M07e Cells Research Articles

ID2 Promotes Leukemogenesis and Chemoresistance of Acute Myeloid Leukemia By Modulating Cellular Oxidative Metabolism

Acute myeloid leukemia (AML) relies on quiescent leukemia stem cells (LSCs) to evade chemotherapy and drive disease relapse. Recent research indicates that a specific subset of LSCs, characterized by elevated oxidative phosphorylation and/or increased senescence, is responsible for chemotherapy resistance and relapse. While numerous genes associated with LSC maintenance and resistance have been identified, the underlying molecular mechanisms remain elusive. Inhibitor of DNA binding (ID) proteins, a group of Helix-Loop-Helix (HLH) transcription regulators, play a dominant role in regulating other HLH proteins (E proteins) required for the normal development of hematopoietic, muscle, neuronal, and other cells. Our previous investigation revealed that ID2 protects quiescent hematopoietic stem cells (HSCs) activation and exhaustion. Analysis of TCGA data sets demonstrated that high levels of ID2 expression are linked to reduced overall survival in AML patients, suggesting ID2 may be required to maintain some LSCs. Consistent with murine cell findings, we found that knocking down (KD) ID2 expression in human cord blood cells resulted in a loss of CD34+CD38- progenitors. Similarly, ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-Patient Derived Xenograft (AML-PDX) cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. In conclusion, it can be said that ID2 plays a critical role in maintaining normal murine and human HSCs, and it likely contributes to leukemia development and chemotherapy resistance. Therefore, targeting ID2 may offer a promising therapeutic approach to selectively tackle LSCs, followed by chemotherapy and bone marrow transplantation to achieve complete remission.

Platelet-derived exosomes as the nano-carrier for miR-150 to modulate gene expression and cell cycle in the M07-e cell line

Aims and objectivePlatelet-derived exosomes are the most abundant Nano-particles in the plasma of healthy individuals. They have been wildly used as drugs, vectors, and miRs carriers. The present study was designed to hope its results will be used to treat patients with refractory thrombocytopenia. To follow the study's aims, we examined the capacity of platelet-derived exosomes to carry miR-150 and their influence on the expression of c-myb (the target gene of miR-150) and mitosis induction in the megakaryocytic model of M07-e. Methods and materialIn this experimental study, platelets were obtained via the platelet concentrates (PCs) prepared by the platelet-rich plasma (PRP) method. Then the exosomes were isolated from the platelets by the isolation kit. The isolated particles were loaded with miR-150 through the modified CaCl2 method. The efficiency of the transfection process was evaluated via qRT-PCR. The effect of loaded exosomes on the gene expression profile (c-myb) and the cell cycle of the megakaryocytic model (M07-e) was assessed via qRT-PCR and flow cytometry methods. ResultsExosomes successfully mediated miR-150 transfer into the cells. As a result, they down modulated c-myb expression significantly (p-value ≤0.05). It means miRs were successfully transferred and did not lose their function; on the other hand, they could not induce cell cycle transition. ConclusionThe findings of the present study shed light on the development of autologous Nano-carriers, which could be a step in advancing the aims of personalized medicine and reducing the side effects of the exogenous carrier.

Effects of LSD1 Inhibition with Img-7289 on the Leukemia Initiating Cell Population with Induction of Differentiation and Cell Death in Pediatric Relapsed/Refractory AML

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3

To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.

Abstract 3504: QL415, a tumor targeted IL-15 fusion protein stimulating both lymphoid and myeloid immune cells for optimal anti-tumor immune response

Abstract Cytokines are potent stimulators of the immune system and have long been investigated and used as therapeutics to treat cancer. However, free cytokines have short half-life and the lack of tumor targeting often results in systemic toxicity. IL-15 is a stimulatory cytokine that shares the common beta and gamma receptors with IL-2. In contrast to IL-2, IL-15 is more selective for NK and effector memory T cells, with less proliferative effect on Tregs, making it a more preferred therapeutic candidate for cancer. QL415 is an anti-PD-L1 x IL-15 fusion protein designed to enhance tumor accumulation and prolong blood circulation, thereby widening the therapeutic window. IL-15 and IL-15 receptor alpha sushi domain are covalently linked to the C-terminus of our proprietary PD-L1 antibody. Fc-mediated effector functions were ablated using mutations in the CH2 domain, to prevent depletion of effector immune cells. In vitro studies showed QL415 to be highly potent in promoting proliferation and pSTAT5 signaling of IL-15 -responding NK92 and M07e cells. QL415 selectively induced proliferation of NK and CD8+ T cells from human PBMC in vitro and mouse CD8+ and NK1.1 cells in vivo. In a MC38 mouse colon cancer model expressing human PD-L1, QL401 was highly effective in suppressing tumor growth, in contrast to PD-L1 monoclonal antibody or a non-targeted IL-15 control. Follow up tumor rechallenge study showed protective memory against cognate MC38 but not B16F10. In a separate MC38 tumor model using a mouse surrogate molecule of QL415, immunophenotyping of antigen presenting cells offered preliminary evidence of enrichment of conventional dendritic cells 1 in the tumor draining lymph nodes. Therefore, QL415 not only directly stimulates effector immune cells, but also indirectly through antigen presenting cells, promotes robust anti-tumor response. In non-human primates, Q415 was tolerated up to 1.5 mg/kg, well above the effective therapeutic dose. In light of these favorable preclinical efficacy and safety results, a phase 1 dose escalation and expansion study has been initiated to evaluate the safety, tolerability, and early efficacy of QL415. Citation Format: Irene Tang, Lauren Schwimmer, Monica Jin, Shenda Gu, Wei Wei Prior, Arianne Capacio, Hieu V. Tran, Allan Chan, Anna McClain, Shihao Chen, Chuanzeng Cao, Xiaoran Wu, Yanling Xu, Dongmei Guan, Xi Chen, Xianli Su. QL415, a tumor targeted IL-15 fusion protein stimulating both lymphoid and myeloid immune cells for optimal anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3504.

Epigenetically Enhanced MED12L in ETO2-GLIS2 Positive Pediatric Acute Megakaryoblastic Leukemia Is Associated with Resistance to the CDK8 Inhibitors

Introduction. ETO2-GLIS2 (aka CBFA2T3-GLIS2) is the most common (30%) alteration in pediatric de novo acute megakaryoblastic leukemia (AMKL). These patients have poor response to induction therapy, a high incidence of relapse (~90%), and dismal 5-year survival rates (<20%). Previous studies suggest that ETO2-GLIS2 induces leukemia through abnormal enhancer formation as a single oncogenic “hit”. Because ETO2-GLIS2 expression induces formation of leukemia-specific neo-superenhancer (SE) elements, we hypothesize that mediator (MED) proteins are involved in linking neo-SE elements to distal gene expression, and thus could be a therapeutic target. Here, we analyzed expression of MED-family genes in ETO2-GLIS2 positive AMKL patients in combination with enhancer-histone marks, and evaluated impact of MED-kinase inhibition with selective CDK8 inhibitors (CDKi) against cell viability.Methods. To address our hypothesis, we analyzed published RNA sequencing dataset (Smith et al. 2020) for human MED-genes expression from a pan-pediatric AML cohort (N=1476). The cohort consisted of subgroups expressing fusions of ETO2-GLIS2 (N=40), CBFB-MYH11 (N=174), DEK-NUP214 (N=49), KMT2A-ELL (N=50), KMT2A-MLLT10 (N=86), KMT2A-MLLT3 (N=114), KMT2A-MLLT4 (N=49), NUP98-NSD1 (N=107), RUNX1-RUNX1T1 (N=210) and no detectable fusions (N=526), compared to normal bone marrow (NBM) samples (N=71). Enrichment of histone marks overlapping MED-genes was analyzed from published chromatin immunoprecipitation (ChIP) sequencing in ETO2-GLIS2 positive M-07e AMKL line (Thirant et al. 2017). We tested efficacy of CDK8 inhibition with BI-1347 and CCT251545 against M-07e cells to determine their activity in the context of marked MED12L overexpression.Results. We examined expression of 29 MED-genes comprising the 4 major (head, middle, tail, and kinase) MED-modules. MED gene expression was variable across AML subtypes and NBM. However, MED genes were more commonly over-expressed in the ETO2-GLIS2 group, in particular MED 17, MED1, MED10, MED27, and MED12L (paralog of MED12) were upregulated in the subgroup. Most notably, we noted exceptional upregulation of MED12L (FC 4.9, log2), compared to NBM (Figure 1). Because MED12/12L plays an intrinsic biological role in establishing oncogenic enhancer-expression loops in hematopoietic stem or leukemic cells, we investigated the overlap of enhancer bound histones such as H3K27ac and H3K4me1 to MED12L in M-07e cells, compared to umbilical cord blood-derived normal megakaryoblasts (MK) (S004BT; Blueprint epigenome database). We found enrichment of H3K4me1 at MED12L transcription start site (TSS) and upstream promoter both in MK and M-07e cells. In addition, we observed a large region of H3K27ac enrichment spanning 89 Kb (16 kb upstream and 73 kb downstream) across MED12L TSS in M-07e cells, suggesting neo-enhancer activity at this locus. Considering the dependency of MED12 on CDK8 for MED-kinase activities (Klatt et al. 2020), we treated M-07e cells with CDK8i(s), to test our hypothesis if perturbation of epigenetically enhanced MED12L expression can impact leukemic growth. However, we observed a poor correlation between M-07e cell viability and IC 50 of BI-1347 (IC 50: 0.87 µM, R 2: 0.36) or CCT251545 (IC 50: 0.4 µM, R 2: 0.48). In contrast, ETO2-GLIS2 negative MV4-11 AML cells were susceptible to both BI-1347 (IC 50: 0.44 µM, R 2: 0.88) and CCT251545 (IC 50: 0.12 µM, R 2: 0.87). Given the inefficacy of CDK8i against M-07e, and cooperativity of bromodomain extra-terminal (BET)-BRD4 and MED12/12L in forming enhancer complexes, we tested possible inhibitory impact of BRD4 inhibitor JQ1 on MED12L expression and leukemic growth in target cells. We observed effective reduction in M-07e cell viability (IC 50:0.3 µM, R 2: 0.93) with concomitant reduction not only in BRD4 protein-expression, but diminished MED12L protein expression at IC 50 and higher doses of JQ1.Conclusion. Our findings revealed that MED12L is highly overexpressed and overlapped with strong neo-enhancer chromatin-marks in ETO2-GLIS2 positive cells, while maintaining resistance to CDK8i. Future studies will contribute to deeper insights into the preferential recruitment and role of MED12L in ETO2-GLIS2 bound enhancers and potential mechanisms of resistance to CDK8 inhibition in the disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-Apoptotic Effect of Angelica Polysaccharide (APS) on Cryopreservation of Platelets

Background: Angelica Polysaccharide (APS) is from the root of Radix Angelicae Sinensis (Danggui). Danggui has been used for centuries to treat blood-deficiency related diseases. The hematopoietic effect of Danggui may be related to its constituent, polysaccharide. The effects of angelica polysaccharide on cryopreservation of platelets and megakaryocytes have not been well studied. This study focused on anti-apoptotic effect of APS and TPO on cryopreservation of platelets and megakaryocytes and provided new methods for prolonging the preservation time of platelets in vitro.Methods: The expression of platelet membrane glycoprotein CD41 and CD61, as well as the platelet apoptotic rate, Caspase 3 expression and mitochondrial membrane potential (MMP) were detected by flow cytometry; the anti-apoptotic mechanism of APS by PI3K /AKT signaling pathway was analyzed by Western blot assay. CFU assays were used to determine the effects of APS on megakaryocytic progenitor cells. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were conducted in megakaryocytic cell line M-07e. The effects of APS on cells treated with Ly294002, PI3K inhibitor and the effect of APS on the p-AKT were also studied.Results: The platelets were divided into 4 group: control group (4 ℃ stored platelets), APS group (APS-treated platelets stored at 4 ℃), LY294002 group (LY294002-treated platelets stored at 4 ℃) and LY294002+APS group (LY294002+APS treated platelets stored at 4 ℃). The apoptotic rate of platelets in LY294002 group was obviously increased. Compared with control group, the expression of CD41 and CD61 gradually decreased along with the enhancement of LY294002 concentrations (r=-0.953). The apoptotic rate of platelets in LY294002 group was enhanced significantly (P<0.05). While in LY294002+APS group, the apoptotic rate of platelets was significantly reduced (P<0.05) as compare with LY294002 group, which suggest that APS has an anti-apoptotic effect on the cryopreserved platelets. APS decreased the expression of Caspase-3 and inhibited the reduction of mitochondrial membrane potential induced by LY294002. Moreover, APS increased the activation of PI3K /AKT pathway in Platelets . We further analyzed the in vitro effect of APS on CFU-MK formation. APS (50 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK formation (p=0.06, n=4). APS also significantly enhanced PDGF, bFGF and VEGF-induced CFU-MK formation (n=4). Moreover, the anti-apoptotic effect of APS in M-07e cells was also demonstrated by Annexin-V, Caspase-3, and JC-1 assays. Adding LY294002 alone increased the percentage of cells undergoing apoptosis. However, additional of APS to LY294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the p-AKT.Conclusion: APS, like TPO, has an anti-apoptotic effect on the cryopreserved platelets and megakaryocytes through activating PI3K/AKT, decreasing the expression of Caspase-3 and inhibiting the reduction of MMP. DisclosuresNo relevant conflicts of interest to declare.

Effect of Thrombospondin-1 on Apoptosis of Human Megakaryocytic Leukemia Cells

Background: Thrombospondin 1 (TSP-1) is an extracellular matrix protein that interacts with a wide array of ligands including cell receptors, growth factors, cytokines, and proteases to regulate various physiological and pathological processes. TSP-1 induces apoptosis of endothelial and cancer cells via its receptor CD36. This study was to investigate the effect of TSP-1 on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism.Methods: The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations, to investigate the effect of TSP-1 on the growth of megakaryocytes. The early apoptosis and the activity of the apoptotic protease caspase-3 were detected by flow cytometry. Bone marrow cells of mice were cultured for CFU-MK and stained with acetylcholine to detect the differentiation of megakaryocytes.Results: CD36 antigen was detected on the surface of Meg-01 cells by flow cytometry and immunocytochemistry. TSP-1 (5 μg/mL) inhibited the proliferation of Meg-01 cells, but not M-07e cells (CD36 -). After the addition of CD36 antibody FA6-152 (5, 10 and 25 μg/mL), the inhibitory effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5 and 7.5 μg/mL) exerted a pro-apoptotic effect by increasing the expression of Annexin V (P<0.01) and caspase-3 activation (P<0.01). Addition of FA6-152 (25 μg/mL) can significantly reduce the apoptosis induced by TSP-1 in Meg-01 cells. In addition, TSP-1 (5, 10 and 25 μg/mL) repress the formation of CFU-MK in mouse bone marrow cells, while β-TG did not. This repression was relieved efficiently by FA6-152 (25 μg/mL).Conclusion: TSP-1 could inhibit the proliferation of Megakaryocyte cell line Meg-01 cells, and induce it's apoptosis. TSP-1 may induce apoptosis of Meg-01 cells via CD36 or caspase-3, which provides a potential new drug choice for clinical treatment of megakaryocytic leukemia. DisclosuresNo relevant conflicts of interest to declare.

Induction of apoptosis in retinoid-refractory acute myelogenous leukemia by a novel AHPN analog

AbstractAcute myelogenous leukemia (AML) is a heterogeneous disease consisting of a variety of different leukemic subtypes. While acute promyelocytic leukemia displays marked sensitivity to the differentiating effects of trans-retinoic acid (tRA), other subtypes of AML display resistance. We now describe a novel compound (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC/MM002) that induces apoptosis in the tRA-resistant leukemia cell lines M07e, KG-1, and HL-60R, and in tRA-resistant patient leukemic blasts. The 3-Cl-AHPC totally inhibits leukemia colony formation at concentrations that inhibit committed human bone marrow stem cell proliferation, that is, granulocyte/macrophage colony-forming units (CFU-GMs) by only 30%. Exposure to 3-Cl-AHPC results in caspase activation and the cleavage of poly(adenosine diphosphate) (poly(ADP)) ribose polymerase. While activation of the extracellular signal-regulated kinase (ERK) and p38 pathways is not necessary for 3-Cl-AHPC-mediated apoptosis, maximal apoptosis requires c-Jun N-terminal kinase (JNK) activation. The 3-Cl-AHPC-mediated cleavage of the antiapoptotic B-cell leukemia XL (Bcl-XL) protein to a proapoptotic 18-kDa product is found in both the M07e cell line and patient leukemic blasts. The 3-Cl-AHPC treatment of mice bearing the AML 1498 cell line results in a 3.3-log kill in the leukemic blasts. While 3-Cl-AHPC does not activate retinoic nuclear receptors, it is a potent inducer of apoptosis in AML cells and may represent a novel therapy in the treatment of this disease. (Blood. 2003; 102:3743-3752)

AML Associated Mesenchymal Stroma Cells Support Growth of AML Cells As a Result of Activated Notch Signaling and This Can be Targeted By Dexamethasone

Introduction: Acute myeloid leukemia (AML) is a hematological malignancy characterized by poor prognosis. Interaction with bone marrow mesenchymal stromal cells (BM-MSC) supports leukemic cells to evade chemotherapy-induced apoptosis. Of particular interest, massive parallel transcriptome sequencing of MSCs from mice and patients with preleukemic syndromes identified inflammatory signaling in inducing genotoxic stress, leukemogenesis, and clinical outcome. Recently, Dexamethasone (Dex), an anti-inflammatory drug, demonstrated antileukemic activity and improved clinical outcome in AML patients. These observations intrigued us to further investigate leukemia-BM niche-associated signaling and to identify signaling cascades supporting leukemic cells and BM-MSC interaction. Methods: BM-MSC from AML patients at diagnosis, or in remission were cultured alone or together with human AML cell lines (Kasumi-1 and M-07e) in direct contact or separated by transwells. Following transplantation experiments, BM-MSC were analyzed by microarray analysis. Gene set enrichment analysis (GSEA) was used to identify the hallmark gene sets overrepresented in AML MSC. The functional characterization of BM-MSC and AML cell interaction was investigated using in vitro (cell proliferation and apoptosis) and in vivo (leukemia development, drug response, and survival) analysis. Results: To functionally evaluate the role of BM-MSC, we co-cultured MSCs from AML (AML-MSC) or non-leukemic (non-AML-MSC) patients with Kasumi-1 or M-07e cells. AML-MSCs significantly supported proliferation of AML cells by altering cell cycle distribution from G0/G1 to S phase (30.5 vs 51.3%, p&amp;lt;0.01). Strikingly, the cell cycle profile was unchanged when the cells were separated by a transwell. Consistently, in vivo,MLL-AF9 leukemic cells (Lin-cells transduced with an MLL-AF9 oncogene) and AML-MSCs succumbed to leukemia only when cotransplanted (40 vs 143 days, p&amp;lt;0.05). To gain further insights into leukemic cell-induced changes in MSC, we performed gene expression profiling in MSCs sorted from secondary transplanted leukemic (AML-MSC, n=3) and healthy (HD-MSC, n=3) mice. GSEA analysis identified hallmark-Notch signaling to be significantly overrepresented in AML-MSC as compared to controls (p&amp;lt;0.005, NES: 1.7). Increased Notch-1 and -2 expression could be verified at both the mRNA and protein levels in AML-MSCs. To functionally determine the contribution of Notch activation in MSC, we ectopically expressed the Notch intracellular domain (Notch-ICN) in a murine stromal cell line, MS-5 (Notch-ICN). Underscoring a role for Notch signaling in AML-MSC interaction, Notch-ICN expression significantly supported the proliferation of both MLL-AF9 leukemic cells and MSCs. A role for Notch signaling in the modulation of the inflammatory response has been previously reported. Dex an anti-inflammatory corticosteroid enhanced clinical outcome in AML patients. We, therefore, investigated whether Notch-dependent AML-MSC interaction could be therapeutically targeted using Dex. Interestingly, Dex treatment affected Notch1 expression (52%; p&amp;lt;0.005) and proliferation of leukemia cells (63%; p&amp;lt;0.001). Finally, we evaluated the therapeutic efficacy of Dex by treating mice transplanted with MLL-AF9 leukemic cells. Interestingly, mice treated with Dex exhibited significantly prolonged survival (73 vs 21 days, p&amp;lt;0.01) and had a lower percentage of blasts (50%, p&amp;lt;0.01) and MSC compared to leukemic mice receiving vehicle treatment. Our data thus identified Dex mediated abrogation of AML-MSC interaction is in part mediated by affecting Notch signaling. Conclusion: Our study demonstrated distinct functional differences between AML and non-AML-MSC and direct AML-MSC interaction as an indispensable factor in influencing cell proliferation, leukemogenesis, and survival in mice. We further identified and confirmed the role of Notch signaling in AML-MSC interaction. Therapeutically, Dex abrogated AML-MSC interaction, decreasing AML cell viability, and leukemic mice survival. Further studies are warranted to better understand how aberrant Notch signaling in MSC contributes to AML development and whether the use of Dex alone or in combination with Notch inhibitors could circumvent chemoresistance and relapse in AML patients. Disclosures Lenz: Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy; Agios: Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria, Research Funding; AQUINOX: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem: Research Funding; BMS: Consultancy. Khandanpour:Astra Zeneca: Research Funding; Sanofi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy.

Human granulocyte-colony stimulating factor (G-CSF)/stem cell factor (SCF) fusion proteins: design, characterization and activity.

BackgroundStem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are well-characterized vital hematopoietic growth factors that regulate hematopoiesis. G-CSF and SCF synergistically exhibit a stimulatory effect on hematopoietic progenitors. The combination of G-CSF and SCF has been used for mobilization of peripheral blood progenitor cells in cancer and non-cancerous conditions. To overcome challenges connected with the administration of two cytokines, we developed two fusion proteins composed of human SCF and human G-CSF interspaced by an alpha-helix-forming peptide linker.MethodsThe recombinant proteins SCF-Lα-GCSF and GCSF-Lα-SCF were purified in three steps using an ion-exchange and mixed-mode chromatography. The purity and quantity of the proteins after each stage of purification was assessed using RP-HPLC, SDS-PAGE, and the Bradford assays. Purified proteins were identified using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the Western blot analyses. The molecular weight was determined by size exclusion HPLC (SE-HPLC). The activity of heterodimers was assessed using cell proliferation assays in vitro. The capacity of recombinant fusion proteins to stimulate the increase of the absolute neutrophil count in rats was determined in vivo. The binding kinetics of the proteins to immobilized G-CSF and SCF receptors was measured using total internal reflection ellipsometry and evaluated by a standard Langmuir kinetics model.ResultsThe novel SCF-Lα-GCSF and GCSF-Lα-SCF proteins were synthesized in Escherichia coli. The purity of the heterodimers reached >90% as determined by RP-HPLC. The identity of the proteins was confirmed using the Western blot and HPLC/ESI-MS assays. An array of multimeric forms, non-covalently associated dimers or trimers were detected in the protein preparations by SE-HPLC. Each protein induced a dose-dependent proliferative response on the cell lines. At equimolar concentration, the heterodimers retain 70–140% of the SCF monomer activity (p ≤ 0.01) in promoting the M-07e cells proliferation. The G-CSF moiety in GCSF-Lα-SCF retained 15% (p ≤ 0.0001) and in SCF-Lα-GCSF retained 34% (p ≤ 0.01) of the monomeric G-CSF activity in stimulating the growth of G-NFS-60 cells. The obtained results were in good agreement with the binding data of each moiety in the fusion proteins to their respective receptors. The increase in the absolute neutrophil count in rats caused by the SCF-Lα-GCSF protein corresponded to the increase induced by a mixture of SCF and G-CSF.

Tannic acid attenuated irradiation-induced apoptosis in megakaryocytes

Ionizing radiation (IR) triggers the generation of reactive oxygen species (ROS), which shows potential roles in damaging the DNA and proteins at the nucleus, and eventually results in apoptosis and even cell death. Antioxidant agents can inhibit the generation of ROS after IR exposure. Tannic acid (TA), has an antioxidant activity involving in preventing cardiovascular and cerebrovascular diseases. However, little is known about the effects of TA on irradiation-induced apoptosis in megakaryocytes. Here, we evaluated the anti-radiation activity of TA in megakaryocytes. Our results showed that TA protected megakaryocytes from apoptosis induced by IR, attenuated IR-induced increases in the production of ROS, and inhibited the changes of mitochondrial membrane potential (MMP). Moreover, TA down-regulated NAPDH oxidase 1 (Nox1) expression, and decreased the phosphorylated levels of JNK and p38. Furthermore, JNK inhibitor could reduce apoptosis induced by X-irradiation in M07e cells. In vivo experiments confirmed that TA could promote the platelet recovery, reduce the percentage of apoptosis CD41+ megakaryocytes in bone marrow and raise survival during 30 days in mice by total body irradiation. In conclusion, TA can protecte the megakaryocytes from apoptosis caused by IR through inhibiting Nox1 expression to reduce ROS generation and repressing JNK/p38 MAPK pathway activation.

Notoginsenoside R1 Has Thrombopoietic Effect in Irradiated Mice

Objective: Chinese herbs have been reported to be effective and safe as a treatment for thrombocytopenia. Sanqi, Radix Notoginseng, is the dried roots of Panax notoginseng (Burk.) F. H. Chen (Araliaceae). It has been prescribed in several Chinese formulas including "Yunnan Bai Yao", which is used for treatment of trauma and bleeding due to internal and external injury. Its main constituents are ginsenosides (a kind of saponins), as well as notoginsenosides (only rich in Notoginseng species). Although Sanqi is a well-known haemostatic drug, its effects and mechanisms on megakaryocyte/platelet production have not been well studied. The objective of this study was to compare the effect of a purified notoginsenoside R1 (NR1) and thrombopoietin (TPO) on thrombopoiesis in irradiated mice.Methods: NR1 (2.5 mg) and TPO (0.25 ug) were dissolved in distilled water and given by intra-peritoneal injection daily for 14 days starting from the day after radiotherapy. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from NR1, TPO, and vehicle control groups on day 0, 7 and 14. On day 14, the mice were sacrificed and bone marrow cells were harvested for CFU-MK, CFU-GM, BFU-E and CFU-F (fibroblastoid) assays.Results: Our results showed that NR1 enhanced the recovery of platelets, WBC, and RBC count. Moreover, NR1 also promoted the CFU-F (12 + 0.8 vs 20 + 0.4 colonies/2 x 106 cells, p=0.003), CFU-MK (22 + 1.8 vs 26 + 3.6 colonies/2 x 105 cells, p=0.025), CFU-GM (26 + 5.0 vs 38 + 4.2 colonies/2 x 105 cells, p=0.002), and BFU-E (12 + 2.6 vs 18 + 1.8 colonies/2 x 105 cells, p=0.003) formation. Similar results were obtained in TPO-treated group. In in-vitro study, we further analyzed the effect of NR1 (0-50mM) on mouse CFU-MK formation using a plasma clot colony assay. The results showed that NR1 (20 mM) enhanced TPO (50 ng/ml)-induced CFU-MK formation (18 + 2.2 vs 30 + 6.0 colonies/2 x 105 cells, p=0.022, n=6). Furthermore, the effect of NR1 (5-50 mM) on the growth of bone marrow stromal cells was also investigated using CFU-F assay. NR1 (50mM) had a promoting effect on CFU-F growth (18 + 3.6 vs 24 + 1.8 colonies/2 x 106 cells, p=0.04, n=6). Our studies showed that NR1 enhances thrombopoiesis in vivo and the growth of bone marrow stromal cells as well as megakaryocytes in vitro. Therefore, we speculate that the thrombopoietic activity of NR1 may be mediated via promoting the progenitor of platelet, megakaryocytes, and bone marrow stromal cells. Consequently, we tested the anti-apoptotic effect of NR1 using megakaryocytic cell line M-07e. Data demonstrated that nutrient deprivation significantly increased cell death (n = 6, control vs. normal), and NR1 significantly reduced apoptosis and total cell death (n = 6, NR1 vs. control). TPO treatment also significantly reduced M-07e cell death (n = 6, TPO vs. control). Data also demonstrated that nutrient deprivation increased caspase-3 expression (n = 6, normal vs. control), and NR1 (NR1 vs. control) or TPO (TPO vs. control) treatment significantly reduced caspase-3 expression. M-07e cells were stained with JC-1 reagent. Control cultures had increased proportion of cells containing JC-1 monomers, the percentage of R2 populations are significant higher in the normal, NR1 and TPO treated groups comparing to those of the control group (n = 6). R1, a transitional cell subset containing both monomers and aggregates, was not significantly affected by these reagents. R2: a cell subset showing mitochondria membrane damage.Conclusions: Here we reported that the effect of NR1 is comparable with TPO on the production of platelets in irradiated mice. Therefore, the effect of NR1 on thrombopoiesis may be mediated via anti-apoptosis on megakaryocytic cells. DisclosuresYang:National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

Blockage of miR-92a-3p with locked nucleic acid induces apoptosis and prevents cell proliferation in human acute megakaryoblastic leukemia.

MicroRNAs (miRNAs) are non-coding RNAs involved in post-transcriptional regulation of gene expression. In many cancers, up- or downregulation of different miRNAs is reported. In acute myeloid leukemia, upregulation of miR-92a-3p was reported in human in vitro studies. We performed blockage of miR-29a-3p in human acute megakaryoblastic leukemia cell line (M-07e) by using locked nucleic acid (LNA) and cell proliferation; apoptosis and necrosis were assessed. At different time points after LNA-anti-miR92a-3p transfection, miR-92a-3p quantitation was assessed by qRT-real-time PCR, MTT assay and annexin/propidium iodide staining were performed. The data were processed using the ANOVA test. At all three time points, the expression of miR-92a-3p was lower in the LNA-anti-miR group compared with the control groups. Cell viability between LNA-Anti-miR and the control group was statistically significant. Blockage of miR-92a-3p was associated with increment of the ratio of apoptotic cells in the LNA-anti-miR group was higher than the other group. The ratio of necrotic cells in the LNA-antimiR group was higher than the other groups. These assessments indicate that miR-92a-3p blockage can decrease the viability of M-07e cells, which is mainly due to induction of apoptosis and necrosis. Our findings could open up a path to a miRNA based therapeutic approach for treatment of acute megakaryoblastic leukemia.

CAMP regulates expression of the cyclic nucleotide transporter MRP4 (ABCC4) through the EPAC pathway.

Multidrug resistance protein 4 (MRP4/ABCC4) has been established as an independent regulator of cyclic AMP (cAMP) levels particularly in vascular smooth muscle cells and in hematopoietic cells. Here, we assessed whether cAMP in turn regulates MRP4. A significant upregulation of MRP4 mRNA and protein by long-term treatment with cAMP-enhancing agents was observed in HeLa cells, smooth muscle cells, and megakaryoblastic leukemia M07e cells. This upregulation was not affected by inhibition of protein kinase A, but could be reverted by inhibitors and siRNA of an alternative cAMP-signaling route involving exchange proteins activated by cyclic AMP (EPAC) and mitogen-activated protein kinases. A selective EPAC activator could equally induce MRP4. The transcriptional regulation was confirmed in a luciferase reporter gene assay using a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene. Our results suggest that enhanced cAMP levels upregulate MRP4 expression, which can result in increased cAMP efflux.

Astragalus Polysaccharide has hematopoietic and thrombopoietic activities in an irradiation mouse model

A herbal decoction (Danggui Buxue Tong, DBT) is a traditional Chinese medicine formula comprising Danggui (Radix Angelicae) and Huang Qi (Radix Astragali), which has been used for promotion of “blood production” for centuries and have shown favorable effects on thrombocytopenia. Previously, we have showed that it has significant effects on promoting hematopoiesis and thrombopoiesis (Yang M et al. J Ethnopharmacol, 2009). Many chemical components have been identified in DBT. Among them is the polysaccharides fraction of Angelica sinensis and Radix Astragali. Our study has demonstrated that Angelica Polysaccharide (APS) promotes hematopoiesis and platelet recovery in a mouse model (Liu et al, BMC Complement Altern Med, 2010). In this present study, we further assess the effect of Astragalus Polysaccharide (ASPS) on hematopoiesis and thrombopoiesis in-vivo and in vitro. A myelosuppression mouse model (4-Gy-irradiated) was treated with ASPS (10 mg/kg/day) and TPO (1 mg/kg/day). Peripheral blood cells from ASPS, TPO and vehicle-treated samples were counted on days 0, 7, 14 and 21. Using the colony-forming unit (CFU) assays, we determine the effects of ASPS on the hematopoietic stem/progenitor cells and megakaryocytic lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were also conducted in HL-60 cells and megakaryocytic cell line M-07e. The effects of ASPS on cells treated with Ly294002, a PI3K inhibitor and the effect of ASPS on the phosphorylation of AKT were further studied. Results showed that ASPS, like TPO, significantly enhanced the recovery of WBC and platelet count, and bone marrow CFU-GM and CFU-MK formation (n=8) in this model. Morphological examination of bone marrows also showed that ASPS treatment significantly increased the recovery of hematopoietic stem/progenitor cells and megakaryocytic series. We further analyzed the in vitro effect of ASPS on CFU-MK and CFU-GM formation. ASPS (50-100 ug/ml) enhanced TPO (50 ng/ml) -induced CFU-MK (p=0.05, n=4), and CFU-GM formation (p=0.04, n=4). However, ASPS alone (1-500 ug/ml) did not show significant effect on CFU-MK and CFU-GM proliferation (n=4). Moreover, the anti-apoptotic effect of ASPS in HL-60 and M-07e cells were also demonstrated by using Annexin-V, Caspase-3, and JC-1 assays. Addition of Ly294002 alone increased the percentage of cells undergoing apoptosis. However, additional of ASPS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of ASPS significantly increased the phosphorylation of AKT. Here, we showed that ASPS, the polysaccharide from the root of Radix Astragali, has hematopoietic and thrombopoietic activities in a mouse model. This effect is likely to result from the stimulation of cell proliferation through the activation of PI3K/AKT pathway, the activation of which prevents cells from undergoing apoptosis. Disclosures:No relevant conflicts of interest to declare.

APS From the Root of Angelica Sinesis Promotes Megakaryocytopoiesis and Thrombopoiesis Through PI3k/Akt Pathway

AbstractAbstract 4648We have demonstrated the thrombopoietic effects of Chinese Medicine Dang Gui Bu Xue Tong (DBT), a formula composed of Radix Angelicae Sinensis and Radix Astragali in a thrombocypenic mouse model (Yang M et al, J Ethnopharm 2009). In this study, we further test the effect of polysaccharide (APS) from the root of Radix Angelicae Sinensis (APS) in in-vivo thrombopoiesis and in in-vitro megakaryocytopoiesis. A myelosuppression and thrombocytopenia mouse model was treated with APS (10 mg/kg/day), thrombopoietin (TPO) and saline as control. Peripheral blood cells from APS, TPO and vehicle-treated samples were then counted in different time-points. Using the colony-forming unit (CFU) assays, we determined the effects of APS on the hematopoietic stem/progenitor cells and megakaryocytic lineages. Analyses of Annexin V, Caspase-3, and Mitochondrial Membrane Potential were also conducted in megakaryocytic cell line M-07e. Lastly, the effects of APS on cells treated with Ly294002, a Phosphatidylinositol 3-Kinse inhibitor and the effect of APS on the phosphorylation of AKT were further studied in megakaryocytic cells. Our results showed that APS significantly enhanced the platelets and other blood cells recovery and CFU formation in this model. Morphological examination of bone marrows showed that APS treatment significantly increased the recovery of the megakaryocytic series and other hematopoietic progenitor cells. We observed the antiapoptotic effects of APS on M-07e cells. Addition of Ly294002 alone increases the percentage of cells undergoing apoptosis. In contrast, additional of APS to Ly294002-treated cells reversed the percentage of cells undergoing apoptosis. Furthermore, addition of APS significantly increased the phosphorylation of AKT. Our studies demonstrate that APS promotes platelet recovery in the mouse model and has anti-apoptosis effect in megakaryocytic cells. This effect is likely to be mediated by the PI3K/AKT pathway. Disclosures:No relevant conflicts of interest to declare.

Effect of plasma membrane cholesterol depletion on glucose transport regulation in leukemia cells.

GLUT1 is the predominant glucose transporter in leukemia cells, and the modulation of glucose transport activity by cytokines, oncogenes or metabolic stresses is essential for their survival and proliferation. However, the molecular mechanisms allowing to control GLUT1 trafficking and degradation are still under debate. In this study we investigated whether plasma membrane cholesterol depletion plays a role in glucose transport activity in M07e cells, a human megakaryocytic leukemia line. To this purpose, the effect of cholesterol depletion by methyl-β-cyclodextrin (MBCD) on both GLUT1 activity and trafficking was compared to that of the cytokine Stem Cell Factor (SCF). Results show that, like SCF, MBCD led to an increased glucose transport rate and caused a subcellular redistribution of GLUT1, recruiting intracellular transporter molecules to the plasma membrane. Due to the role of caveolae/lipid rafts in GLUT1 stimulation in response to many stimuli, we have also investigated the GLUT1 distribution along the fractions obtained after non ionic detergent treatment and density gradient centrifugation, which was only slightly changed upon MBCD treatment. The data suggest that MBCD exerts its action via a cholesterol-dependent mechanism that ultimately results in augmented GLUT1 translocation. Moreover, cholesterol depletion triggers GLUT1 translocation without the involvement of c-kit signalling pathway, in fact MBCD effect does not involve Akt and PLCγ phosphorylation. These data, together with the observation that the combined MBCD/SCF cell treatment caused an additive effect on glucose uptake, suggest that the action of SCF and MBCD may proceed through two distinct mechanisms, the former following a signalling pathway, and the latter possibly involving a novel cholesterol dependent mechanism.

The small peptide OGP(10-14) reduces proliferation and induces differentiation of TPO-primed M07-e cells through RhoA/TGFbeta1/SFK pathway

SummaryBackgroundOsteogenic growth peptide (OGP) is a 14-mer peptide found in relevant concentration in blood, and its carboxy-terminal fragment [OGP(10-14)] represents the active portion of the full-length peptide. In addition to stimulating bone formation, OGP(10-14) shows hematological activity. In fact, it highly enhances hematopoiesis-affecting stem progenitors. Moreover, OGP(10-14) reduces the growth and induces the differentiation of the hematological tumour cell line trombophoietin(TPO)-primed M07-e by interfering with RhoA and Src kinase pathways. In the present report, we went deeper into this mechanism and evaluated the possible interference of the OGP(10-14) signal pathway with TGFβ1 and TPO receptor Mpl.Material/MethodsIn OGP(10-14)-treated M07-e cells cultured with or without RhoA and Src kinases inhibitors (C3 and PP2), expression of TGFβ1, Mpl, and Src kinases was analyzed by immunoperoxidase technique. Activated RhoA expression was studied using the G-LISA™ quantitative test.ResultsIn M07-e cells, both OGP(10-14) and PP2 activate RhoA, inhibit Src kinases, reduce Mpl expression and increase TGFβ1 expression. OGP(10-14) and PP2 show the same behavior, causing an additive effect when associated.ConclusionsOGP(10-14) induces TPO-primed M07-e cells differentiation through RhoA/TGFβ1/SFKs signalling pathway. In particular OGP(10-14) acts as a Src inhibitor, showing the same effects of PP2.

High-throughput combinatorial cell co-culture using microfluidics

Co-culture strategies are foundational in cell biology. These systems, which serve as mimics of in vivo tissue niches, are typically poorly defined in terms of cell ratios, local cues and supportive cell-cell interactions. In the stem cell niche, the ability to screen cell-cell interactions and identify local supportive microenvironments has a broad range of applications in transplantation, tissue engineering and wound healing. We present a microfluidic platform for the high-throughput generation of hydrogel microbeads for cell co-culture. Encapsulation of different cell populations in microgels was achieved by introducing in a microfluidic device two streams of distinct cell suspensions, emulsifying the mixed suspension, and gelling the precursor droplets. The cellular composition in the microgels was controlled by varying the volumetric flow rates of the corresponding streams. We demonstrate one of the applications of the microfluidic method by co-encapsulating factor-dependent and responsive blood progenitor cell lines (MBA2 and M07e cells, respectively) at varying ratios, and show that in-bead paracrine secretion can modulate the viability of the factor dependent cells. Furthermore, we show the application of the method as a tool to screen the impact of specific growth factors on a primary human heterogeneous cell population. Co-encapsulation of IL-3 secreting MBA2 cells with umbilical cord blood cells revealed differential sub-population responsiveness to paracrine signals (CD14+ cells were particularly responsive to locally delivered IL-3). This microfluidic co-culture platform should enable high throughput screening of cell co-culture conditions, leading to new strategies to manipulate cell fate.