CD34-positive Hematopoietic Stem Cells Research Articles

In vitro generation of epidermal keratinocytes from human CD34-positive hematopoietic stem cells.

The epidermis is largely composed of keratinocytes (KCs), and the proliferation and differentiation of KCs from the stratum basale to the stratum corneum is the cellular hierarchy present in the epidermis. In this study, we explore the differentiation abilities of human hematopoietic stem cells (HSCs) into KCs. Cultured HSCs positive for CD34, CD45, and CD133 with prominent telomerase activity were induced with keratinocyte differentiation medium (KDM), which is composed of bovine pituitary extract (BPE), epidermal growth factor (EGF), insulin, hydrocortisone, epinephrine, transferrin, calcium chloride (CaCl2), bone morphogenetic protein 4 (BMP4), and retinoic acid (RA). Differentiation was monitored through the expression of cytokeratin markers K5 (keratin 5), K14 (keratin 14), K10 (keratin 10), K1 (keratin 1), transglutaminase 1 (TGM1), involucrin (IVL), and filaggrin (FLG) on day 0 (D0), day 6 (D6), day 11 (D11), day 18 (D18), day 24 (D24), and day 30 (D30) using immunocytochemistry, fluorescence microscopy, flow cytometry, qPCR, and Western blotting. The results revealed the expression of K5 and K14 genes in D6 cells (early keratinocytes), K10 and K1 genes in D11-D18 cells (mature keratinocytes) with active telomerase enzyme, and FLG, IVL, and TGM1 in D18-D24 cells (terminal keratinocytes), and by D30, the KCs were completely enucleated similar to cornified matrix. This method of differentiation of HSCs to KCs explains the cellular order exists in the normal epidermis and opens the possibility of exploring the use of human HSCs in the epidermal differentiation.

Humanization with CD34-positive hematopoietic stem cells in NOG-EXL mice results in improved long-term survival and less severe myeloid cell hyperactivation phenotype relative to NSG-SGM3 mice.

NSG-SGM3 and NOG-EXL mice combine severe immunodeficiency with transgenic expression of human myeloid stimulatory cytokines, resulting in marked expansion of myeloid populations upon humanization with CD34+ hematopoietic stem cells (HSCs). Humanized NSG-SGM3 mice typically develop a lethal macrophage activation syndrome and mast cell hyperplasia that limit their use in long-term studies (e.g., humanization followed by tumor xenotransplantation). It is currently unclear to what extent humanized NOG-EXL mice suffer from the same condition observed in humanized NSG-SGM3 mice. We compared the effects of human CD34+ HSC engraftment in these two strains in an orthotopic patient-derived glioblastoma model. NSG-SGM3 mice humanized in-house were compared to NOG-EXL mice humanized in-house and commercially available humanized NOG-EXL mice. Mice were euthanized at humane or study endpoints, and complete pathological assessments were performed. A semiquantitative multiparametric clinicopathological scoring system was developed to characterize chimeric myeloid cell hyperactivation (MCH) syndrome. NSG-SGM3 mice were euthanized at 16 weeks after humanization because of severe deterioration of clinical conditions. Humanized NOG-EXL mice survived to the study endpoint at 22 weeks after humanization and showed less-severe MCH phenotypes than NSG-SGM3 mice. Major differences included the lack of mast cell expansion and limited tissue/organ involvement in NOG-EXL mice compared to NSG-SGM3 mice. Engraftment of human lymphocytes, assessed by immunohistochemistry, was similar in the two strains. The longer survival and decreased MCH phenotype severity in NOG-EXL mice enabled their use in a tumor xenotransplantation study. The NOG-EXL model is better suited than the NSG-SGM3 model for immuno-oncology studies requiring long-term survival after humanization.

Targeting SF3B1 in High-Risk B-Cell Acute Lymphoblastic Leukemia

Introduction B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer. The outcomes of young adults with B-ALL are poor, with an event-free survival (EFS) rate of <50%. The prognosis of children with certain types of B-ALL is also poor with an EFS rate of 70%. Therefore, there is a need for new treatments for these children with high-risk B-ALL. RNA splicing alternations have been suggested to contribute to the initiation and/or maintenance of cancer. In B-ALL, dysregulated splicing is reported to be associated with drug resistance and disease relapse. SF3B1 is a core component of the spliceosome and an essential protein in the RNA splicing process. SF3B1 inhibition is therapeutic in many cancers, including myeloid malignancies and T-cell ALL. However, there have been no reports studying the efficacy and the function of spliceosome inhibitors including SF3B1 inhibitors in B-ALL. In this study, we determined the therapeutic potential of pladienolide B (Plad-B), an SF3B1 inhibitor, in B-ALL. Methods SF3B1 mRNA and protein expressions were determined in the cell lines Reh and JM1 and 14 patient samples (standard-risk: 7 samples, high-risk: 7 samples), by qRT-PCR and Western blotting, respectively. Efficacy of Plad-B was tested in two B-ALL cell lines, CD34 positive hematopoietic stem cells (HSCs), and three high-risk patient-derived xenograft (PDX) samples (sample#1: age < 1 year old, sample#2: age > 10 years old, sample#3: relapse), by an MTS assay. In vivo efficacy of Plad-B was tested using an Reh xenograft model and a PDX model. Function of Plad-B was assessed in cell lines by a cell cycle assay, Annexin V assay with flow cytometry, and Western blotting of cleaved PARP and cleaved caspase-3. To determine the changes in the splicing events and apoptosis-associated genes, we are performing RNA-seq at 15min, 30min, 60min, and 24 hours after the treatment. Results SF3B1 mRNA and protein expressions were significantly higher in Reh and JM1 than in hematopoietic monocular cells and CD34-positive HSCs (p < 0.05). SF3B1 protein, but not mRNA, expressions were higher in primary patient samples than in hematopoietic monocular cells and CD34-positive HSCs. There was no significant difference in the mRNA and protein expressions between standard-risk and high-risk groups. Plad-B treatments showed significant cytotoxicity in both Reh and JM1 cell lines with IC50 of 1.2nM and 0.6nM, respectively, at 48 hours after treatment. Plad-B did not show cytotoxicity at the same tested concentrations in CD34-positive HSCs. Plad-B treatments showed similarly significant cytotoxicity in three harvested PDXs with IC50 of 0.7nM, 2.5nM, and 1.1nM, respectively, at 48 hours after the treatment. Plad-B, at a dose of 10 mg/kg 2 times on 1 and 2 days, significantly prolonged survival in the Reh xenograft mouse model, with a median survival time of 33.5 days compared with 25.0 days in the control group (p < 0.05) (Figure 1). Plad-B, at a dose of 10 mg/kg 4 times on 1, 3, 5, and 7 days, prolonged survival in the PDX mouse model (sample#1), with a median survival time of 38.0 days compared with 32.0 days in the control group (p = 0.14). Cell cycle arrest was observed at 24 hours after Plad-B treatment in both Reh (Control: 12.6%, Plad-B 3nM: 23.8% in G2/M phase, p < 0.001) and JM1 (Control: 20.6%, Plad-B 3nM: 35.1% in G2/M phase, p < 0.01). Plad-B caused significant cell apoptosis in Reh (Control: 3.2%, Plad-B 3nM: 24.1% with Annexin V positive cells, p < 0.001) and JM1 (Control: 5.9%, Plad-B 3nM: 31.2% with Annexin V positive cells, p < 0.001) cells at 24 hours after treatment. Plad-B also caused significant cell apoptosis in harvested PDX sample #1 (Control: 29.8.%, Plad-B 3nM: 47.6% with Annexin V positive cells, p < 0.001) at 24 hours after treatment. Western blotting of anti-cleaved PARP and anti-cleaved caspase-3 showed that the expression of cleaved caspase-3 and cleaved PARP were significantly augmented in Reh and JM1 cells after Plad-B treatment. RNA seq data is currently being analyzed. Conclusions SF3B1 is highly expressed in B-ALL cell lines and in primary patient samples regardless of the risk group. Plad-B showed significant cytotoxicity in B-ALL leading leukemia cells to G2/M cycle arrest and apoptosis. Plad-B treatment demonstrated in vivo therapeutic efficacy in pre-clinical B-ALL animal models. These data suggest therapeutic potential of SF3B1 inhibition in B-ALL including high-risk patients.

A-075 Development and Validation of Image-based Cell Counter Microscanner C3 for CD34-positive Hematopoietic Stem Cell Enumeration

Abstract Background Accurate measurement of CD34-positive hematopoietic stem cells (HSCs) is critical for successful peripheral blood stem cell transplantation (PBSCT). Flow cytometry is by far the gold standard for measuring CD34-positive stem cells. However, its complicated procedure requires time, cost and highly-trained operators, thus the necessity of simpler and cheaper alternative method is emerging. We have developed a new image-based cell counter, Microscanner C3 (MSC3, Biozentech, South Korea) as an alternative method for enumeration of CD34-positive HSCs and evaluated its performance. Methods 100 µl of apheresis product was stained with CD45-BB515 and CD34-PE antibodies (Becton Dickinson, USA) at room temperature in the dark. Red blood cell lysis with 500 µl of OptiLyze C solution (Beckman Coulter, USA) was followed by centrifugation. After the removal of supernatants, the sample was resuspended with 500 µL PBS, which then infused to filter paper imbedded BZ-1 microchip (Biozentech, South Korea). Thirty-fields of the loaded microchip was auto-scanned by MSC3 and its dedicated software analyzed the images and yielded CD34-positive cell fraction out of CD45-positive cells as percentage. Results of thirty-six samples were compared to that of FacsLyric (Beckton Dickinson, USA) flow cytometry and accuracy and imprecision (pentaplicate measurements for three samples), repeatability (two experimenters performed triplicate measurements for three samples each), linearity (triplicate measurements for five levels), limit of detection (hexaplicate measurements for five levels) and specificity (using whole blood specimen from three donors with monoclonal gammopathy, thrombocytosis or monocytosis) was evaluated. Results CV% values of inaccuracy, imprecision and repeatability did not exceed 15%. The method was found to be linear across the measuring range. LOD was evaluated to be 7 cells/µL and no background interference was detected. The difference between the measured fraction of MSC3 and FacsLyric was within allowable limit, with an R-value greater than 0.98. Conclusion We developed a reliable method for quantifying CD34-positive HSCs based on image analysis. The limitation involved in MSC3 applications in CD34-positive HSCs is that, like dual-platform flow cytometry, the results should be combined with the results of conventional hematology analyzer in order to define absolute HSC numbers. However, with its simpler and faster procedures, MSC3 exhibited a great potential to be used as an alternative to conventional flow cytometry in CD34-positive HSCs enumeration and other applications as well.

Correlation between SDF-1α, CD34 positive hematopoietic stem cells and CXCR4 expression with liver fibrosis in CCl4 rat model

BackgroundOne of the most frequent disorders is liver fibrosis. An improved understanding of the different events during the process of liver fibrosis & its reversibility could be helpful in its staging and in finding potential therapeutic agents.AimThe goal of this research was to evaluate the relationship among CD34 + HPSCs, SDF-1α, and CXCR4 receptor expression with the percentage of the area of hepatic fibrosis.Materials and methodsThirty-six male Sprague-Dawley rats were separated into the control group, liver injury group & spontaneous reversion group. The liver injury was induced by using 2 ml/kg CCl4 twice a week. Flow cytometric examination of CD34 + cells in the blood & liver was performed. Bone marrow & liver samples were taken for evaluation of the SDF-1α mRNA by PCR. Liver specimens were stained for histopathological and CXCR4 immuno-expression evaluation.ResultsIn the liver injury group, the hepatic enzymes, fibrosis area percentage, CXCR4 receptor expression in the liver, CD34 + cells in the blood and bone marrow & the level SDF-1α in the liver and its concentration gradient were statistically significantly elevated with the progression of the liver fibrosis. On the contrary, SDF-1α in the bone marrow was statistically significantly reduced with the development of liver fibrosis. During the spontaneous reversion group, all the studied parameters apart from SDF-1α in the bone marrow were statistically substantially decreased compared with the liver injury group. We found a statistically substantial positive correlation between fibrosis area and all of the following: liver enzymes, CXCR4 receptor expression in the liver, CD34 + cells in the blood and liver, and SDF- 1α in the liver and its concentration gradient. In conclusion, in CCl4 rat model, the fibrosis area is significantly correlated with many parameters in the blood, bone marrow, and liver, which can be used during the process of follow-up during the therapeutic interventions.

Glucose-6-phosphate dehydrogenase is dispensable for human erythroid cell differentiation in vitro

Glucose-6-phosphate dehydrogenase (G6PD) deficiency impairs cellular processes under oxidative stress. Individuals with severe G6PD deficiency still produce sufficient numbers of erythrocytes. Nevertheless, the G6PD independence of erythropoiesis remains questionable. This study elucidates the effects of G6PD deficiency on the generation of human erythrocytes. Peripheral blood-derived CD34-positive hematopoietic stem and progenitor cells (HSPCs) of human subjects with normal, moderate, and severe G6PD activities were cultured in two distinct phases: erythroid commitment and terminal differentiation. Regardless of G6PD deficiency, HSPCs were able to proliferate and differentiate into mature erythrocytes. There was no impairment in erythroid enucleation among the subjects with G6PD deficiency. To our knowledge, this study is the first report of effective erythropoiesis independent of G6PD deficiency. The evidence firmly indicates that the population with the G6PD variant could produce erythrocytes to an extent similar to that in healthy individuals.

Comparison of Efficacy and Safety of Generic Plerixafor Vs Original Plerixafor in the Mobilization of Myeloma Patients

Introduction The commonest indication for an Autologous Stem Cell Transplantation (ASCT) is still Multiple Myeloma. A successful mobilization of hematopoietic stem cells (HSC) is a sine qua non of ASCT. The introduction of Plerixafor, which is a partial agonist of the alfa-chemokine receptor CXCR4 has added an important value and impact on mobilization. Plerixafor is successfully integrated into both growth factor-only and cyclophosphamide and growth factor mobilization strategies with significantly reducing the mobilization failure rate in myeloma patients. In addition, plerixafor + G-CSF has also been shown to successfully mobilize the majority of patients who previously failed to mobilize with either growth factor alone or in combination with chemotherapy. Even a Just-in-Time algorithm which induces plerixafor in patients who lacks a certain threshold of CD34 positive HSCs on the day of mobilization led to a cost-effective and successful mobilization with highly restricted rates of mobilization failure. In this study we tried to demonstrate the efficacy and safety of a novel generic Plerixafor (Pleksor - Gen Ilac) and to compare it with original one (Mozobil - Sanofi) in a retrospective manner. Method Patients who were transplanted in two centers who adopted the same mobilization standard operating procedures (SOP) were included in the study. An age and sex matched cohort of patients who received Mozobil (from 2020-2022 - Group A) were compared with the ones who received Pleksor (2021-2022 Group B) as a Just-in-Time conjunct to GCSF alone or chemo mobilization. Poor mobilization was defined as a final yield of 2 million CD34 positive HSCs per kg. Our aim was to collect enough stem cells for at least two ASCTs, thus our current SOP's indicated a minimum CD34 positive HSC threshold of at least 4 million per kg and an ideal HSC threshold of 6 million per kg. Results A total of 28 patients were included and they were equally distributed among Group A (n=14) and B (n=14). Median age of the patients at the time of mobilization were as follows, 60 (35-72) in Group A and 61 (38-70) in Group B. 14 patients who received Pleksor achieved a median yield of 8.40 million CD34 positive HSCs per kg (4.8-21) and the patients who received Mozobil have ended with a yield of 6.7 million CD34 positive HSCs per kg (4.5-13) (p=0.210). None of the patients in both groups were named to be a poor mobilizer according to the threshold of 2 million CD34 positive HSCs per kg but 3 of the patients in Group A and 2 of the patients in Group B ended with a yield of 6 million CD34 positive cells which was below to the ideal threshold for two transplants. Regarding lenalidomide exposure before mobilization, history of radiotherapy, line of the therapies received before mobilization, number of leukapheresis and the mobilization policy (chemo vs gcsf alone) there were no statistically significant difference between two groups (p=0.120, 0.702, 0.842, 0.769 and 0.420 respectively). The median neutrophil engraftment time in days were as follows for Group A and B, 11(10-14) vs 11 (10-16), p=0.541 and the median platelet engraftment time in days were 17 (10-30) in Group A and 16 (10-28) in Group B with a p value of 0.571. In none of the cases any specific side effects were noted which could be attributable to Pleksor or Mozobil. Conclusion Our study demonstrated a comparable efficacy of a generic form of Plerixafor when compared with the originator. This would lead to a decrease in the cost of total process of mobilization with a similar efficacy and toxicity profile. We are now planning to initiate a prospective trial to validate these results in a larger patient population. Up to our knowledge this is the first study comparing the efficacy of a generic Plerixafor in a sole myeloma patient cohort.

Autologous stem cell transplantation in tandem with Anti-CD30 CAR T-cell infusion in relapsed/refractory CD30+ lymphoma

BackgroundLong-term outcome is unfavourable for relapsed/refractory (r/r) lymphoma patients who are resistant to salvage chemotherapy, even after subsequent autologous stem-cell transplantation (ASCT). Although anti-CD30 chimeric antigen receptor (CAR30) T-cell therapy induces high response rates in these patients, the duration of response is relatively limited.MethodsThis open-label, single-center and single-arm pilot study investigated the safety and efficacy of ASCT in tandem with CAR30 T-cell infusion in r/r CD30+ lymphoma. The primary endpoint was safety and key secondary endpoint was overall response rate, overall survival, progression-free survival, and duration of response.ResultsFive classical Hodgkin lymphoma (cHL) patients and 1 anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) patient were enrolled. The median age was 24 years. No patient had prior ASCT. Three patients (50.0%) relapsed for ≥ 2 times and 3 patients (50.0%) had primary refractory diseases. All had a Deauville score of 4 or 5, and 5 patients (83.3%) had a stable or progressive disease (SD/PD) at enrollment. All patients received myeloablative chemotherapy and infused CD34-positive hematopoietic stem cells (HSCs) and CAR30 T cells in tandem, with a median dose of 3.9 × 106/kg and 7.6 × 106/kg, respectively. Five paitents presented with cytokine release syndrome (CRS), all of which were grade 1. No neurotoxicity was observed. All patients had successful HSCs engraftment and reached an objective response, including 5 (4 cHL and 1 ALCL, 83.3%) with a complete response (CR) and 1 with a partial response (PR). With a median follow-up of 20.4 (range, 12.1–34.4) months, all remained alive and maintained their responses.ConclusionOur work demonstrates the combined administration of ASCT and CAR30 T-cell therapy is well-tolerate and highly effective in r/r cHL and ALCL, even in PET-positive or chemorefractory patients who are expected to have inferior outcome after ASCT, although further large-scaled validation in prospective clinical trial is warranted.Trial registration The trial was registered with the Chinese Clinical Trial Registry (ChiCTR, number ChiCTR2100053662).

Effect of Replacement of Wharton Acellular Jelly With FBS on the Expression of Megakaryocyte Linear Markers in Hematopoietic Stem Cells CD34.

Animal environments for the growth of stem cells cause the transmission of some diseases and immune problems for the recipient. Accordingly, replacing these environments with healthy environments, at least with human resources, is essential. One of the media that can be used as an alternative to animal serums is Wharton acellular jelly (AWJ). Therefore, in this study, we intend to replace FBS with Wharton jelly and investigate its effect on the expression of megakaryocyte-related genes and markers in stem cells. In this study, cord blood-derived CD34 positive HSCs were cultured and expanded in the presence of cytokines including SCF, TPO, and FLT3-L. Then, the culture of expanded CD34 positive HSCs was performed in two groups: 1) IMDM culture medium containing 10% FBS and 100 ng / ml thrombopoietin cytokine 2) IMDM culture medium containing 10% AWJ, 100 ng / ml thrombopoietin cytokine. Finally, CD41 expressing cells were analyzed with the flow cytometry method. The genes related to megakaryocyte lineage including FLI1 and GATA2 were also evaluated using the RT-PCR technique. Results: The expression of CD41, a specific marker of megakaryocyte lineage in culture medium containing Wharton acellular jelly was increased compared to the FBS group. Additionally, the expression of GATA2 and FLI1 genes was significantly increased related to the control group. This study provided evidence of differentiation of CD34 positive hematopoietic stem cells from umbilical cord blood to megakaryocytes in a culture medium containing AWJ.<br />.

Abstract 6070: Functional natural killer cells derived from engineered hiPSC with hypoimmunity gene combo demonstrate hypoimmunity features in evading host attacks

Abstract Engineered human induced pluripotent stem cells (hiPSC)-derived allogeneic Natural Killer (NK) cells are emerging as promising safe and effective off-the-shelf cell therapy drugs. Repeated dosing of NK cells can partly compensate the relatively shorter persistence of NK cells compared with chimeric antigen receptor T cells (CAR-T); However, with each repeat, the persistence and efficacy are increasingly challenged by activation of host innate and adaptive immunity. Thus, reducing immunogenicity of allogeneic NK cells holds promise for better efficacy. In this study, we report a combination of “hypoimmunity” gene edits including multiple transgene knock-ins and both class I and class II MHC knockouts (2KO). We show that hiPSC engineered by the hypoimmunity combo can be efficiently differentiated into NK cells, comparable to the non-engineered ones. This is contrary to the notion from mouse studies that NK cells devoid of class I molecules are hypo-responsive, these engineered NK cells can be expanded by aAPC (artifitial Antigen Presenting Cells, a K562 cell line with surface expression of mbIL-21 and CD137L) and are capable of killing tumor cells, Additionally, we show that the engineered NK cells can evade T cell immunity, as examined by in vitro T cell proliferation and cytotoxicity assays. A humanized mouse model with transplanted CD34-positive human hematopoietic stem cells confirmed that the engineered NK cells can persist longer than WT and 2KO NK cells. Hypoimmune-QN-019 NK cells are generated by incorporating hypoimmunity gene combo edits into our hiPSC-derived NK cell clinical candidate QN-019 which has 3 transgenes including membrane-bound IL-15, high affinity non-cleavable CD16 and CAR targeting CD19. Comparing to QN-019, hypoimmune-QN-019 displayed better efficacy in humanized CDX mouse model in clearing CD19-positive cancer cells. Taken together, we have engineered hiPSC-derived allogeneic NK cells with hypoimmunity features, which hold great promise in becoming an off-the-shelf drug with improved in vivo persistence and efficacy for repeated dosing. Citation Format: Jiabiao Hu, Xiangjun He, Jing Xu, Yixuan Zhou, Yanan Yue, Yangbin Gao, George Church, Luhan Yang. Functional natural killer cells derived from engineered hiPSC with hypoimmunity gene combo demonstrate hypoimmunity features in evading host attacks [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 6070.

Physical interaction between BAALC and DBN1 induces chemoresistance in leukemia

BAALC is identified as a leukemia-associated gene and is highly expressed in CD34-positive hematopoietic stem cells. High BAALC expression is associated with poor prognosis in several types of acute myeloid leukemia. We explored binding partner proteins of BAALC by means of mass spectrometry and analyzed biological properties of BAALC-expressing leukemic cells. We found that BAALC physically interacts with DBN1, which is an actin-binding protein and promotes cell adhesion and mobility by forming cell membrane spines during cell-cell interactions. Drebrin1 downregulation impeded cell adhesion to bone marrow stromal cells, resulting in improvement of sensitivity to cytarabine. Taken together, our findings suggest that BAALC-DBN1 interaction contributes to the anchoring of BAALC-expressing cells in the bone marrow, which in turn leads to resistance to cytotoxic drugs. Therefore, the BAALC-DBN1 interaction provides us with an opportunity to overcome the chemotherapy resistance in BAALC-activated leukemia via functional blockage of these genes.

Profiling of Metabolic Differences between Hematopoietic Stem Cells and Acute/Chronic Myeloid Leukemia.

Although many studies have been conducted on leukemia, only a few have analyzed the metabolomic profiles of various leukemic cells. In this study, the metabolomes of THP-1, U937, KG-1 (acute myelogenous leukemia, AML), K562 (chronic myelogenous leukemia, CML), and cord blood-derived CD34-positive hematopoietic stem cells (HSC) were analyzed using gas chromatography-mass spectrometry, and specific metabolic alterations were found using multivariate statistical analysis. Compared to HSCs, leukemia cell metabolomes were found to have significant alterations, among which three were related to amino acids, three to sugars, and five to fatty acids. Compared to CML, four metabolomes were observed specifically in AML. Given that overall more metabolites are present in leukemia cells than in HSCs, we observed that the activation of glycolysis and oxidative phosphorylation (OXPHOS) metabolism facilitated the incidence of leukemia and the proliferation of leukemic cells. Analysis of metabolome profiles specifically present in HSCs and leukemia cells greatly increases our basic understanding of cellular metabolic characteristics, which is valuable fundamental knowledge for developing novel anticancer drugs targeting leukemia metabolism.

CD133 mRNA-Loaded Dendritic Cell Vaccination Abrogates Glioma Stem Cell Propagation in Humanized Glioblastoma Mouse Model

Cancer stem cells are initiating cells of cancer and propagate its growth through self-renewal and differentiation of its daughter cells. CD133 is a cell surface antigen that is present on glioma stem cells and has been used to prospectively isolate glioma stem cells. We hypothesized that a major histocompatibility complex (MHC)-independent and long-lasting immune response against CD133 could be generated by transfecting CD133 mRNA into dendritic cells and vaccinating animals with experimental gliomas. To test this hypothesis, we developed a novel humanized mouse model using CD34-positive hematopoietic stem cells. We confirmed the robust simultaneous activation of CD8- and CD4-positive T cells by dendritic cell vaccination with modified CD133 mRNA leading to a potent and long-lived immune response, with subsequent abrogation of CD133-positive glioma stem cell propagation and tumor growth. This study for the first time demonstrates in both a humanized mouse model and in a syngeneic mouse model of glioblastoma that targeting a glioma stem cell-associated antigen is an effective strategy to target and kill glioma stem cells. This novel and simple humanized mouse model for immunotherapy is a significant advance in our ability to test human-specific immunotherapies for glioblastoma.

The Mir-99b/Let-7e/Mir-125a cluster Is Associated with Platelet Count and Mean Platelet Volume and Regulates Megakaryocyte Proplatelet Formation

Platelet count (PC) and mean platelet volume (MPV) are critical measurements when considering the role of platelets in hemostasis and thrombosis, abnormalities in PC and MPV have been shown to be predictors of ischemic cardiovascular disease. Ischemic vascular disease has a known genetic component, and we and others have shown high heritability for both PC and MPV. Many inherited, and most immune, thrombocytopenias show an inverse relationship between PC and MPV, and such a relationship has also been reported in healthy subjects. Although genome-wide association studies have identified numerous single nucleotide variants in protein-coding genes associated with PC and MPV (Geiger Nat Genet 2011), non-coding genes have had rather little consideration. The goal of our studies was to identify microRNAs (miRs) regulating PC and MPV and to begin to understand the molecular mechanisms of this inverse platelet number-volume relationship. Initially, we queried our Platelet RNA and Expression-1 data set and observed that PC and MPV are inversely correlated in these 154 healthy subjects (r=-0.45, p=3.98E-09). We next determined which of the 734 platelet-expressed miRs were significantly (p&lt;0.05) correlated with both PC and MPV. Of these miRs, only miR-125a-5p, miR-99b-5p, let-7e-5p and miR-150-5p had an inverse relationship with PC and MPV. The Table shows the correlations between these miRs and complete blood count data. Subsequent studies focused on miR-125a-5p, miR-99b-5p, let-7e-5p because they (1) were associated with only platelet parameters and not WBC or Hb, (2) were more highly expressed than miR-150-5p and (3) showed the same directionality (e.g., positive r value for PC). We were also intrigued by the fact that these 3 miRs exist as a genomic cluster within 727 bp on chr19q13.41. The expression of each of the 3 miRs in this cluster was strongly correlated with each other in platelets (r=0.93, p=1.02E-68 for miR-125a and let-7e; r=0.91, p=8.53E-61 for miR-99b and let-7e; r=0.89, p=1.19E-52 for miR-125a and miR-99b), consistent with a single, polycistronic transcript containing all genes. To begin to address the potential functionality of this miR cluster, we overexpressed and knocked down their expression in megakaryocytes (MKs), since MK proplatelet formation (PPF) is a necessary step in the generation of peripheral blood platelets. We utilized human umbilical cord blood derived CD34-positive hematopoietic stem and progenitor cells differentiated along the MK lineage with thrombopoietin. Quantification of miR levels with qPCR demonstrated all 3 miRs in the cluster progressively increased from culture day 6 to day 9 to day 13 (the time during which MKs mature in culture). Compared to a scrambled control, when miR activity was knocked down with locked nucleic acids (LNAs), we observed a 61% reduction in PPF for miR-125a-5p (p&lt;0.05), a 54% reduction for let-7e-5p (p&lt;0.05) and a ~28% reduction for miR-99b-5p (p=0.33). PPF was quantified blinded as to treatment group. When all 3 miRs in the cluster were inhibited simultaneously by LNAs, we observed a 74% reduction in PPF (p&lt;0.05) (Figure 1). LNA inhibition of two miRs not associated with PC or MPV did not alter MK PFF. To date, we have only tested the effect of overexpression of one miR, but observed that lentiviral overexpression of miR-125a-5p induced an increase in MK PPF (p&lt;0.05). In summary, we have identified a genomic cluster of miRs (miR-99b/let-7e/miR-125a) that regulates PPF in cultured human MKs and whose expression is positively correlated with human PC and negatively correlated with MPV. We speculate that platelet mass homeostasis, which is determined by PC and MPV, may be controlled in part by this novel genomic locus. Disclosures No relevant conflicts of interest to declare.

Differentiation potential of human CD133 and CD34 positive hematopoietic stem cells into motor neuron- like cells; an in vitro study

Differentiation potential of human CD133 and CD34 positive hematopoietic stem cells into motor neuron- like cells; an in vitro study

GPI-80 Defines Primitive Human Cord Blood-Derived CD34-Positive and Negative Hematopoietic Stem Cells

Background. We identified very primitive CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) in human cord blood (CB) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). These CD34- SRCs possess myeloid-biased differentiation potential, which suggests that they are a distinct class of primitive hematopoietic stem cell (HSC) and that they reside at the apex of the human HSC hierarchy (Blood Cancer J 2015:5;e290). We recently developed high-resolution purification methods for CD34- SRCs using 18 Lineage (18Lin)-specific antibodies, which can enrich CD34- SRC at the 1/1,000 level in 18Lin- CD34- fractions (Exp Hematol 2011:39:203). In addition, we previously identified CD133 as a positive marker for CD34- SRCs as well as for CD34+ SRCs (Leukemia 2014:28;1308). The results showed that CD34+/- SRCs were enriched to approximately 1/100 and 1/140 in 18Lin- CD34+/- CD133+ fractions, respectively.Aim. In order to further elucidate the details of the characteristics of human CD34+/- HSCs, we aimed to identify additional positive markers for the high-level purification of CB-derived CD34+/- SRCs.Materials and Methods. First, weextensively analyzed the candidate positive markers, including cell adhesion molecules and homing receptors that are expressed on 18Lin- CD34+ CD38- and 18Lin- CD34- cells by multicolor FACS. Finally, we discovered that glycosylphosphatidylinositol-anchored protein GPI-80, which has recently been reported as a marker for human fetal liver hematopoietic stem/progenitor cells (HSPCs) (Cell Stem Cell 2015:16;80), was also expressed on human full-term CB-derived 18Lin- CD34+ CD38- and 18Lin- CD34- cells. Next, we sorted 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells from human CB. The HSPC characteristics of the 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells were assessed as follows: (1) the in vitro maintenance/production capacities of CD34+ and CD34+ CD38- cells were examined in co-cultures with mesenchymal stroma cells (MSCs) established from human bone marrow-derived CD45- Lin- CD271+ SSEA-4+ cells (Stem Cells 2015:33;1554); (2) an SRC assay was performed using NOD/Shi-scid/IL-2Rγcnull (NOG) mice; (3) limiting dilution analyses (LDAs) were performed to determine the SRC frequencies in these four fractions of cells.Results. In the CB-derived 18Lin- CD34+ CD38- and 18Lin- CD34- fractions, 10.1% and 14.4% of cells expressed GPI-80, respectively. The 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells were then co-cultured with human MSCs for 7 days in the presence of SCF and TPO. As a result, the 18Lin- CD34+ CD38- GPI-80+ cells maintained significantly higher percentages of CD34+ (86.4%) and CD34+ CD38- cells (24.8%) in comparison to 18Lin- CD34+ CD38- GPI-80- cells (78.7% and 14.3%, respectively). However, 18Lin- CD34- GPI-80+/- cells produced comparable levels of CD34+ (50.3% and 50.8%) and CD34+ CD38- cells (4.4% and 5.3%). These four fractions of cells were then transplanted into NOG mice using the IBMI method. All of these four fractions of cells showed long-term repopulating SRC activities with multi-lineage differentiation potential in mouse bone marrow. However, LDAs demonstrated that the frequencies of SRC in the 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- fractions were 1/21, 1/35 and 1/28, 1/874, respectively. These data clearly demonstrate that both CD34+/- SRCs are enriched in GPI-80+ fractions. Surprisingly, a number of mice received a limited number of 18Lin- CD34+ CD38- GPI-80+ (2 cells) and 18Lin- CD34- GPI-80+ cells (10 cells), and thus also showed multi-lineage long-term human hematopoietic cell repopulation.Conclusion. These observations clearly demonstrated that GPI-80 defines CB-derived human primitive HSCs. Furthermore, these results indicate that GPI-80 is a useful marker for the high-level purification of human CB-derived CD34+/- SRCs (HSCs). DisclosuresNo relevant conflicts of interest to declare.

Novel Targeted Therapy for Precursor B Cell Acute Lymphoblastic Leukemia: Anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugates

Precursor B cell (preB) acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite aggressive treatment, current approaches for preB ALL have significant limitations with a cure rate of only 30% in certain subtypes. In addition, long-term survivors are at risk for late effects that include development of secondary malignancies and toxicity to visceral organs. Therefore, there is a desperate need for more effective and less toxic therapy. [Display omitted] Previously, we demonstrated that the transcription factor MXD3 is a critical regulator of preB ALL cell proliferation. Knockdown of MXD3 expression in preB ALL cells resulted in cell death as determined by annexin V and caspase assays (Satake et al., British Journal of Haematology, in press). We hypothesize that targeted delivery of an MXD3 therapeutic to preB ALL cells will increase therapy effectiveness and decrease off target effects and toxicity, thus increasing the quality of life.In the current study, we developed an antisense oligonucleotide (ASO) that specifically targets MXD3. Taking advantage of anti-CD22 antibody (aCD22 Ab), which specifically targets B cell ALL, we developed aCD22 Ab-MXD3 ASO conjugates as a potential combined phenotypic and molecularly targeted therapeutic. In in vitro tests, using the Reh ALL cell line, we confirmed successful delivery of the aCD22 Ab-MXD3 ASO compound to leukemia cells. This correlated with MXD3 knockdown at the protein level, and inhibition of leukemia cell growth. Cytotoxicity was tested on anonymized healthy donor normal blood cells, including CD34 positive hematopoietic stem cells (HSCs), B cells, and non-B cells from mobilized peripheral blood mononuclear cells. As expected, cytotoxicity was observed in normal B cells, but CD34 HSCs and non-B cells were unaffected. In combination with conventional preB ALL chemotherapy drugs (vincristine or doxorubin), we observed additive cytotoxic effects of aCD22 Ab-MXD3 ASO conjugates on cells in vitro.We determined the compound effectiveness in pre-clinical xenograft animal models of preB ALL, using both the Reh cell line as well as a primary leukemia sample. Age matched female NOD/SCID/IL2Rg-/- (NSG) mice were randomly assigned to 4 treatment groups: 1) PBS, 2) MXD3 ASO and aCD22 Ab unconjugated at the equivalent dose of the high dose group, 3) aCD22 Ab-ASO conjugates at 0.2mg of Ab/kg/dose, and 4) at 1mg of Ab/kg/dose. Five million leukemia cells were inoculated either intravenously (iv) (Reh) or intra-bone marrow (primary cells) to each mouse. Twenty-four hours following leukemia inoculation, animals started receiving twice weekly iv treatments for 3 weeks. All mice treated with PBS and unconjugated MXD3 ASO & aCD22 Ab, died of leukemia at approximately day 21 in the Reh model and at day 30 in the primary leukemia model (Figure). Animals treated with aCD22 Ab-MXD3 ASO conjugates, either at low or high dose, had significantly prolonged survival both in the Reh (p<0.0084, n=4/group) and primary leukemia (p=0.0001, n=8/group) xenograft models (Log-rank (Mantel-Cox) test). Leukemia-related death was confirmed by necropsy. Harvested leukemia cells were HLA and CD22 positive. During treatment, the mice in all the treatment groups remained healthy and active, and did not lose weight. Toxicity was assessed in the primary leukemia model with weekly CBC and chemistry panels and revealed no significant toxicity.In conclusion, we have demonstrated the therapeutic efficacy of the aCD22 Ab-MXD3 ASO conjugates in preB ALL. This is the first study to demonstrate effective direct ASO-conjugated monoclonal Ab-mediated delivery for the treatment of leukemia or other malignancies. Future studies will focus on the further characterization of the compound, effectiveness of combination therapy with standard chemotherapeutic drugs and toxicological profile. DisclosuresNo relevant conflicts of interest to declare.

Targeted therapy with MXD3 siRNA, anti-CD22 antibody and nanoparticles for precursor B-cell acute lymphoblastic leukaemia.

Conventional chemotherapy for precursor B-cell (preB) acute lymphoblastic leukaemia (ALL) has limitations that could be overcome by targeted therapy. Previously, we discovered a potential therapeutic molecular target, MDX3 (MAX dimerization protein 3), in preB ALL. In this study, we hypothesize that an effective siRNA therapy for preB ALL can be developed using antiCD22 antibody (αCD22 Ab) and nanoparticles. We composed nanocomplexes with super paramagnetic iron oxide nanoparticles (SPIO NPs), αCD22 Abs and MXD3 siRNA molecules based on physical interactions between the molecules. We demonstrated that the MXD3 siRNA-αCD22 Ab-SPIO NP complexes entered leukaemia cells and knocked down MXD3, leading the cells to undergo apoptosis and resulting in decreased live cell counts in the cell line Reh and in primary preB ALL samples in vitro. Furthermore, the cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes were significantly enhanced by addition of the chemotherapy drugs vincristine or doxorubicin. We also ruled out potential cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes on normal primary haematopoietic cells. Normal B cells were affected while CD34-positive haematopoietic stem cells and non-B cells were not. These data suggest that MXD3 siRNA-αCD22 Ab-SPIO NP complexes have the potential to be a new targeted therapy for preB ALL.

Epigenetic Dysregulation Of ZNF423 Impedes B-Cell Differentiation and Is Linked To Adverse Outcome Of Childhood B-Precursor ALL

▪Despite increasing treatment success, acute lymphoblastic leukemia continues to be the most common cancer-related cause of death in childhood. Structural and numerical chromosomal aberrations are considered to be the primary event in disease development followed by a variety of secondary mutational incidents. However, in a large proportion of B-precursor ALL (B-ALL) cases, the cause of differentiation arrest -a hallmark of acute leukemias- has not been attributed to defined genetic aberrations.To identify differentially regulated genes in B-ALL, comparative intra-individual transcriptome profiling of leukemic versus immunologically matched normal lymphoblasts was employed. In this study, we found the multifunctional transcription modulator Zinc Finger Protein 423 (ZNF423) to be upregulated in the leukemic blasts. Subsequent expression analysis in a cohort of 200 patients corroborated a substantially increased abundance of ZNF423 transcripts compared to normal lymphopoietic cells. Aberrant expression of ZNF423 is maintained in relapsed ALL of initially positive samples. Thus, the transcriptional activity of ZNF423 represents a stable leukemia-associated phenomenon.Our evaluation of the mechanistic basis of aberrant ZNF423 activation in B-ALL revealed a perturbed epigenetic and transcriptional regulation of ZNF423 as a novel mechanism interfering with B-cell differentiation. Cancer cells show global DNA hypomethylation with concomitant abnormal methylation of cytosines preceding guanosine residues in so-called CpG islands (CGI) (Feinberg and Tycko, 2004). We detected three of these CGIs in the upstream region of the ZNF423 gene locus. Bisulfite sequencing of primary ALL samples (n=58) revealed a significantly lower degree of methylation at multiple CpG positions of the analyzed CGI than in mononuclear control cells. Hence, the hypomethylation of ZNF423 regulatory sequences reflects a dysregulated epigenetic state.ZNF423 is known to direct bone morphogenetic protein (BMP)-dependent signaling in a ternary SMAD1-SMAD4 transcription factor complex in Xenopus (Hata et al., 2000). Our data demonstrate that ZNF423 not only acts as a binding partner of this complex but is also regulated along BMP2-induced pathways, as BMP2 treatment causes a marked increase of ZNF423 expression.Moreover, ZNF423 has been implicated as a potent inhibitor of the Early B-cell Factor 1 (EBF-1) in the olfactory system (Tsai & Reed, 1997). In hematopoiesis, EBF-1 controls the expression of essential components of the pre-B-cell receptor (preBCR). We demonstrate that enforced expression of ZNF423 in CD34-positive hematopoietic stem or progenitor cells leads to a significant reduction of transcriptional activation of the EBF-1 target genes CD79a and CD79b, validating the repressive function of ZNF423 on EBF-1 in the hematopoietic context.In addition, the influence of aberrant ZNF423 expression was assessed in vivo in a xenograft hematopoietic stem cell transplantation model. Analyzing the immunological phenotypes of ZNF423-positive versus control cells after engraftment, a lower frequency of cells expressing mature BCR was found within the ZNF423-transduced cell population. Hence aberrant ZNF423 expression leads to a B-cell maturation arrest in vivo.Given the impact of ZNF423 on B-cell maturation, we investigated whether presence of ZNF423 is associated with event-free survival of ALL patients. Statistical analyses performed in B-ALL patients (n=190) uncovered high ZNF423 expression to be associated with poor outcome of EVT6-RUNX1-negative patients, whereas low levels of ZNF423 expression were linked to favorable outcome.We reasoned that -beside the repressive effect of ZNF423 on EBF-1 target gene activation- the heterodimerization of aberrantly expressed ZNF423 with EBF-1 could induce a shift in the target gene spectrum of EBF-1 in leukemic blasts, resulting in a substantial distortion of transcriptional programs in B-ALL patients. Our ongoing studies focus on the identification of the target spectrum of EBF-1 in presence or absence of ZNF423, as well as putative transcriptional targets of the ZNF423-EBF-1 heterodimer and ZNF423 alone, using ChIP-Seq analysis. Disclosures:No relevant conflicts of interest to declare.

Associations among Darbepoetin-α, CD34+ Cells and Cardiovascular Disease Events in Patients on Hemodialysis

Background and Objectives: Erythropoiesis-stimulating agents (ESAs) might moderate circulating CD34-positive hematopoietic stem (CD34⁺) cells. We assessed associations between ESA therapy and CD34⁺ cells and their impact on cardiovascular disease (CVD) events in patients on prevalent hemodialysis (HD). Design, Setting, Participants and Measurements: We analyzed 95 patients on prevalent HD who received the ESAs epoetin-β (n = 22), darbepoetin-α (n = 60), or neither (control; no ESA, n = 13). Baseline values for CD34⁺ cells, high-sensitivity C-reactive protein, interleukin-6, vascular endothelial growth factor, inter-cellular adhesion molecule-1, and carotid intima-media thickness were determined. The numbers of CD34⁺/erythropoietin receptor (EPOR)⁺ cells were determined in 35 and 8 patients in the darbepoetin-α and control groups, respectively. CD34⁺ cells were counted after 6 and 12 months of darbepoetin-α treatment (n = 35). All patients were followed up for a mean of 28 months. Results: Hemoglobin levels were lower, carotid intima-media thickness was more pronounced, and the ESA dose was higher in patients with a low, than with a high, CD34⁺ cell count. The ratio of CD34⁺/EPOR⁺ to CD34⁺ cells positively correlated with the darbepoetin-α dose. A low, but not a high, dose of darbepoetin-α for 6 and 12 months was associated with more CD34⁺ cells. Although high-dose darbepoetin-α therapy was an independent predictor of composite CVD events, this association disappeared when adjusted for the CD34⁺ cell count with other confounders. Conclusions: High-dose ESA therapy is associated with a low CD34⁺ cell count and comprises a risk factor for CVD events in patients on prevalent HD.