Primary human CD34+ cells isolated from umbilical cord blood (CB) mononuclear cells (MNCs) include hematopoietic stem and progenitor cells. CD34+ cells are isolated from cord blood using positive immunomagnetic separation techniques, and citrate-phosphate-dextrose (CPD) is added to the cord blood as an anticoagulant. High-resolution HLA typing for Class I and Class II alleles is available upon request.
CD34+ stem and progenitor cells can be used together with a number of products including StemSpan™, MyeloCult™, MethoCult™, and MegaCult™ S for the expansion and differentiation of these rare cell types.
Cells were collected using consent forms and protocols approved by either an Institutional Review Board, the Food and Drug Administration, the U.S. Department of Health and Human Services, and/or an equivalent regulatory authority.
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Targeting Self-Renewal Function in Normal Hematopoietic and Leukemic Stem Cells
Humanized Mouse Models for Hematopoietic Stem Cell Research: Principles and Pitfalls
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BACKGROUND Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and also that insulators can improve FV vector safety. However, in a previous analysis of insulator effects on FV vector safety, strong viral promoters were used to elicit genotoxic events. In the present study, we developed and analyzed the efficacy and safety of a high-titer, clinically relevant FV vector driven by the housekeeping promoter elongation factor-1alpha$ and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). METHODS Human CD34+ cord blood cells were exposed to an enhanced green fluorescent protein expressing vector, FV-EGW-A1, at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. RESULTS FV-EGW-A1 resulted in high-marking, multilineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. CONCLUSIONS An FV vector with an elongation factor-1alpha$ promoter and an A1 insulator is a promising vector design for use in the clinic.
Nature medicine 2018 AUG
DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.
D. C. Johnson et al.
Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells, Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b, which in turn activates pro-caspase-1 to mediate cell death3, but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples, but not in cells from many other lineages, and that these inhibitors inhibit human AML progression in mouse models. Overall, this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML.
Journal of leukocyte biology 2017 MAY
A neutralizing anti-G-CSFR antibody blocks G-CSF-induced neutrophilia without inducing neutropenia in nonhuman primates.
Scalzo-Inguanti K et al.
Neutrophils are the most abundant WBCs and have an essential role in the clearance of pathogens. Tight regulation of neutrophil numbers and their recruitment to sites of inflammation is critical in maintaining a balanced immune response. In various inflammatory conditions, such as rheumatoid arthritis, vasculitis, cystic fibrosis, and inflammatory bowel disease, increased serum G-CSF correlates with neutrophilia and enhanced neutrophil infiltration into inflamed tissues. We describe a fully human therapeutic anti-G-CSFR antibody (CSL324) that is safe and well tolerated when administered via i.v. infusion to cynomolgus macaques. CSL324 was effective in controlling G-CSF-mediated neutrophilia when administered either before or after G-CSF. A single ascending-dose study showed CSL324 did not alter steady-state neutrophil numbers, even at doses sufficient to completely prevent G-CSF-mediated neutrophilia. Weekly infusions of CSL324 (%10 mg/kg) for 3 wk completely neutralized G-CSF-mediated pSTAT3 phosphorylation without neutropenia. Moreover, repeat dosing up to 100 mg/kg for 12 wk did not result in neutropenia at any point, including the 12-wk follow-up after the last infusion. In addition, CSL324 had no observable effect on basic neutrophil functions, such as phagocytosis and oxidative burst. These data suggest that targeting G-CSFR may provide a safe and effective means of controlling G-CSF-mediated neutrophilia as observed in various inflammatory diseases.
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 2017 MAR
Mast cells are permissive for rhinovirus replication: potential implications for asthma exacerbations.
Akoto C et al.
BACKGROUND Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations, with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity. OBJECTIVE In view of the emerging role of MCs in innate immunity and increased localization to the asthmatic bronchial epithelium, we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection. METHODS The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-β or IFN-λ. After 24 h, innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay. RESULTS HRV infection of LAD2 MCs induced expression of IFN-β, IFN-λ and IFN-stimulated genes. However, LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralization of the type I IFN receptor had minimal effects on viral shedding, suggesting that endogenous type I IFN signalling offered limited protection against HRV. However, augmentation of these responses by exogenous IFN-β, but not IFN-λ, protected MCs against HRV infection. CONCLUSION AND CLINICAL RELEVANCE MCs are permissive for the replication and release of HRV, which is prevented by exogenous IFN-β treatment. Taken together, these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbations.
Cell death and differentiation 2017 DEC
Necroptotic signaling is primed in Mycobacterium tuberculosis-infected macrophages, but its pathophysiological consequence in disease is restricted.
Stutz MD et al.
Mixed lineage kinase domain-like (MLKL)-dependent necroptosis is thought to be implicated in the death of mycobacteria-infected macrophages, reportedly allowing escape and dissemination of the microorganism. Given the consequent interest in developing inhibitors of necroptosis to treat Mycobacterium tuberculosis (Mtb) infection, we used human pharmacologic and murine genetic models to definitively establish the pathophysiological role of necroptosis in Mtb infection. We observed that Mtb infection of macrophages remodeled the intracellular signaling landscape by upregulating MLKL, TNFR1, and ZBP1, whilst downregulating cIAP1, thereby establishing a strong pro-necroptotic milieu. However, blocking necroptosis either by deleting Mlkl or inhibiting RIPK1 had no effect on the survival of infected human or murine macrophages. Consistent with this, MLKL-deficiency or treatment of humanized mice with the RIPK1 inhibitor Nec-1s did not impact on disease outcomes in vivo, with mice displaying lung histopathology and bacterial burdens indistinguishable from controls. Therefore, although the necroptotic pathway is primed by Mtb infection, macrophage necroptosis is ultimately restricted to mitigate disease pathogenesis. We identified cFLIP upregulation that may promote caspase 8-mediated degradation of CYLD, and other necrosome components, as a possible mechanism abrogating Mtb's capacity to coopt necroptotic signaling. Variability in the capacity of these mechanisms to interfere with necroptosis may influence disease severity and could explain the heterogeneity of Mtb infection and disease.
PloS one 2016 DEC
Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.
Shah SN et al.
Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.
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