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Acute myeloid leukaemia (AML) is thought to be maintained by a small population of leukemic progenitor cells. To define the phenotype of such cells with long-term proliferative capacity in vitro and in vivo,we have used the production of leukemic clonogenic cells (CFU) after 2 to 8 weeks in suspension culture as a measure of these cells in vitro and compared their phenotype with that of cells capable of engrafting nonobese diabetic severe combined immune deficient (NOD/SCID) mice. Leukemic blast peripheral blood cells were evaluated for expression of CD34 and Thy-1 (CD90) antigens. The majority of AML blast cells at diagnosis lacked expression of Thy-1. Most primary CFU-blast and the CFU detected at up to 8 weeks from suspension cultures were CD34+/Thy-1-. AML cells that were capable of engrafting NOD/SCID mice were also found to have the CD34+/Thy-1- phenotype. However,significant engraftment was achieved using both CD34+/Thy-1- and CD34- subfractions from one AML M5 patient. These results suggest that while heterogeneity exists between individual patients,the leukemic progenitor cells that are capable of maintaining the disease in vitro and in vivo differ from normal hematopoietic progenitor cells in their lack of expression of Thy-1. View Publication

Simultaneous ex vivo expansion of different progenitor cell types may be beneficial for cord blood (CB) transplantation,to overcome a potential limitation due to restricted cell numbers. Therefore,1.5 x 10(6) CD34+ cells isolated from fresh or thawed CB samples were inoculated in a large-scale stirred suspension bioreactor and cultured in the presence of Flt3-L,SCF and IL-3. At days 0,7,10,14,21 and 28,the spinner cultures were analyzed for viable cells,colony-forming cells (CFC),including erythroid burst-forming unit (BFU-E),granulocyte-macrophage colony-forming unit (CFU-GM) and granulocyte-erythrocyte-megakaryocyte-monocyte colony-forming unit (CFU-GEMM) as well as long-term culture-initiating cells (LTC-IC). Expansion of thawed CD34+ cells resulted in a substantial amplification of total cells (maximal at day 28: 154 +/- 132-fold),CFC (maximal at day 14: 45 +/- 36-fold),CFU-GM (maximal at day 14: 88 +/- 85-fold),CFU-GEMM (maximal at day 7: 4 +/- 2-fold) and of LTC-IC (maximal at day 10: 8 +/- 3-fold). There was no significant difference between fresh and thawed CD34+ cells. These results demonstrate that simultaneously committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified from CD34+-enriched CB samples in large-scale stirred suspension cultures within 7-14 days without exhausting the proliferative potential and,thus,it may be possible to improve CB transplantation by ex vivo generated cells. View Publication

Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable,long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation. View Publication

In order to realize the practical use of human pluripotent stem cell (hPSC)-derived cardiomyocytes for the purpose of clinical use or cardiovascular research,the generation of large numbers of highly purified cardiomyocytes should be achieved. Here,we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC-derived cardiomyocytes using a reporter system. Regulation of the Wnt/β-catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939,a tankyrase inhibitor and IWP2,a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%),it was difficult to suppress differentiation into non-cardiac cells,Therefore,we applied a lentiviral reporter system,wherein green fluorescence protein (GFP) and Zeocin-resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT),a cardiac-specific protein,to exclude non-cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC-derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re-plated after single-cell dissociation. Purified cells specifically expressed GFP,cTnT,displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC-derived cardiomyocytes for application in regenerative medicine and biomedical research. View Publication

The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4,which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [(3)H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [(3)H]ERGO uptake. On the other hand,exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker βIII-tubulin,but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP),with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly,edaravone and ascorbic acid did not affect such differentiation of NPCs,in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP,but decreased the number immunoreactive for βIII-tubulin,with concomitant down-regulation of Math1 in P19-NPCs. Thus,OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress,and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action. View Publication

In culture conditions,human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks,which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons,the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study,we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. View Publication

The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols,immaturity,and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional,and thereby stable,optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons,and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level. View Publication

Neurotrophins and their receptors are frequently expressed in malignant gliomas,yet their functions are largely unknown. Previously,we have shown that p75 neurotrophin receptor is required for glioma invasion and proliferation. However,the role of Trk receptors has not been examined. In this study,we investigated the importance of TrkB and TrkC in survival of brain tumor-initiating cells (BTICs). Here,we show that human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas express the neurotrophin receptors TrkB and TrkC,not TrkA,and they also express neurotrophins NGF,BDNF,and neurotrophin 3 (NT3). Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 enhances tumor-initiating cell viability through activation of ERK and Akt pathways. Conversely,TrkB and TrkC knockdown or pharmacologic inhibition of Trk signaling decreases neurotrophin-dependent ERK activation and BTIC growth. Further,pharmacological inhibition of both ERK and Akt pathways blocked BDNF,and NT3 stimulated BTIC survival. Importantly,attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling,and neurotrophin signaling is sufficient for long term BTIC growth as spheres in the absence of EGF and FGF. Our results highlight a novel role for neurotrophin signaling in brain tumor and suggest that Trks could be a target for combinatorial treatment of malignant glioma. View Publication

BACKGROUND Glioma is the most common form of primary malignant brain tumor in adults,with approximately 4 cases per 100 000 people each year. Gliomas,like many tumors,are thought to primarily metabolize glucose for energy production; however,the reliance upon glycolysis has recently been called into question. In this study,we aimed to identify the metabolic fuel requirements of human glioma cells. METHODS We used database searches and tissue culture resources to evaluate genotype and protein expression,tracked oxygen consumption rates to study metabolic responses to various substrates,performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates,and employed an animal model of malignant glioma to evaluate a new therapeutic intervention. RESULTS We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition,we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover,inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma. CONCLUSIONS Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition,the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice. View Publication

Human embryonic stem cell-derived cardiovascular progenitor cells (hESC-CVPCs) hold great promise for cell-based therapies of heart diseases. However,little is known about their niche microenvironment and in particular the required extracellular matrix (ECM) components. Here we screened combinations of surface-immobilized ECM proteins to identify substrates that support the attachment and survival of hESC-CVPCs. Covalent immobilization of ECM proteins laminin (Lm),fibronectin (Fn),collagen I (CI),collagen III (CIII),and collagen IV (CIV) in multiple combinations and concentrations was achieved by reductive amination on transparent acetaldehyde plasma polymer (AAPP) interlayer coatings. We identified that CI,CIII,CIV,and Fn and their combinations were important for hESC-CVPC attachment and survival,while Lm was dispensable. Moreover,for coatings displaying single ECM proteins,CI and CIII performed better than CIV and Fn,while coatings displaying the combined ECM proteins CIII + CIV and Fn + CIII + CIV at 100 µg/mL were comparable to Matrigel in regard to supporting hESC-CVPC attachment and viability. Our results identify ECM proteins required for hESC-CVPCs and demonstrate that coatings displaying multiple immobilized ECM proteins offer a suitable microenvironment for the attachment and survival of hESC-CVPCs. This knowledge contributes to the development of approaches for maintaining hESC-CVPCs and therefore to advances in cardiovascular regeneration. textcopyright 2017 Wiley Periodicals,Inc. J Biomed Mater Res Part A: 105A: 1094-1104,2017. View Publication

Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system,acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study,we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox,and not on gp91phox/NOX2,as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover,we revealed that NOX5 expression was strongly increased during Mo-DC differentiation,but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC,and at a lower level in plasmacytoid DC. Interestingly,NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation,and thus could be critical for immunity and inflammation. View Publication

Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders such as cystic fibrosis. However,limited understanding of human airway patterning has made this goal challenging. Here,we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach,we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases. View Publication