scholarly journals IFN-γ Inhibitors and Ruxolitinib Therapy for Acquired Severe Aplastic Anemia in an Ex-Vivo Model

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1123-1123
Author(s):  
Edo Schaefer ◽  
Yanling Liao ◽  
Bernadette Fallon ◽  
Janet Ayello ◽  
Melanie Peters ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Severe Aplastic Anemia ◽  
Ex Vivo Model ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Tnf Α ◽  
Proliferation And Differentiation ◽  
Ifn Γ

Abstract Background: Severe aplastic anemia (SAA) is a life-threatening disorder that is associated with multiple etiologies, both inherited and acquired. In acquired SAA, oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Currently, the first line treatment for patients who have a suitable HLA matched donor is a hematopoietic progenitor cell transplant (HPCT). When HPCT is not possible, due to lack of a closely matched HLA donor and/or concomitant co-morbidities, then the treatment of choice is immunosuppression with anti-thymocyte globulin, cyclosporine and eltrombopag (ELT)(Georges et al, Blood, 2018). Alvarado et al (Blood, 2019) recently demonstrated that ELT bypasses the inhibitory effect of IFN-γ by alternatively activating TPO signaling. However, ELT cannot overcome other IFN-γ mediated effect through JAK-STAT1 phosphorylation or apoptosis via Fas/FasL. Alternative therapies are in great need for patients with aSAA as treatment response is sub-optimal. Objective: To determine the effects of IFN-γ neutralizing antibodies or Ruxolitinib on HSPCs survival, proliferation and differentiation in an ex vivo culture of human CD34+ cells in the presence of IFN-γ and TNF-α. Design/Methods: Human CD34+ HSPCs were isolated from cord blood, based on CD34 microbeads magnetic selection (Miltenyl Biotec, Germany). The CD34+ cells were cultured in StemSpan Serum-Free Medium II (STEMCELL Technologies) supplemented with 5 ng/mL human stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and 5 ng/mL recombinant human TPO. A 1x10 5 CD34+ HSPCs were seeded in a 96 well plate. HSPC alone, with and without IFN-γ (100 ng/mL) and TNF-α (10 ng/mL), were the negative and positive control, respectively. Specific IFN-γ neutralizing antibody, B27 (BD Pharmingen), MD1 (BioLegend) and B133.5 (ImmunoTools) or Ruxolitinib (Jakafi, Incyte) were added to the culture and HSPC were harvested and assayed for their survival at day 7 and 14. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO 2. Also, we assessed the multi-lineage differentiation capacity with a selective colony forming units (CFU) assay. Fourteen days after co-culture of each experimental treatment, 500 CD34+ cells were seeded in 6-well plates Smart Dish™ (STEMCELL Technologies). Big burst forming units of erythroid (BFU-E), CFU of granulocyte and megakaryocyte (GM) and granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) were counted and compared between the experimental groups. The signaling pathways were determined using phospho flow-cytometric analysis of pSTAT1, pSTAT3, pSTAT5. All statistically analyzed data is represented as mean ± SD. Differences between groups were analyzed by multiple 2-tailed unpaired Student t tests using Excel. Statistically significant differences were represented as *P <0.05, ** P <0.01 and *** P<0.001. Results: The number of CD34+ cells at day 7 and day 14 of culture in the presence of IFN-γ and TNF-α was significantly lower (21% ± 3 (p<0.0006) and 15% ± 6 (p<0.02), respectively, than that of the control (Fig 1). Importantly, the myelosuppressive effect of IFN-γ and TNF-α was significantly rescued by the addition of Ruxolitinib (at day 14, p<0.05) or IFN-γ neutralizing antibodies (B27 and B133.5, respectively) (day 7 and 14, p<0.01). Improvement in HSPC survival ranged between 1.5-3.3 fold compared to our negative control. Based on the CFU analysis, the CD34+ cells cultured in the presence of B27, B133.5 or Ruxolitinib, were able to produce more CFU at day 7 (p<0.01, p<0.01 and p<0.05 respectively)(Fig 2A). Additionally, Both B27 (p < 0.01) and Ruxolitinb (p ≤ 0.001) were found to produce more CFU-GM on day 14 (Fig 2B). Phospho flow-cytometry demonstrated a significant decrease in STAT1 phosphorylation of CD34+ cells in the presence of B27 and B133.5 (p<0.05, p<0.001, respectively). Conclusions: Our preliminary studies supports the potential benefits of utilizing IFN-γ neutralizing antibodies or Ruxolitinib to improve HSPC survival, proliferation and differentiation in aSAA. Future studies will need to be done to investigate the exact mechanisms of action and the effects of IFN-γ neutralizing antibodies in an animal model of aSAA. Figure 1 Figure 1. Disclosures Cairo: Amgen: Speakers Bureau; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau. OffLabel Disclosure: Ruxolitinib was used to inhibit JAK-STAT signaling pathway in an Ex-Vivo model of Aplastic Anemia. Drug wasn't supplied by drug company.

scholarly journals Rapid Engraftment, Immune Recovery, and Resolution of Transfusion Dependence in Treatment-Refractory Severe Aplastic Anemia Following Transplantation with Ex Vivo Expanded Umbilical Cord Blood (Omidubicel)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 37-38
Author(s):  
Mohamed Samour ◽  
Georg Aue ◽  
Joseph Clara ◽  
Jennifer Wilder ◽  
Robert Reger ◽  
...  
Keyword(s):  
Cord Blood ◽  
Aplastic Anemia ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Severe Aplastic Anemia ◽  
Immune Recovery ◽  
Transplant Outcomes ◽  
Cd34 Cells ◽  
Matched Donor

Introduction Severe Aplastic Anemia (SAA) is a life-threatening bone marrow failure disorder associated with pancytopenia, serious infections, and transfusion dependence. Although long term survival for SAA patients (pts) can be achieved with immunosuppressive therapy (IST), one quarter to one third will fail to respond and about half of responders relapse. Many refractory SAA pts lack an HLA matched donor for salvage allogeneic stem cell transplantation. Although umbilical cord blood transplantation (UCBT) is an alternative approach for SAA pts, the procedure is associated with delayed engraftment and high rejection rates. Further, a substantial portion of UCB units have an insufficient number of TNCs/CD34+ cells for optimal transplant outcomes. To address the need to improve UCBT outcomes for SAA, we instituted a phase II trial exploring the use of nicotinamide (NAM) ex-vivo expanded UCB [omidubicel] to transplant pts with refractory SAA. Methods Eligible pts were 4-55 years and had transfusion dependent SAA. Other criteria included a) failure or intolerance to IST, b) lack of an HLA matched donor, c) having a ≥ 4/8 HLA matched UCB unit with a minimum of 1.8 x 109 and at least 1.8x107/kg TNCs and at least 8 x 106 CD34+ cells, and d) absence of donor specific antibodies to mismatched alleles on the UCB unit. The study was designed to enroll up to 6 pts receiving omidubicel and CD34+ selected haploidentical cells (cohort 1), followed by enrollment of up to 23 pts receiving omidubicel only (cohort 2). The conditioning regimen consists of hATG (40 mg/kg) on D -11 to -8, cyclophosphamide (60 mg/kg) on D -7 and -6, fludarabine (25 mg/m2) on D -5 to -1, and 2 Gy TBI on D -1. Tacrolimus and mycophenolate mofetil were given as GVHD prophylaxis. The primary end point of the study is sustained early engraftment. Secondary endpoints include treatment-related mortality, and standard transplant outcomes. Results A total of 8 pts with SAA refractory to IST and eltrombopag with a median age of 23 years (range 6-45) have been transplanted to date. Pts were heavily transfused (median ferritin 3745 µg/dL), had severe neutropenia with a median pre-transplant ANC of 115 (range 0-680), with 5 (63%) pts having HLA alloantibodies and 1 pt having an invasive fungal infection treated with multiple antifungals and granulocyte transfusions in the peri-transplant period. In cohort 1, the first 3 pts received omidubicel and ~3 x106 CD34+ cells/kg from a haploidentical donor. Since all 3 pts in cohort 1 had sustained engraftment with omidubicel, the study moved to cohort 2, where so far 5 pts have received omidubicel alone. UCB units were matched at a median of 5/8 HLA alleles (range 4-6). Before expansion, units contained a median 1.6 x105 CD34+ cells/kg. At time of transplantation, the expanded units contained a median 75.3 x105 CD34+ cells/kg, representing a median 42-fold expansion. With a median follow-up of 10 months (range 1-35), 7/8 (88%) pts have had early and sustained cord engraftment. One pt in cohort 2 failed to engraft and was salvaged with a haploidentical transplant. Another pt in cohort 2 died on D+ 62 from disseminated adenovirus infection. This pt also developed grade 2 acute GVHD and his IST was stopped early in an effort to boost immunity against adenovirus. The other 7 pts are alive without evidence for acute or chronic GVHD and are transfusion independent. CMV reactivation requiring treatment occurred in 3/6 (50%) of pts at risk. Neutrophil and platelet recovery have been remarkably quick, occurring at a median of 10 days (range 6-14) and 31 days (15-40) respectively. Among the 7 pts with sustained engraftment, six pts had ≥ 95% cord myeloid chimerism by D+ 14 and ≥ 95% T-cell chimerism by D+ 26. Immune recovery has also been remarkably brisk: at D+ 100, the median absolute CD4 count was 186/µL (IQR, 118-340) and mean (±SD) IgG level was 522(±161) mg/dL, respectively (Figure). Conclusions These results provide the first evidence that omidubicel can result in rapid engraftment and sustained hematopoiesis in heavily transfused and allo-immunized SAA pts who are at high risk for graft failure with conventional UCBT. Data showing rapid recovery of IgG levels and rapid reconstitution of CD4, CD8, and NK cell subsets suggests immune recovery may occur quicker with omidubicel compared to conventional UBCT. Taken altogether, our preliminary data suggest omidubicel represents a promising new alternative graft source for SAA pts who lack HLA matched donors. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Engraftment and Retroviral Marking of CD34+ and CD34+CD38− Human Hematopoietic Progenitors Assessed in Immune-Deficient Mice

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1243-1255 ◽  
Author(s):  
Mo A. Dao ◽  
Ami J. Shah ◽  
Gay M. Crooks ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Low Levels ◽  
Ex Vivo Culture ◽  
Daunting Challenge

Abstract Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

Involvement of TNF-α and IFN-γ in Inflammation-Mediated Cochlear Injury

2019 ◽  
Vol 128 (6_suppl) ◽  
pp. 8S-15S ◽  
Author(s):  
Sung K. Moon ◽  
Jeong-Im Woo ◽  
David J. Lim
Keyword(s):  
Cell Injury ◽  
Ex Vivo ◽  
Organ Of Corti ◽  
Blue Staining ◽  
Sensory Cells ◽  
Spiral Ligament ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Ifn Γ ◽  
High Concentrations

Objectives: Inflammation is crucial for the pathogenesis of acquired sensorineural hearing loss, but the precise mechanism involved remains elusive. Among a number of inflammatory mediators, tumor necrosis factor-alpha (TNF-α) plays a pivotal role in cisplatin ototoxicity. However, TNF-α alone is cytotoxic to cochlear sensory cells only at the extremely high concentrations, suggesting the involvement of other factors that may sensitize cells to TNF-α cytotoxicity. Since interferon gamma (IFN-γ) importantly contributes to the cochlear inflammatory processes, we aim to determine whether and how IFN-γ affects TNF-α cytotoxicity to cochlear sensory cells. Methods: TNF-α expression was determined with western blotting in RSL cells and immunolabeling of mouse temporal bone sections. HEI-OC1 cell viability was determined with MTT assays, cytotoxicity assays, and cytometric analysis with methylene blue staining. Cochlear sensory cell injury was determined in the organotypic culture of the mouse organ of Corti. Results: Spiral ligament fibrocytes were shown to upregulate TNF-α in response to pro-inflammatory stimulants. We demonstrated IFN-γ increases the susceptibility of HEI-OC1 cells to TNF-α cytotoxicity via JAK1/2-STAT1 signaling. TNFR1-mediated Caspase-1 activation was found to mediate the sensitization effect of IFN-γ on TNF-α cytotoxicity. The combination of IFN-γ and TNF-α appeared to augment cisplatin cytotoxicity to cochlear sensory cells ex vivo. Conclusions: Taken together, these findings suggest the involvement of IFN-γ in the sensitization of cochlear cells to TNF-α cytotoxicity, which would enable us to better understand the complex mechanisms underlying inflammation-mediated cochlear injury.

Soluble Interleukin-6 (IL-6) Receptor With IL-6 Stimulates Megakaryopoiesis From Human CD34+ Cells Through Glycoprotein (gp)130 Signaling

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2525-2532 ◽  
Author(s):  
Xingwei Sui ◽  
Kohichiro Tsuji ◽  
Yasuhiro Ebihara ◽  
Ryuhei Tanaka ◽  
Kenji Muraoka ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Ex Vivo ◽  
Serum Free ◽  
Human Cd34 ◽  
Receptor Component ◽  
Cd34 Cells ◽  
Interleukin 6 Receptor ◽  
Clonal Cultures

Abstract We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34+ cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34+ cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34+ cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34+ cells were subfractionated into two populations of IL-6R–negative (CD34+ IL-6R−) and IL-6R–positive (CD34+ IL-6R+) cells by fluorescence-activated cell sorting. The CD34+IL-6R− cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34+ IL-6R+cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.

scholarly journals Rupintrivir reduces RV-induced TH-2 cytokine IL-4 in precision-cut lung slices (PCLS) of HDM-sensitized mice ex vivo

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Olga Danov ◽  
Lisa Lasswitz ◽  
Helena Obernolte ◽  
Christina Hesse ◽  
Armin Braun ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Antiviral Drug ◽  
Cytokine Response ◽  
Experimental Conditions ◽  
Tnf Α ◽  
Dust Mite ◽  
Ifn Γ ◽  
Inflammatory Immune Response ◽  
Precision Cut Lung Slices

Abstract Background Antiviral drugs such as rupintrivir may have an immune-modulatory effect in experimentally induced allergic asthma with subsequent RV infection. We infected lung slices of house-dust mite (HDM)-sensitized asthmatic mice ex vivo with human rhinovirus (RV) and investigated the effect of the antiviral drug rupintrivir on RV-induced cytokine response in lung tissue of HDM-sensitized mice ex vivo. Methods Mice were sensitized with HDM. Precision-cut lung slices (PCLS) were prepared from HDM-sensitized or non-sensitized mice. Lung slices were infected ex vivo with RV or RV together with rupintrivir. Modulation of immune responses was evaluated by cytokine secretion 48 h post infection. Results In vivo HDM sensitization resulted in a TH-2/TH-17-dominated cytokine response that persisted in PCLS ex vivo. RV infection of PCLS from non-sensitized mice resulted in the induction of an antiviral and pro-inflammatory immune response, as indicated by the secretion of IFN-α, IFN-β, IFN-γ, TNF-α, MCP-1, IP-10, IL-10, and IL-17A. In contrast, PCLS from HDM-sensitized mice showed an attenuated antiviral response, but exaggerated IL-4, IL-6, and IL-10 secretion upon infection. Rupintrivir inhibited exaggerated pro-inflammatory cytokine IL-6 and TH-2 cytokine IL-4 in HDM-sensitized mice. Conclusions In summary, this study demonstrates that treatment with rupintrivir influences virus-induced IL-4 and IL-6 cytokine release under experimental conditions ex vivo.

Homing of in vitro expanded Stro-1- or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3313-3319 ◽  
Author(s):  
Morad Bensidhoum ◽  
Alain Chapel ◽  
Sabine Francois ◽  
Christelle Demarquay ◽  
Christelle Mazurier ◽  
...  
Keyword(s):  
Scid Mouse ◽  
Dna Analysis ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Quantitative Polymerase Chain Reaction ◽  
Severe Combined Immunodeficiency ◽  
Egfp Gene ◽  
Human Cd34 ◽  
Mouse Tissues ◽  
Cd34 Cells

Abstract The Stro-1 antigen potentially defines a mesenchymal stem cell (MSC) progenitor subset. We here report on the role of human ex vivo-expanded selected Stro-1+ or Stro-1- MSC subsets on the engraftment of human CD34+ cord blood cells in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. The data show that cotransplantation of expanded Stro-1- cells with CD34+ cells resulted in a significant increase of human CD45, CD34, CD19, and CD11b cells detected in blood or in bone marrow (BM) and spleen as compared with the infusion of CD34+ cells alone. Infusion into mice of expanded Stro-1+ and Stro-1- cells (without CD34+ cells) showed that the numbers of Stro-1+-derived (as assessed by DNA analysis of human β-globin with quantitative polymerase chain reaction [PCR]) were higher than Stro-1--derived cells in spleen, muscles, BM, and kidneys, while more Stro-1--derived than Stro-1+-derived cells were found in lungs. The transduction of expanded Stro-1+ cells with an enhanced green fluorescent protein (eGFP) gene did not modify their cytokine release and their homing in NOD/SCID mouse tissues. The difference between the hematopoietic support and the homing capabilities of expanded Stro-1+ and Stro-1- cells may be of importance for clinical therapeutic applications: Stro-1+ cells may rather be used for gene delivery in tissues while Stro-1- cells may rather be used to support hematopoietic engraftment. (Blood. 2004;103:3313-3319)

Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4099-4099
Author(s):  
Zhenhua Qiao ◽  
Xiujuan Zhao
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Hematopoietic Cell ◽  
Control Group ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

In Megakaryocytic Differentiation, the Aryl Hydrocarbon Receptor (AhR) Transcription Factor Regulates Hes1 Expression and Megakaryocytic Polyploidization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2519-2519
Author(s):  
Stephan Lindsey ◽  
Eleftherios Papoutsakis
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Notch Signaling ◽  
Cell Cycle Regulation ◽  
Ex Vivo ◽  
Megakaryoblastic Leukemia ◽  
Aryl Hydrocarbon ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cycle Regulation

Abstract Abstract 2519 Poster Board II-496 Understanding the mechanisms underlying megakaryocytic (Mk) differentiation and maturation is vital to the discovery of novel approaches to treating Mk and platelet disorders such as thrombocytopenia, megakaryoblastic leukemia, and thrombocythemia. The number of platelets released is proportional to the amount of DNA present in a given Mk, so insights into the molecular basis of Mk polyploidization could inspire improved ex vivo culturing methods to promote Mk commitment, expansion, and differentiation, leading to improved autologous transfusion protocols to offset thrombocytopenia associated with HSC transplants following high-dose chemotherapy or MDS progression. Microarray analyses on ex vivo Mk-differentiated primary human CD34+ cells showed that mRNA levels of the Aryl Hydrocarbon Receptor (AhR) increased during Mk differentiation and was elevated 4–6 fold in Mks compared to isogenic granulocytic cultures. These data were further confirmed by quantitative(Q)-RT-PCR analysis of differentiating Mks derived from primary human CD34+ cells as well as from CHRF cells (human megakaryoblastic leukemia). We have shown that CHRF cells are a valid model of human Mk differentiation (Fuhrken PG et al. Exp Hematol, 2007; 35:476–489). Thus, we hypothesized that AhR may act as a novel Mk transcription factor, possibly by influencing or regulating Mk polyploidization. Known as a “toxin sensor”, AhR is involved in the mechanism of action of environmental toxins, likely by altering cell cycle regulation. Epidemiological studies of toxic waste spills and Vietnam veterans suggest that exposure to known AhR ligands may result in increased platelet counts proportional to dioxin exposure level (Webb K et al. Am J Ind Med, 1987;11:685–691, Michalek JE Arch Environ Health, 2001; 56:396–405). These studies offer the intriguing possibility that AhR activation modulates megakaryocyte differentiation and/or platelet production. Interestingly, AhR influences the differentiation of other myeloid lineages including monocytes (Hayashi S et al. Carcinogenesis, 1995; 16:1403–1409) and is upregulated after leukocyte activation (Crawford RB et al. Mol Pharmacol, 1997; 52:921–927). Western blot analyses determined that although initially expressed in both the cytoplasm and nucleus, AhR became solely nuclear in differentiating CHRF cells. EMSA analysis using CHRF nuclear extracts demonstrated that AhR binding to a consensus binding sequence increased as megakaryopoiesis progressed (n=3). Increased AhR-DNA binding during CHRF Mk differentiation correlated with 4.6-fold increased mRNA expression of the AhR transcriptional target Hes1 (n=3, p<0.005), a known cell-cycle regulator and mediator of notch signaling. In order to examine the functional role of AhR in megakaryopoiesis, we generated 3 independent AhR knockdown (KD) CHRF cell lines. Depending on the day of culture, AhR-KD CHRF cell lines differentiated into Mk cells expressed 2-3 fold less AhR mRNA (n=3; p<0.02), 40–60% less AhR protein (n=3), 2.7 times less Hes1 mRNA (n=3; p=0.018), displayed Mk-ploidy distributions shifted towards lower ploidy classes, and were incapable of reaching higher ploidy classes (i.e., ≥32n) seen in control cells. Ploidy levels on day 7 (maximal ploidy in control cells) were 3-fold lower in AhR-KD CHRF cells (n=3; p=0.012 or p=0.005 depending on KD cell line). AhR KD resulted in increased DNA synthesis of low ploidy (<8n; n=3; p<0.05) without influencing apoptosis (n=3, p=0.37). These data suggest that AhR may regulate the cell cycle differently in Mks compared to other cell types, where loss of AhR results in cell cycle blockage and increased apoptosis. As such, AhR deregulation provides a mechanistic explanation for chemical-induced thrombocytopenia, including chemotherapy, and suggests that AhR agonists may provide novel therapies for megakaryoblastic leukemia. AhR-mediated expression of Hes1, an established regulator of the Notch signaling pathway, provides a novel molecular model of endomitotic entry and Mk polyploidization; in drosophila, Notch cell-cycle regulation controls the initial switch toward endomitosis. Disclosures: No relevant conflicts of interest to declare.

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