects (e.g impaired ERK signaling pathway has been detected as a possible cause of hematopoietic defects in Caspase-3 mutant murine HSCs, Janzen et al, Cell Stem Cell 2008). Furthermore, we could show that the technique is also applicable to human BM cells and that the human hematopoietic stem cell marker CD34 is also preserved by our fixation and permeabilization protocol. Preliminary results suggest that cytokines induce similar signaling activation in human CD34+BM cells collected from healthy donors. As observed in mouse KSL BM cells, stimulation of human CD34+cells with human stem cell factor (hSCF) induced activation of the ERK but not the Akt pathway. Ongoing experiments analyse the stimulatory effects of other cytokines such as thrombopoietin (TPO) and fms-related tyrosine kinase 3 (Flt-3) and their corresponding pathways. Moreover, comparative studies are underway analyzing cross-reactivity between mouse and human cytokines, aiming to provide insights into cytokine-induced biases in commonly used xenotransplantation models.

Differential Effects of a Novel Non-Peptidic Thrombopoietin Mimetic on Proliferation and Differentiation of Human CD34+ Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2888-2888 ◽  
Author(s):  
Juha Punnonen ◽  
Marcus O. Muench ◽  
Jeffrey R. Spencer
Keyword(s):  
Molecular Weight ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Aqueous Solubility ◽  
Similar Proportion ◽  
Low Molecular Weight ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Megakaryocyte Differentiation

Abstract Thrombopoietin (TPO) and TPO mimetics have been shown to be beneficial in the treatment of thrombocytopenia. Low molecular weight, orally available compounds offer the advantage of convenience and lack of immunogenicity when compared to protein-based drugs. STS-T4 is a novel low-molecular weight (<500 Da), non-peptidic TPO mimetic compound that is a 2nd generation agent in preclinical development with promising in vitro potency and aqueous solubility. We have studied the effects of STS-T4 on the growth and differentiation of primary human CD34+ progenitor cells. A dose-dependent proliferation of highly purified human CD34+ cells was observed with EC50 value of less than 1 μM. A bell-shaped dose-response curve was observed, which is consistent with previous reports on compounds stimulating homodimeric cytokine receptors. In addition, the phenotype of the cultured cells was analyzed by flow cytometry and monoclonal antibodies specific for markers of megakaryocyte differentiation. During a culture period of 10 days, 85% of the cells cultured in the presence of STS-T4 differentiated into CD41+ megakaryocytes in the absence of any other cytokines or mimetics. In addition, 30% of these CD41+ cells coexpressed CD42b as a marker of more mature cells. When compared to recombinant human TPO (rhTPO), a similar proportion of the cultured cells expressed CD41 and CD42b in response to STS-T4 and the levels of expression of these antigens on the surface of the megakaryocytes were similar, suggesting that the effects of STS-T4 and rhTPO on human megakaryocyte differentiation are comparable. However, the level of cell proliferation induced by STS-T4 in cultures of CD34+ cells was 50–60% of that induced by rhTPO, supporting the conclusion that the signals mediating proliferation and differentiation of CD34+ progenitor cells are differentially regulated. In addition to the activity profile, the physical properties of STS-T4 are desirable for further development. Aqueous solubility and gastrointestinal permeability are major contributors to oral absorption, and high solubility and permeability generally also reduce the risk of food effects. The aqueous solubility of STS-T4 was measured by a kinetic method with HPLC and visual detection and was determined to be >1 mM. These data suggest that further evaluation of safety and efficacy of STS-T4 for the treatment of thrombocytopenia is warranted. In addition, the results imply that CD34+ progenitor cells can differentiate into megakaryocytes expressing high levels of CD41 and CD42b in the absence of a full rhTPO-like proliferative response, which may support further investigation of new therapies for the treatment of thrombocytopenia that provide the benefit of megakaryocyte maturation while avoiding excessive expansion of hematopoietic stem cells.

SFK Inhibition with Dasatinib Results In Selective Targeting of Primitive Human Acute Myeloid Leukemia Stem and Progenitor Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1053-1053
Author(s):  
Cedric Emmanuel Dos Santos ◽  
Tinisha McDonald ◽  
Liang Li ◽  
Allen Lin ◽  
Ya-Huei Kuo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Growth And Survival ◽  
Cd34 Cells ◽  
Dependent Inhibition ◽  
Stem And Progenitor Cells ◽  
Control Versus ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Abstract Abstract 1053 The Src family tyrosine kinases (SFKs) are abnormally activated in AML compared to normal CD34+ hematopoietic progenitors. Studies using pharmacological and siRNA approaches indicate an important role for the SFK Lyn in AML progenitor cell growth and survival (Dos Santos et al., 2008). However the role of SFKs in AML leukemic stem cell (LSC) growth and survival is not clear. SFK activity in Lin- CD34+ CD38- primitive stem/progenitor cells, Lin- CD34+ CD38+ committed progenitors, and Lin-CD34- cells from primary human AML (n=14) and cord blood (CB) (n=6) samples was measured by analyzing SFK phosphorylation using flow cytometry. We observed significant increase of SFK phosphorylation in AML compared to CB Lin- CD34+CD38- cells (3.8±1.9 versus 1.9±0.7, p=0.006), Lin- CD34+ CD38+ (3.9±1.7 versus 1.9±0.6, p=0.013) and Lin- CD34- cells (3.4±1.8 versus 1.1±0.5, p=0.0005). Dasatinib, a potent SFK and ABL kinase inhibitor, is approved for clinical use in chronic myeloid leukemia. We evaluated the effect of SFK inhibition using Dasatinib on the growth and viability of AML stem and progenitor cells. Dasatinib exposure resulted in dose-dependent inhibition of SFK phosphorylation in each subpopulation (in CD34+ CD38-, 3.0 for the control versus 1.9 with 100nM and 1.6 with 500nM, in CD34+ CD38+, 2.8 for the control versus 1.9 with 100nM and 1.6 with 500nM) after 30 minutes of drug treatment). The addition of Dasatinib (10-500nM) to methylcellulose progenitor assays resulted in dose-dependent inhibition of AML colony forming cell (CFC) growth (83.9±16.1% inhibition with 500nM, and 48.1±6.8% inhibition with 10nM Dasatinib, n=8), to a greater extent than CB CFC to (CFU-GM inhibition 62.6±1.5% with 500nM, and 1.1±16.3% with 10nM Dasatinib, n=4). Short-term exposure to Dasatinib (10-500nM) for 48 hours also resulted in significantly greater inhibition of AML CFC (73.6±13% with 500nM, and 41.7±10.8% with 10nM Dasatinib, n=8) compared to CB CFC (CFU-GM inhibition 23±9.1% with 500nM, and 1.3±11.3% with 10nM Dasatinib, n=4). Importantly Dasatinib treatment (200nM) also resulted in reduction of AML stem/primitive progenitor growth in long term culture-initiating cells (LTC-IC) assays (56±23, 8 % inhibition, p=0.003, n=4), suggesting that SFK inhibition may inhibit AML stem cell maintenance. The effect of Dasatinib on apoptosis was evaluated by labeling cells with Annexin V and DAPI. Treatment with Dasatinib resulted in significant increase in apoptosis of Lin- AML cells (41.5% ±10.7 of apoptosis with 200nM Dasatinib versus 25%±10.8 for the control, p=0,004, n=5) We studied the effects of Dasatinib on differentiation of Lin- CB (n=3) and AML cells (N=5) cultured with SCF, IL-3, GM-CSF, G-CSF and EPO. In normal CD34+, Dasatinib (100 nM) treatment resulted in increased CD33+ and CD14+ cells and reduced CD34+, CD11b+, CD15+, GPA+ and CD71+ cell numbers, indicating that SFK increased monocytic but reduced granulocytic and erythroid differentiation. Treatment of AML cells with Dasatinib resulted in markedly reduced numbers of CD34+, CD33+ and CD71+ cells, but increased numbers of CD11b cells, in 3 of 5 samples, indicating a trend towards increased granulocytic differentiation in contrast to normal progenitors. Our results indicate that SFK activity is increased in primary human AML stem and progenitor cells and suggest that SFK blockade with Dasatinib may reduce maintenance of AML LSC/ primitive progenitors, through inhibition of progenitor proliferation, induction of apoptosis and enhancement of differentiation. These results support further evaluation of SFK blockade with Dasatinib for targeting of AML stem and progenitor cells in preclinical and clinical studies. Disclosures: Bhatia: Novartis: Consultancy, Honoraria.

Ultrastructural effects of recombinant human hematopoietic stem cell factor on mast cells and basophils from human CD34+ pluripotent progenitor cells

1991 ◽  
Vol 49 ◽  
pp. 310-311
Author(s):  
J.P. Goff ◽  
A. S. Kirshenbaum ◽  
J. P. Albert ◽  
S.W. Kessler ◽  
K. M. Zsebo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mast Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem Cell ◽  
Stem Cell Factor ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
3T3 Fibroblasts ◽  
Proliferation And Differentiation ◽  
Cells Cultured

Hematopoietic stem cell factor (SCF) is the product of Sl locus in the mouse and is the ligand for the protooncogene c-kit. The human homologue has recently been cloned and recombinant protein (rhSCF) expressed and purified to homogeneity. rhSCF is synergistic with human cytokines in promoting the proliferation and differentiation of human progenitor cells.Human mast cells and basophils have been shown to originate from human bone marrow-derived CD34+ pluripotent progenitor cells cultured in the presence of rhIL-3, and develop granule scroll patterns only when cocultured with mouse 3T3 fibroblasts. Mast cells, from CD34+ cells cultured over agarose surfaces, in the presence of rhIL-3 had granules that contained only homogeneously dense material. To determine the effect of rhSCF on the appearance of mast cells and basophils in culture, highly purified human CD34+ progenitor cells were grown in the presence of rhIL-3, rhSCF, or rhIL-3 in combination with rhSCF over agarose surfaces.

Neuropeptides Orexin A and B Are Funktionally Aktive in CD34+ Hematopoietic Stem and Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4593-4593
Author(s):  
Ron-Patrick Cadeddu ◽  
Akos G. Czibere ◽  
Sebastian Büst ◽  
Johannes C Fischer ◽  
Ulrich Steidl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Calcium Influx ◽  
Receptor Stimulation ◽  
Orexin A ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Orexin Receptors ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 4593 Orexin receptors are involved in the regulation of sleep-wake-rhythm, food intake and energy homeostasis and it was still recently believed that their expression is restricted to the nervous system. But, during the last years orexin receptors have been detected in an increasing number of peripheral tissues. We have earlier found orexin receptor 1 and 2 expression on human CD34+ hematopoietic stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seemed so far to be restricted to the central nerve system. Ca2+-dependent signaling and activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways are considered as main downstream signaling pathways of the orexin receptors. In this study, we investigated the signaling and functional role of orexin receptors in CD34+ hematopoietic stem and progenitor cells. Using confocal fluorescence microscopy and flow cytometry we found that stimulation of purified CD34+ cells with orexin A and B led to an increase of the intracellular calcium concentration due to both calcium influx and calcium release from intracellular stores. Of interest, incubation with orexin reduces the SDF-1β-induced calcium influx. Furthermore orexin receptor stimulation led to a decrease of the intracellular cAMP concentration. Following orexin receptor stimulation with orexin A and B, we observed an initial increase of ERK1/2 phosphorylation up to 30 minutes upon incubation with orexin followed by a decrease at several time points up to 8 hours in comparison to the unstimulated control. To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. Remarkably, stimulation with orexin A and B led to a significant higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors. A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significant higher frequency of LTC-IC indicating also a more immature phenotype of orexin-stimulated cells. In line, orexin receptor stimulation led to a significant increase of the proportion of Lin-, CD34+, CD38- HSC in the G0-phase of the cell cycle. Furthermore, stimulation with orexin A and B increased the number of apoptotic cells in the Lin-, CD34+, CD38- HSC fraction and the total hematopoietic stem and progenitor population determined by flowcytometric analysis of intracellular cleaved caspase 3 content. The adhesive capacity of CD34+ cells to fibronectin and collagen coated dishes and the migratory capacity was significantly decreased upon orexin receptor stimulation. Concurrent incubation with the selective Gi-protein inhibitor pertussis toxin abrogated these effects. Given the functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using FACS analysis, immunfluorescent staining and western blotting we could detect prepro-Orexin in CD34+ cells and using ELISA orexin was found in the serum obtained by bone marrow biopsies and peripheral blood. Taken together, the phenotype of orexin-stimulated hematopoietic stem and progenitor cells suggest a mobilizing effect of the orexin receptor stimulation as well as an increased repopulation capacity which might be of relevance in clinical stem cell mobilization and transplantation and is currently verified in murine models. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4619-4619
Author(s):  
Susan Hilgendorf ◽  
Hendrik Folkerts ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Liquid Culture ◽  
Erythroid Differentiation ◽  
Apoptotic Response ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract In recent clinical studies, it has been demonstrated 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). Mutation of ASXL1 leads to truncation of the protein and thereby to a 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 hematopoietic system in vitro mimics the changes observed in MDS-patients. We down regulated ASXL1 in CD34+ cord blood (CB) cells using a lentiviral approach and obtained a 40-50% reduction of ASXL1 expression. Colony forming (CFC) assays revealed that erythroid colony formation was significantly impaired (p=0.01) and, to some extent, granulocytic and macrophage colony formation (p=0.09, p=0.05 respectively). As MDS can affect all hematopoietic lineages, we first stimulated cell differentiation along the myeloid or erythroid lineage in liquid culture. Upon culturing shASXL1 CB CD34+ cells in suspension, we observed a modest reduction in expansion (two-fold at week1) 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 of cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. This phenotype is similar to what has been observed in patients, where increased cell death of progenitors occurs, and suggests that ASXL1 loss may reflect an MDS-like phenotype in this culture setting. Furthermore, as MDS is considered a hematopoietic stem cell (HSC)-driven disorder, 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, we performed cultures on stromal layers with or without cytokines. shASXL1 CB CD34+ cells cultured on MS5 stromal layer 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. In patients, mutations in ASXL1 are frequently accompanied by a loss of p53. Possibly, loss of p53 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 first data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies compared to shASXL1/shSCR cells, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, when using erythroid liquid conditions, a decrease in apoptosis compared to the ASXL1 single mutation could be observed. Nevertheless, no transformation occurred and ASXL1 mutated cells were eventually lost in the double hit model despite reduced apoptosis, suggesting that the p53 axis might not be sufficient as the second hit for full transformation. In conclusion, our data indicate that mutations in ASXL1 may lead to an increase in cell death and reduced progenitor output in vitro, which may reflect disease development and progression as seen in patients. Unexpectedly, MS5 stromal did not alter the negative phenotype caused by ASXL1 knock down. Therefore, studies are ongoing to investigate whether an already established MDS microenvironment will influence ASXL1 mutation positively. To this end, we are using healthy human mesenchymal stem cells (MSC) and patient derived MDS MSCs. Disclosures No relevant conflicts of interest to declare.

JAK/STAT Signal Transduction Pathway Activation During Hematopoietic Differentiation From Human Embryonic Stem Cells (hESC)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2346-2346
Author(s):  
Chun Fan ◽  
Richard Yunkang Liu ◽  
Kristine Li ◽  
Kenneth S. Zuckerman
Keyword(s):  
Signal Transduction ◽  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Undifferentiated Hesc

Abstract Abstract 2346 The ability to produce hematopoietic cells from human embryonic stem cells (hESC) has been demonstrated, using different multistage culture systems with multiple growth factor combinations. However, very little is understood about the molecular mechanisms that regulate the differentiation from hESC to hematopoietic stem and progenitor cells and further to specific lineages of differentiated hematopoietic cells. Among many signaling pathways involved in stem and progenitor cell differentiation, the JAK/STAT pathways are known to play critical roles in hematopoietic stem cell maintenance and hematopoietic differentiation. STAT3 activation is known to be essential for maintenance of murine ESC, but not human ESC, but it appears not to play a major role in myeloid cell differentiation. Although different levels of JAK2 and STAT5 signaling are important for erythroid and megakaryocytic differentiation, JAK/STAT signaling is not thought to play a role in hESC maintenance or differentiation and is not known to be essential for early stages of differentiation to hematopoietic stem and progenitor cells (HSC/HPC). We have established a serum-free, feeder cell-free system for maintaining hESC (H1 and H9 cells) and for differentiating the hESC to embryoid bodies (EB), from which end-stage hematopoietic cells, notably megakaryocytes and platelets, are produced. We used a multi-stage culture system to produce megakaryocytes and platelets from EBs, including 2 days with vascular endothelial growth factor (VEGF) and bone morphogenic protein (BMP4), 2 more days with VEGF, BMP4, stem cell factor (SCF), Flt3 ligand (FL), and thrombopoietin (TPO), 10 days with VEGF, BMP4, TPO, SCF, FL, IL3, IL6, and IL11, and 2–6 weeks with TPO, SCF, FL, IL3, IL6, and IL11. We used serial western blots, immunofluorescence with confocal microscopy and systematically observed changes of JAK/STAT signal transduction molecule activation. We found a consistent, dynamic change of STAT5 protein phosphorylation during the hematopoietic differentiation process. Interestingly, although JAK2, STAT3 and STAT5 protein were present, and JAK2 and STAT3 were phosphorylated in hESC, there was no evidence of STAT5 phosphorylation/activation in the undifferentiated hESC. During the early phases of differentiation of hESC-derived EBs toward hematopoietic progenitors in the presence of hematopoiesis-related cytokines, STAT5 was phosphorylated and activated in CD34+ HSCs and in CD61+/CD235a (glycophorin A)+ or CD41+/CD235a+ early megakaryocytic/erythroid progenitor cells (MEP). Although there was no detectable change in total STAT5 protein expression levels through hematopoietic differentiation, there was a slowly progressive decrease in phosphorylated/activated STAT5 with further maturation to megakaryocytes that express CD42b+, platelet factor 4, and von Willebrand factor and form proplatelets and platelets. Thus, in spite of hESC containing abundant phosphorylated JAK2, which is a known activator of STAT5, there was no phosphorylation/activation of STAT5 in undifferentiated hESC or early EBs. However, STAT5 became phosphorylated/activated early in hematopoiesis and declined over the course of progressive differentiation along the megakaryocytic lineage. These findings imply that activated JAK2 does not drive the activation of STAT5 that is an early event in differentiation from EBs and mesoderm to HSC and HPC in vitro. Disclosures: No relevant conflicts of interest to declare.

R4 Rgs Subfamily Proteins Negatively Regulates SDF-1/CXCR4 Signaling in CD34+ Hematopoietic Stem and Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5048-5048
Author(s):  
Kam Tong Leung ◽  
Yorky Tsin Sik Wong ◽  
Karen Li ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Rgs Proteins ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract RGS family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to stromal cell-derived factor-1 (SDF-1) are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 10) expressed 7 out of 10 R4 RGS proteins at mRNA level (RGS1-3, 5, 13, 16 and 18), whereas expressions of RGS4, 8 and 21 were undetectable. Exposure of CB CD34+ cells to SDF-1 significantly increased RGS1, 2, 13 and 16 expressions and decreased RGS3 and 18 expressions (P ≤ 0.0402, n = 5). Expressions of RGS1, 13 and 16 were significantly higher in bone marrow (BM, n = 10) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 5) CD34+ cells (P ≤ 0.0160), while RGS3 and 18 expressions were lower in BM CD34+ cells (P ≤ 0.0471), suggesting a SDF-1- and niche-dependent regulation of RGS expressions. To investigate the potential involvement of RGS proteins in SDF-1-mediated homing-related functions, we introduced RGS overexpression constructs into CB CD34+ cells by lentiviral transduction. With >80% transduction efficiency, we showed that overexpression of RGS1, 13 and 16 but not RGS2 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.0391, n = 4-5). Similarly, RGS1, 13 and 16 overexpression suppressed SDF-1-induced Akt phosphorylation (n = 2), but none of them affected SDF-1-mediated actin polymerization (n = 3). In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing was impaired in RGS1- (16.3% reduction), RGS13- (12.7% reduction) or RGS16-overexpressing CD34+ cells (33.7% reduction). Taken together, we provided the first evidence that expressions of R4 RGS proteins are regulated by the SDF-1/CXCR4 axis in human CD34+ HSC. We also presented evidence that specific R4 RGS proteins (RGS1, 13 and 16) negatively regulate in vitro SDF-1-mediated responses and in vivo homing of CD34+ cells, suggesting that RGS proteins may serve as a feedback mechanism to regulate SDF-1/CXCR4 signaling. Strategies to inhibit RGS signaling could thus be a potential method for enhancing efficiency of HSC homing and long-term engraftment, which is particularly important in the setting of CB transplantation. Disclosures No relevant conflicts of interest to declare.

Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1498-1505 ◽  
Author(s):  
Aleksandra Rizo ◽  
Sandra Olthof ◽  
Lina Han ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Oxygen Species ◽  
Self Renewal ◽  
Human Cd34 ◽  
Bmi1 Expression ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Cord Blood Cd34

Abstract High expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34+ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that down-modulation of BMI1 in cord blood CD34+ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34+ cells, down-modulation of BMI1 impaired long-term expansion, whereby self-renewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and self-renewal of normal and leukemic stem and progenitor cells, and that expression of BMI1 protects cells against oxidative stress.

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