Bone Marrow Multipotent Progenitor Research Articles

Recent advances of eosinophils and its correlated diseases.

Eosinophils are differentiated by bone marrow multipotent progenitor cells and are further released into peripheral blood after maturation. Human eosinophils can exhibit unique multi-leaf nuclear morphology, which are filled with cytoplasmic granules that contain cytotoxicity and immune regulatory proteins. In recent years, many studies focused on the origin, differentiation and development process of eosinophils. It has been discovered that the eosinophils have the regulatory functions of innate and adaptive immunity, and can also function in several diseases, including asthma, chronic obstructive pulmonary diseases, acute respiratory distress syndrome, malignant tumors and so on. Hence, the role and effects of eosinophils in various diseases are emphasized. In this review, we comprehensively summarized the development and differentiation process of eosinophils, the research progress of their related cytokines, diseases and current clinical treatment options, and discussed the potential drug target, aiming to provide a theoretical and practical basis for the clinical prevention and treatment of eosinophil-related diseases, especially respiratory diseases. To conclude, the guiding significance of future disease treatment is proposed based on the recent updated understandings into the cell functions of eosinophils.

ALLO-819, an Allogeneic Flt3 CAR T Therapy Possessing an Off-Switch for the Treatment of Acute Myeloid Leukemia

▪Patients with relapsed acute myeloid leukemia (AML) have poor prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented clinical efficacy in hematological malignancies, leading to durable responses in heavily pretreated patients. Adoptive immunotherapies using T cells redirected against AML cells are being pursued as one option with potential curative intent. However, the development of autologous CAR T therapies presents a significant logistical and clinical challenge in a rapidly progressing disease setting such as AML due to the lag time of cell manufacturing. Additionally, harvesting sufficient numbers of healthy T cells from patients with AML may not always be possible. For these reasons the development of an off-the-shelf CAR T cell product may be of benefit. This work details the preclinical evaluation of ALLO-819, an allogeneic CAR T therapy targeting the receptor tyrosine kinase Flt3 (CD135), an AML target with high prevalence in all AML subtypes and limited expression outside of the hematopoietic tissue.To construct a Flt3 CAR, a panel of high affinity (KD values of 0.19 to 233 nM, determined at 37°C) fully-human antibodies was generated using phage display technology. Single-chain variable fragments (scFvs) recognizing different immunoglobulin domains of the extracellular region of Flt3 were inserted into second-generation CAR constructs and tested for their ability to redirect T cell specificity and effector function towards AML cells. A lead CAR exhibiting minimal tonic signaling and potent antitumor activity in orthotopic mouse models of AML (2.5x106 and 1x107 CAR T cells for Eol-1 and Molm-13, respectively) was selected for further engineering to incorporate a safety off-switch in cis. To accomplish this, short amino acid stretches mimicking epitopes for the FDA-approved antibody rituximab were inserted between the hinge and target-binding regions of the CAR. The CAR T cell phenotype and antitumor efficacy were not affected by the presence of the off-switch. In the presence of rituximab, Flt3 CAR T cells were efficiently lysed via complement-dependent cytotoxicity (~ 80 % CAR T cell depletion in 3 hours) in vitro and eliminated in peripheral blood and bone marrow of NSG mice (>100-fold and >300-fold, respectively).Allogeneic ALLO-819 Flt3 CAR T cells with a lower risk of TCR-mediated graft-versus-host disease and resistant to anti-CD52 antibody (alemtuzumab)-mediated lysis were generated by disruption of the T-cell receptor alpha chain (TRAC) and the CD52 loci using TALEN® gene-editing technology. Transient expression of TALEN® in Flt3 CAR T cells resulted in high-efficiency inactivation of both loci and had no impact on T cell phenotype or antitumor efficacy. ALLO-819 Flt3 CAR T cells co-cultured with primary AML blasts ex vivo displayed target-dependent activation, cytokine secretion and cytotoxic activity.Consistent with previous reports, we detected Flt3 expression on a subset of normal hematopoietic stem and progenitor cells (HSPCs) which also showed susceptibility to CAR T cell cytotoxicity. To evaluate off-tumor effects of Flt3 CAR T cells in vivo, NSG mice were administered T cells expressing a CAR with similar affinity to both mouse and human Flt3. Mouse-cross-reactive Flt3 CAR T cells exhibited off-tumor activity that was limited to a subset of bone marrow multipotent progenitors and correlated with antitumor efficacy. Administration of rituximab led to effective depletion of CAR T cells in peripheral blood that was followed by a rapid repopulation of HSPCs to levels observed in naïve mice.In summary, these results support the development of ALLO-819 Flt3 CAR T as a novel immunotherapy for the treatment of AML. DisclosuresSommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Nguyen:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Djuretic:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Epigenetic silencing of miR-125b is required for normal B-cell development

AbstractDeregulation of several microRNAs (miRs) can influence critical developmental checkpoints during hematopoiesis as well as cell functions, eventually leading to the development of autoimmune disease or cancer. We found that miR-125b is expressed in bone marrow multipotent progenitors and myeloid cells but shut down in the B-cell lineage, and the gene encoding miR-125b lacked transcriptional activation markers in B cells. To understand the biological importance of the physiological silencing of miR-125b expression in B cells, we drove its expression in the B-cell lineage and found that dysregulated miR-125b expression impaired egress of immature B cells from the bone marrow to peripheral blood. Such impairment appeared to be mediated primarily by inhibited expression of the sphingosine-1-phosphate receptor 1 (S1PR1). Enforced expression of S1PR1 or clustered regularly interspaced short palindromic repeats/Cas9–mediated genome editing of the miR-125b targeting site in the S1PR1 3′ untranslated region rescued the miR-125b–mediated defect in B-cell egress. In addition to impaired B-cell egress, miR-125b dysregulation initially reduced pre–B-cell output but later induced pre–B-cell lymphoma/leukemia in mice. Genetic deletion of IRF4 was found in miR-125b–induced B-cell cancer, but its role in oncogenic miR-125b–induced B-cell transformation is still unknown. Here, we further demonstrated an interaction of the effects of miR-125b and IRF4 in cancer induction by showing that miR125b-induced B-cell leukemia was greatly accelerated in IRF4 homozygous mutant mice. Thus, we conclude that physiological silencing of miR-125b is required for normal B-cell development and also acts as a mechanism of cancer suppression.

Abstract 4719: Targeting interdependent signaling pathways to increase the durability and magnitude of response: promising combination therapy with dual mTORC1/2 inhibitors and CDK4/6 inhibitors

Abstract Two emerging mechanisms of endocrine resistance in estrogen receptor positive (ER+) breast cancers include the activation of the phosphatidylinositol 3-kinase (PI3K) / mammalian target of rapamycin (mTOR) pathway and the de-coupling of cell cycle control from ER signaling via de-regulation of the cyclin D/cyclin dependent kinase (CDK4/6) pathway. In this study, we hypothesized that combining inhibitors of both pathways using the dual mTORC1 and mTORC2 inhibitor AZD2014 and the CDK4/6 inhibitor palbociclib would elicit an improved tumor response over agents that inhibit either pathway alone. Moreover, we hypothesized that combined inhibition of CDK4/6 and TORC1/2 together with inhibition of ER signaling, would cause a profound anti-tumor effect in breast cancer models. In breast cancer cell lines, the combination of AZD2014 and palbociclib caused a synergistic inhibitory effect on cell growth. These effects occurred under conditions where addition of 300nM AZD2014 resulted in significant blockade on both TORC1 and TORC2 downstream effectors (>80% inhibition of p-AKT, p-S6 and p-4EBP1) as well as significant down-regulation of cyclin D1 levels (>70%). Similarly, inhibition of CDK4/6 using palbociclib (300nM) caused >80% inhibition of p-RB and subsequent cell cycle blockade.The effects observed in breast cancer cell lines were recapitulated in vivo using the MCF7 xenograft model, where tumor regressions (>105%) were observed with the combination. Furthermore, combining AZD2014, palbociclib and fulvestrant in this model also caused tumor regressions.To assess bone marrow tolerability of this combination, we investigated the response of human bone marrow multipotent progenitors (CD34+) in vitro. Palbociclib caused transient cell cycle G1 arrest. The combination of palbociclib with AZD2014 delayed entry into cell cycle, but did not have significant impact on the cell viability (<15%). We hypothesized that inhibition of CDK4/6 in combination with AZD2014 would also significantly affect pathway outputs and the transcriptional events downstream of the transcription factors E2F (e.g. CENPE, CCNA2, CDC6 and E2F1) and ER (e.g. PR). We therefore measured specific gene signatures downstream of these receptors in breast cancer cell lines. The ability to dose AZD2014 intermittently compared with rapalogues, together with its ability to block signaling from both TORC1 and 2, make this compound an ideal candidate for combining with CDK4/6 inhibitors such as palbociclib. Furthermore, addition of anti-hormonal therapies such as fulvestrant to this combination may provide additional benefit to breast cancer patients. Citation Format: Claire Crafter, Jon Curwen, Oona Delpuech, Lenka Oplustilova, Stephen Green, Henry Brown, Cath Trigwell, Sabina Cosulich. Targeting interdependent signaling pathways to increase the durability and magnitude of response: promising combination therapy with dual mTORC1/2 inhibitors and CDK4/6 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4719. doi:10.1158/1538-7445.AM2015-4719

Resveratrol increases the bone marrow hematopoietic stem and progenitor cell capacity.

Resveratrol is a plant-derived polyphenol that has shown protective effects against many disorders including, several types of cancers and other age-associated diseases as well as blood disorders in cultured cells and/or animal models. However, whether resveratrol has any impact specifically on normal blood stem cells remains unknown. Here, we show that a 3-week treatment of resveratrol increases the frequency and total numbers of normal bone marrow hematopoietic stem cells (HSC) without any impact on their competitive repopulation capacity. In addition, we show that resveratrol enhances the bone marrow multipotent progenitor capacity in vivo. These results have therapeutic value for disorders of hematopoietic stem and progenitor cells (HSPC) as well as for bone marrow transplantation settings.

Platelet apoptosis and agonist-mediated activation in myelodysplastic syndromes

Patients with myelodysplastic syndromes (MDS) have a defect in the differentiation of bone marrow multipotent progenitor cells. Thrombocytopenia in MDS patients may be due to premature megakaryocyte death, but platelet apoptotic mechanisms may also occur. This study aimed to study function and apoptotic state of platelets from MDS patients with different platelet count. Reticulated platelets, platelet activation, activated caspases and annexin-V binding were evaluated by flow cytometry. Pro-apoptotic Bax and Bak proteins were determined by western blots and plasma thrombopoietin by ELISA. Microparticle-associated procoagulant activity and thrombin generation capacity of plasma were determined by an activity kit and calibrated automated thrombography, respectively. High plasma thrombopoietin levels and low immature circulating platelet count showed a pattern of hypoplastic thrombocytopenia in MDS patients. Platelets from MDS patients showed reduced activation capacity and more apoptosis signs than controls. Patients with the lowest platelet count showed less platelet activation and the highest extent of platelet apoptosis. On this basis, patients with thrombocytopenia should suffer more haemorrhagic episodes than is actually observed. Consequently, we tested whether there were some compensatory mechanisms to counteract their expected bleeding tendency. Microparticle-associated procoagulant activity was enhanced in MDS patients with thrombocytopenia, whereas their plasma thrombin generation capacity was similar to control group. This research shows a hypoplastic thrombocytopenia that platelets from MDS patients possess an impaired ability to be stimulated and more apoptosis markers than those from healthy controls, indicating that MDS is a stem cell disorder, and then, both number and function of progeny cells, might be affected.

Differential regulation of V(D)J recombination in multipotent progenitors in bone marrow versus thymus (111.8)

Abstract V(D)J recombinase activity is expressed in bone marrow (BM) multipotent progenitors (MPPs) that retain the potential to differentiate into B cells in BM and T cells in thymus. However, it remains unclear whether MPPs in each organ exhibit distinct transcriptional requirements for recombinase activity, or whether thymic MPPs imprint the requirements of upstream BM MPPs from which they originate. Here we use V(D)J recombination reporter mice to examine transcriptional requirements in BM versus thymic multipotent subsets. Within BM, the frequency of recombinase+ cells increases from 5% in MPPs to 15% in common lymphoid progenitors (CLPs) and 20% in pre-pro B cells. Within thymus, 10% of early thymic progenitors (ETPs) are recombinase+. While the E47 transcription factor is expressed in both BM and thymus, E47 deficiency selectively ablates V(D)J recombination in the former. BM MPPs, CLPs and pre-pro B cells from E47 knockouts lack detectable recombinase activity. In the thymus, E47 deficiency reduces ETP numbers but V(D)J recombinase activity is maintained. Loss of the related E protein HEB does not diminish recombinase activity in ETPs while functional inhibition of E47:HEB complexes leads to a 40% reduction in TCRβ rearrangements. Thus, V(D)J recombinase activity is regulated differently in MPPs in BM and thymus. E protein transcription factors are essential for recombinase activity in multipotent BM MPPs and CLPs but are dispensable in multipotent thymic ETPs.

Ox-LDL modifies the behaviour of bone marrow stem cells and impairs their endothelial differentiation via inhibition of Akt phosphorylation

This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL) on the behaviour of bone marrow stem cells and their endothelial differentiation as well as the underlying mechanisms. Adult rat bone marrow multipotent progenitor cells (MAPCs) were incubated with ox-LDL for up to 2 weeks. Ox-LDL treatment resulted in a time- and dose-dependent reduction of MAPC population in culture through a combination of decreased cell proliferation and increased apoptosis. The expression of stem cell marker Oct-4 was significantly suppressed in MAPCs by ox-LDL in a dose- and time-dependant manner. Endothelial differentiation of MAPCs was substantially inhibited by ox-LDL with markedly decreased expression of endothelial markers vWF, Flk-1 and CD31, as well as impaired in vitro vascular structure formation. Ox-LDL-induced apoptosis and inhibition of Oct-4 expression, cell proliferation and endothelial differentiation of MAPCs were associated with significant inhibition of Akt phosphorylation. Akt overexpression in MAPCs transfected with a constitutively active Akt completely reversed the effects of ox-LDL on MAPCs including enhanced apoptosis, decreased cell proliferation, suppressed Oct-4 expression and endothelial differentiation as well as in vitro vascular structure formation. In conclusion, ox-LDL promotes apoptosis and inhibits Oct-4 expression and self-renewal of MAPCs, and impairs their endothelial differentiation via suppression of Akt signalling.

Cardiomyocyte Differentiation of Rat Bone Marrow Multipotent Progenitor Cells Is Associated with Downregulation of Oct-4 Expression

This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.

High glucose attenuates VEGF expression in rat multipotent adult progenitor cells in association with inhibition of JAK2/STAT3 signalling

This study was to investigate the effect of high glucose (HG) on vascular endothelial growth factor (VEGF) expression in bone marrow stem cells and JAK2/STAT-3 signalling. Adult rat bone marrow multipotent progenitor cells (rMAPCs) were cultured to evaluate VEGF expression (both mRNA and protein) with or without exposure to HG for up to 48 hrs using RT-PCR and ELISA. JAK2 and STAT3 phosphorylation in rMAPCs was analysed by Western blotting. With cells in normal media, VEGF mRNA level after 24 hrs of culture was significantly increased by 15 times over baseline (day 0) with detectable level of VEGF protein intracellularly using immunofluorescence staining. Although there was no measurable VEGF in the media after 24 hrs of culture, a significant amount of VEGF was detected in the media after 48 hrs of incubation. VEGF expression was associated with constitutive activation of JAK2 and STAT3 in rMAPCs. However, VEGF mRNA level was significantly reduced without detectable VEGF in the media when rMAPCs exposed to HG for 48 hrs. Tyrosine-phosphorylation of JAK2 and STAT3 and nuclear translocation of phosphorylated STAT3 were significantly decreased in the cells exposed to HG for 48 hrs. When JAK2 and STAT3 phosphorylation was blocked by the selective inhibitor AG490, VEGF mRNA level was significantly decreased in rMAPCs in normal media by 80% with no detectable VEGF in the media. VEGF expression was significantly suppressed in rMAPCs cultured in HG media that was further reduced by AG490. VEGF expression in rMAPCs is impaired by HG possibly through inhibition of JAK2/STAT3 signalling.

Nitric oxide enhances Oct-4 expression in bone marrow stem cells and promotes endothelial differentiation

This study was designed to investigate the role of nitric oxide (NO) in bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells. Oct-4 expression (both mRNA and protein) was significantly increased by up to 68.0% in MAPCs when incubated with NO donors DETA-NONOate or sodium nitroprusside (SNP) in a concentration-dependant manner ( n = 3, P < 0.05). However, the cell proliferation was dramatically decreased by over 3-folds when treated with DETA-NONOate or SNP for 48 h ( n = 3, P < 0.05). When MAPCs were exposed to DETA-NONOate (100 μM) for the first 48 h during differentiation, the expression (both mRNA and protein) of vWF was significantly increased at day 14 in the differentiating cells. The effects of DETA-NONOate or SNP on cell proliferation, Oct-4 expression and endothelial differentiation of MAPCs were not affected by the guanylyl cyclase inhibitor 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one or cGMP analog 8-Br-cGMP. These data indicate that NO may regulate both the pluripotency and differentiation of MAPCs via a cGMP-independent mechanism.

Ox‐LDL Impairs Bone Marrow Stem Cell Differentiation into Endothelial Cells Possibly through Inhibition of Akt Phosphorylation

In this study, experiments were designed to investigate the effects of oxidized low density lipoprotein (ox‐LDL) on bone marrow stem cell differentiation into endothelial cells and underlying mechanisms. Adult rat bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells, and induced to differentiate into endothelial cells with or without ox‐LDL in the presence of VEGF. The differentiation process was closely monitored with cell morphology and expressions of endothelial markers including vWF, Flk‐1, and CD31 with RT‐PCR and Western blot analysis. As expected, the differentiating stem cells started to express vWF, Flk‐1, and CD31 from day 5 of differentiation, and reached their maximum at day 14. Ox‐LDL (at 9, 18, and 36 ug/ml) significantly inhibited the differentiation of MAPCs into endothelial cells in a dose‐dependent manner (p &lt; 0.05) as reflected by markedly decreased mRNA levels and contents of vWF, Flk‐1, and CD31. Interestedly, the decreased expressions of endothelial markers were associated with significantly reduced Akt phosphorylation during the early phase of differentiation in these cells. These results indicate that ox‐LDL inhibits the differentiation of bone marrow stem cells into endothelial cells possibly through preventing Akt phosphorylation. The data from this study provides a novel mechanism for the development of ox‐LDL‐related cardiovascular diseases.

Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization.

We isolated hematopoietic stem cells (HSC) from mice treated with cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). All mobilized multipotent progenitor activity was contained in two populations: Thy-1(lo) Sca-1+ Lin- Mac-1- CD4- c-kit+ long-term reconstituting progenitors and Thy-1(lo) Sca-1+ Lin- Mac-1(lo) CD4- transiently reconstituting progenitors. CY/G-CSF treatment drove both long-term and transient multipotent progenitors into cycle, leading to a more than 12-fold expansion in the number of long-term self-renewing HSC prior to mobilization. After CY and 2 days of G-CSF treatment the number of bone marrow HSC began to decline and the number of blood and splenic HSC increased. HSC continued to proliferate in the bone marrow and spleen through 8 days of G-CSF treatment, but HSC released into the blood tended to be in G0/G1 phase. Mobilized multipotent progenitors isolated from the spleen were less efficient than normal bone marrow multipotent progenitors in engrafting irradiated mice but did not differ in colony forming unit-spleen (CFU-S) activity or single cell in vitro assays of primitive progenitor activity. The data suggest that mobilized HSC isolated from the spleen are less efficient at homing to and engrafting the bone marrow of irradiated recipient mice.

Differential cytotoxicity of deoxyguanosine and 8-aminoguanosine for human leukemic cell lines and normal bone marrow progenitor cells.

The inhibitory effect of deoxyguanosine (GdR) alone or in combination with the purine nucleoside phosphorylase (PNP) inhibitor, 8-aminoguanosine (AG) was tested on human T, B, cALL and myeloid leukaemia cell lines and on normal human bone marrow haemopoietic progenitor cells. GdR was found to be toxic to T-leukaemia cells. AG (100 microM) alone did not have any inhibitory effect, but when used with GdR (2.5 X 10(-5)M) a synergistic effect was seen towards T cells. Incubation with GdR and AG resulted in a marked decrease in cell viability (greater than 90 per cent in three and greater than 75 per cent in four of 5 T leukaemic cell lines tested at 72 h). This drug combination did not inhibit the growth of non-T leukaemic cells and was also non-toxic to normal bone marrow multipotent progenitor cells (CFU-GEMM) in vitro. Adenosine deaminase (ADA) acts consecutively with PNP in purine degradation. The addition of an ADA inhibitor, deoxycoformycin and deoxyadenosine, however, did not enhance the toxicity of GdR and AG for T cell leukaemia. The possibility of using GdR and AG for in vitro removal of residual T leukaemic blasts with the sparing of normal bone marrow cells, prior to autologous bone marrow transplantation should be further explored.