技术资料

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. View Publication

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease,the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study,a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus,preventing it from infecting cells in an in vitro cell-based assay,including multiple viral variants that are currently circulating in the human population. In addition,we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease. View Publication

Ubiquinone and alpha-lipoic acid are natural constituents which are involved in mitochondrial energy metabolism. Their bioenergetic activities require redox-cycling. In the case of alpha-lipoic acid redox-cycling leads to dihydrolipoic acid which occurs in multienzyme complexes involved in the citric acid cycle while UQ recycles through semi- and divalently reduced ubiquinones in the respiratory chain. We have proved the validity of the concept about the antioxidant function of these natural compounds in their reduced form. Ubiquinol was found to interfere with lipid peroxidation of liposomal membranes being itself degradated by two consecutive oxidation steps. Dihydrolipoic acid was found to totally recycle ubiquinone to the antioxidant active divalently reduced form. In contrast to the antioxidative derived reaction products of ubiquinols which in turn promoted lipid peroxidation,the antioxidant derived reaction product of dihydrolipoic acid was the unreactive two electron oxidation product alpha-lipoic acid. Our experiments demonstrate the existence of an dihydrolipoic acid driven recycling of UQ to the antioxidative-active UQH2. The efficiency of the antioxidative capacity of the latter was found to be diminished through prooxidant activities of the antioxidant-derived metabolites. View Publication

In the present study,the trilineage differentiation capacity of human foreskin-derived precursor cells (hSKP) was evaluated upon exposure to various (non)commercial (i and ii) ectodermal,(iii) mesodermal and (iv) endodermal differentiation media. (i) Upon sequential exposure of the cells to keratinocyte growth (CnT-07® or CnT-057®) and differentiation (CnT-02® or Epilife®) media,keratinocyte-like cells (filaggrin(+)/involucrin(+)) were obtained. The preferred keratinocyte differentiation strategy was exposure to CnT-07®. (ii) When hSKP were subsequently exposed to NeuroCult® media,cells underwent a weak neuro-ectodermal differentiation expressing nestin,myelin binding protein (MBP),vimentin and alpha-foetoprotein (AFP). Sequential exposure to NPMM® and NPDM® generated cells with an inferior neuro-ectodermal phenotype (nestin(+)/vimentin(+)/MBP(-)/AFP(-)). (iii) Upon exposure of hSKP to insulin-transferrin-selenite (ITS) and dexamethasone,small lipid droplets were observed,suggesting their differentiation potential towards adipocyte-like cells. (iv) Finally,after sequential exposure to hepatogenic growth factors and cytokines,an immature hepatic cell population was generated. The presence of pre-albumin suggests that a sequential exposure strategy is here superior to a cocktail approach. In summary,a considerable impact of different (non)commercial media on the lineage-specific differentiation efficiency of hSKP is shown. In addition,we demonstrate here for the first time that,in a suitable keratinocyte stimulating micro-environment,hSKP can generate keratinocyte-like progeny in vitro. View Publication

Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology,including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells-based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well-known mechanisms that are activated by chemically-induced oxidative stress is the Nrf2 signaling pathway. Therefore,in the current study,we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)-derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone-induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM),as shown by Nrf2 nuclear translocation and the up-regulation of the Nrf2-downstream antioxidant enzymes,NQO1 and SRXN1. Interestingly,exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover,rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH(+)) cells was observed. The obtained results suggest that hiPSC-derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. View Publication

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene,predominantly expressed in the liver. Two compounds that knockdown TTR,comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx),are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background,this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown,siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (textgreater80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds. View Publication

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation,overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities,which indicates the presence of other mechanisms for Evi-1 activation. In this study,we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population,in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore,gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells. View Publication

Evi-1 is a zinc-finger transcriptional factor whose inappropriate expression leads to leukemic transformation in mice and humans. Recently,it has been shown that Evi-1 regulates proliferation of hematopoietic stem/progenitor cells at embryonic stage via GATA-2 up-regulation; however,detailed mechanisms underlying Evi-1-mediated early hematopoiesis are not fully understood. We therefore evaluated hematopoietic potential of Evi-1 mutants using a cultivation system of murine para-aortic splanchnopleural (P-Sp) regions,and found that both the first zinc finger domain and the acidic domain were required for Evi-1-mediated hematopoiesis. The hematopoietic potential of Evi-1 mutants was likely to be related to its ability to up-regulate GATA-2 expression. We also showed that the decreased colony forming capacity of Evi-1-deficient P-Sp cells was successfully recovered by inhibition of TGF-b signaling,using ALK5 inhibitor or retroviral transfer of dominant-negative-type Smad3. Our findings suggest that Evi-1 promotes hematopoietic stem/progenitor expansion at the embryonic stage through up-regulation of GATA-2 and repression of TGF-beta signaling. View Publication

Evi1 (ecotropic viral integration site 1) is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly,high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. However,mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here,we show that Evi1 directly represses phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription in the murine bone marrow,which leads to activation of AKT/mammalian target of rapamycin (mTOR) signaling. In a murine bone marrow transplantation model,Evi1 leukemia showed modestly increased sensitivity to an mTOR inhibitor rapamycin. Furthermore,we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN down-regulation,which shows a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and ChIPassays with human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1. View Publication

Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17),which are able to indirectly induce the recruitment of neutrophils. Recently,human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors,including CCR2 and CCR6. Herein,we show that highly purified neutrophils cultured with interferon-gamma plus lipopolysaccharide produce the CCL2 and CCL20 chemokines,the known ligands of CCR2 and CCR6,respectively. Accordingly,supernatants from activated neutrophils induced chemotaxis of Th17 cells,which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment,neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally,we report that,although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor,tumor necrosis factor-alpha,and interferon-gamma release,these latter cells cannot be activated by IL-17A and IL-17F,because of their lack of IL-17RC expression. Collectively,our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells,which may represent a useful target for the treatment of chronic inflammatory diseases. View Publication

The sympathetic (SNS) and parasympathetic (PNS) branches of the autonomic nervous system have been implicated in the modulation of the renewal of many tissues,including the intestinal epithelium. However,it is not known whether these mechanisms are direct,requiring an interaction between autonomic neurotransmitters and receptors on proliferating epithelial cells. To evaluate the existence of a molecular framework for a direct effect of the SNS or PNS on intestinal epithelial renewal,we measured gene expression for the main autonomic neurotransmitter receptors in this tissue. We separately evaluated intestinal epithelial regions comprised of the stem,progenitor,and mature cells,which allowed us to investigate the distinct contributions of each cell population to this proposed autonomic effect. Notably,we found that the stem cells expressed the receptors for the SNS-associated alpha2A adrenoreceptor and the PNS-associated muscarinic acetylcholine receptors (M1 and M3). In a separate experiment,we found that the application of norepinephrine or acetylcholine decreases the expression of cyclin D1,a gene necessary for cell cycle progression,in intestinal epithelial organoids compared with controls (P {\textless} 0.05). Together,these results provide evidence of a direct mechanism for the autonomic nervous system influence on intestinal epithelial stem cell proliferation. View Publication

Eosinophils,a type of granulocyte derived from myeloid precursors in the bone marrow,are distinguished by their cytoplasmic granules. They play crucial roles in immunoregulation,tissue homeostasis,and host defense,while also contributing to the pathogenesis of various inflammatory diseases. Although long non-coding RNAs (lncRNAs) are known to be involved in eosinophilic conditions,their specific expression and functions within eosinophils have not been thoroughly investigated,largely due to the reliance on tissue homogenates. In an effort to address this gap,we analyzed publicly available high-throughput RNA sequencing data to identify lncRNAs associated with eosinophilic conditions. Among the identified lncRNAs,ITGB2 antisense RNA 1 (ITGB2-AS1) was significantly downregulated in blood eosinophils from patients with hypereosinophilia. To further explore its role in eosinophil biology,we generated a stable ITGB2-AS1 knockdown in the HL-60 cell line. Interestingly,ITGB2-AS1 deficiency led to impaired eosinophil differentiation,as evidenced by a reduction in cytoplasmic granules and decreased expression of key eosinophil granule proteins,including eosinophil peroxidase (EPX) and major basic protein-1 (MBP-1). Additionally,ITGB2-AS1-deficient cells exhibited compromised eosinophil effector functions,with reduced degranulation and impaired production of reactive oxygen species (ROS). These findings suggest that ITGB2-AS1 plays a pivotal role in eosinophil differentiation and function,positioning it as a novel regulator in eosinophil biology. View Publication