Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Studies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs) of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs.</p> <p>Results</p> <p>From hESCs, we derived NESs, the <it>in-vitro </it>version of brain-derived neurospheres. NES formation was confirmed by increased levels of various NSC marker genes and the emergence of rosette structures in which neuroprogenitors are known to reside. We found that Notch signaling, which maintains stem cell characteristics of <it>in-vivo</it>-derived neuroprogenitors, is active in these hESC-derived NESs, similar to their <it>in-vivo </it>counterpart. Expression levels of Notch signaling molecules such as NICD, DLLs, JAG1, HES1 and HES5 were increased in the NESs. Inhibition of the Notch signaling by a γ-secretase inhibitor reduced rosette structures, expression levels of NSC marker genes and proliferation potential in the NESs, and, if combined with withdrawal of growth factors, triggered differentiation toward neurons.</p> <p>Conclusion</p> <p>Our results indicate that the hESC-derived NESs, which share biochemical features with brain-derived neurospheres, maintain stem cell characteristics mainly through Notch signaling, which suggests that the hESC-derived NESs could be an <it>in-vitro </it>model for <it>in-vivo </it>neurogenesis.</p

The power of bioluminescence imaging in understanding host-pathogen interactions

Infectious diseases are one of the leading causes of death worldwide. Modelling and understanding human infection is imperative to developing treatments to reduce the global burden of infectious disease. Bioluminescence imaging is a highly sensitive, non-invasive technique based on the detection of light, produced by luciferase-catalysed reactions. In the study of infectious disease, bioluminescence imaging is a well-established technique; it can be used to detect, localize and quantify specific immune cells, pathogens or immunological processes. This enables longitudinal studies in which the spectrum of the disease process and its response to therapies can be monitored. Light producing transgenic rodents are emerging as key tools in the study of host response to infection. Here, we review the strategies for identifying biological processes in vivo, including the technology of bioluminescence imaging and illustrate how this technique is shedding light on the host-pathogen relationship

Polarity-tunable magnetic tunnel junctions based on ferromagnetism at oxide heterointerfaces

Complex oxide systems have attracted considerable attention because of their fascinating properties, including the magnetic ordering at the conducting interface between two band insulators, such as LaAlO3 (LAO) and SrTiO3 (STO). However, the manipulation of the spin degree of freedom at the LAO/STO heterointerface has remained elusive. Here, we have fabricated hybrid magnetic tunnel junctions consisting of Co and LAO/STO ferromagnets with the insertion of a Ti layer in between, which clearly exhibit magnetic switching and the tunnelling magnetoresistance (TMR) effect below 10 K. The magnitude and the of the TMR are strongly dependent on the direction of the rotational magnetic field parallel to the LAO/STO plane, which is attributed to a strong Rashba-type spin orbit coupling in the LAO/STO heterostructure. Our study provides a further support for the existence of the macroscopic ferromagnetism at LAO/STO heterointerfaces and opens a novel route to realize interfacial spintronics devices.Comment: 25 pages, 5 figure

Functional Recapitulation of Smooth Muscle Cells Via Induced Pluripotent Stem Cells From Human Aortic Smooth Muscle Cells

Rationale: Generation of induced pluripotent stem (iPS) cells has been intensively studied by a variety of reprogramming methods, but the molecular and functional properties of the cells differentiated from iPS cells have not been well characterized. Objective: To address this issue, we generated iPS cells from human aortic vascular smooth muscle cells (HASMCs) using lentiviral transduction of defined transcription factors and differentiated these iPS cells back into smooth muscle cells (SMCs). Methods and Results: Established iPS cells were shown to possess properties equivalent to human embryonic stem cells, in terms of the cell surface markers, global mRNA and microRNA expression patterns, epigenetic status of OCT4, REX1, and NANOG promoters, and in vitro/in vivo pluripotency. The cells were differentiated into SMCs to enable a direct, comparative analysis with HASMCs, from which the iPS cells originated. We observed that iPS cell-derived SMCs were very similar to parental HASMCs in gene expression patterns, epigenetic modifications of pluripotency-related genes, and in vitro functional properties. However, the iPS cells still expressed a significant amount of lentiviral transgenes (OCT4 and LIN28) because of partial gene silencing. Conclusions: Our study reports, for the first time, the generation of iPS cells from HASMCs and their differentiation into SMCs. Moreover, a parallel comparative analysis of human iPS cell-derived SMCs and parental HASMCs revealed that iPS-derived cells possessed representative molecular and in vitro functional characteristics of parental HASMCs, suggesting that iPS cells hold great promise as an autologous cell source for patient-specific cell therapy. (Circ Res. 2010;106:120-128.)Yu JY, 2007, SCIENCE, V318, P1917, DOI 10.1126/science.1151526Hanna J, 2007, SCIENCE, V318, P1920, DOI 10.1126/science.1152092Takahashi K, 2007, CELL, V131, P861, DOI 10.1016/j.cell.2007.11.019Byrne JA, 2007, NATURE, V450, P497, DOI 10.1038/nature06357Lee TH, 2007, PLOS MED, V4, P1101, DOI 10.1371/journal.pmed.0040186Matsumura H, 2007, NAT METHODS, V4, P23, DOI 10.1038/NMETH973Aoi T, 2008, SCIENCE, V321, P699, DOI 10.1126/science.1154884Dimos JT, 2008, SCIENCE, V321, P1218, DOI 10.1126/science.1158799Barroso-delJesus A, 2008, MOL CELL BIOL, V28, P6609, DOI 10.1128/MCB.00398-08Aasen T, 2008, NAT BIOTECHNOL, V26, P1276, DOI 10.1038/nbt.1503Stadtfeld M, 2008, SCIENCE, V322, P945, DOI 10.1126/science.1162494Okita K, 2008, SCIENCE, V322, P949, DOI 10.1126/science.1164270Tateishi K, 2008, J BIOL CHEM, V283, P31601, DOI 10.1074/jbc.M806597200Zhang JH, 2009, CIRC RES, V104, pE30, DOI 10.1161/CIRCRESAHA.108.192237Soldner F, 2009, CELL, V136, P964, DOI 10.1016/j.cell.2009.02.013Chang SA, 2008, STEM CELLS, V26, P1901, DOI 10.1634/stemcells.2007-0708Park IH, 2008, NATURE, V451, P141, DOI 10.1038/nature06534Lowry WE, 2008, P NATL ACAD SCI USA, V105, P2883, DOI 10.1073/pnas.0711983105Laurent LC, 2008, STEM CELLS, V26, P1506, DOI 10.1634/stemcells.2007-1081Kim JB, 2008, NATURE, V454, P646, DOI 10.1038/nature07061Ross JJ, 2006, J CLIN INVEST, V116, P3139, DOI 10.1172/JCI28184Takahashi K, 2006, CELL, V126, P663Yu JY, 2006, STEM CELLS, V24, P168, DOI 10.1634/stemcells.2005-0292Cowan CA, 2005, SCIENCE, V309, P1369, DOI 10.1126/science.1116447Adhikary S, 2005, NAT REV MOL CELL BIO, V6, P635, DOI 10.1038/nrm1703DALLAFAVERA R, 1982, P NATL ACAD SCI-BIOL, V79, P78241

Oct-4 controls cell-cycle progression of embryonic stem cells

Mouse and human ES (embryonic stem) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the ‘stemness’ of ES cells; however, little is known about how the cell-cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of ES cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell-cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall integrity of the Oct-4 functional domains is important for the stimulation of S-phase entry. We also show in the present study that the p21 gene is a target for Oct-4 repression. Furthermore, p21 protein levels were repressed by Oct-4 and were induced by the down-regulation of Oct-4 in ZHBTc4 ES cells. Therefore the down-regulation of p21 by Oct-4 may contribute to the maintenance of ES cell proliferation

Efficacy of two different self-expanding nitinol stents for atherosclerotic femoropopliteal arterial disease (SENS-FP trial): study protocol for a randomized controlled trial

BACKGROUND: There have been few randomized control trials comparing the incidence of stent fracture and primary patency among different self-expanding nitinol stents to date. The SMART™ CONTROL stent (Cordis Corp, Miami Lakes, Florida, United States) has a peak-to-valley bridge and inline interconnection, whereas the COMPLETE™-SE stent (Medtronic Vascular, Santa Rosa, California, United States) crowns have been configured to minimize crown-to-crown interaction, increasing the stent's flexibility without compromising radial strength. Further, the 2011 ESC (European society of cardiology) guidelines recommend that dual antiplatelet therapy with aspirin and a thienopyridine such as clopidogrel should be administered for at least one month after infrainguinal bare metal stent implantation. Cilostazol has been reported to reduce intimal hyperplasia and subsequent repeat revascularization. To date, there has been no randomized study comparing the safety and efficacy of two different antiplatelet regimens, clopidogrel and cilostazol, following successful femoropopliteal stenting. METHODS/DESIGN: The primary purpose of our study is to examine the incidence of stent fracture and primary patency between two different major representative self-expanding nitinol stents (SMART™ CONTROL versus COMPLETE™-SE) in stenotic or occlusive femoropopliteal arterial lesion. The secondary purpose is to examine whether there is any difference in efficacy and safety between aspirin plus clopidogrel versus aspirin plus cilostazol for one month following stent implantation in femoropopliteal lesions. This is a prospective, randomized, multicenter trial to assess the efficacy of the COMPLETE™-SE versus SMART™ CONTROL stent for provisional stenting after balloon angioplasty in femoropopliteal arterial lesions. The study design is a 2x2 randomization design and a total of 346 patients will be enrolled. The primary endpoint of this study is the rate of binary restenosis in the treated segment at 12 months after intervention as determined by catheter angiography or duplex ultrasound. DISCUSSION: This trial will provide powerful insight into whether the design of the COMPLETE™-SE stent is more fracture-resistant or effective in preventing restenosis compared with the SMART™ CONTROL stent. Also, it will determine the efficacy and safety of aspirin plus clopidogrel versus aspirin plus cilostazol in patients undergoing stent implantation in femoropopliteal lesions. TRIAL REGISTRATION: Registered on 2 April 2012 with the National Institutes of Health Clinical Trials Registry (ClinicalTrials.gov identifier# NCT01570803)

Specification of intermediate mesoderm (IM) from the hESC-derived PS cells.

<p>A. (a) Optimal timing for IM induction was determined by transcriptional expression level of <i>OSR1</i>, comparing with expression values of undifferentiated hESCs, in hESC-derived cells. Relative gene expression was normalized to GAPDH, and the values of fold-changes are represented by mean ± S.E.M (n = 3). (b) Comparison of <i>OSR1</i> transcripts by combinatorial treatments with exogenous growth factors of retinoic acid (RA), BMP7 (B7) and FGF2 (F2). Untreated samples were designated as (C). Transcriptional expression levels were normalized to GAPDH, and results of fold-change are indicated as mean ± S.E.M (n = 3). (c) Comparison of <i>OSR1</i> transcription levels by the concentration of RA treatment, including 0.1, 1 and 10 µM. Relative gene expression was normalized to GAPDH. The values of fold-changes are represented by mean ± S.E.M (n = 3). B. Immunocytochemistry for representative expression of OSR1 (red) in (a) hESC-derived PS and (b) IM cells. Quantification of the number of cells expressing the key markers of specific differentiation stage was performed by manual counting in three randomly chosen fields. Scale bars = 50 µm. C. Transcriptional expression of various IM marker genes were evaluated by (a) real time RT-PCR and (b) RT-PCR in hESC-derived IM cells. Human fetal kidney (HFK) cDNA was used as positive control. Relative transcriptional levels were normalized to GAPDH, and the bars show mean ± S.E.M (n = 3). D. Immunostaining of PAX2 (green) and SALL1 (red) at the end of IM induction in hESC-derivatives. Scale bars = 100 µm.</p

Induction of hESCs into primitive streak (PS) cells.

<p>A. (a) Optimal duration for PS induction was determined by transcription level of <i>T</i> compared with undifferentiated hESCs before treatments. Relative gene expression was normalized to GAPDH, and the values of fold-changes are represented by mean ± S.E.M (n = 3). (b) Comparison of expression levels of PS specific genes by combinatorial treatments with exogenous factors of Activin A, Wnt3a (AW) and BMP4, FGF2 (BF), in hESC-derived PS cells. Untreated samples were designated as (C). Relative transcriptional levels were normalized to GAPDH, and the bars show mean ± S.E.M (n = 3). B. (a) Determination of heterogeneity with other germ layers such as definitive endoderm (DE) and ectoderm by evaluating transcriptional expression of (a) DE and (b) ectoderm marker genes in hESC-derived PS cells. C. Immunofluorescence of T (red), TRA1-81 (green), and OCT4 (red) expression in the cells of induction day 0 (undifferentiated hESCs) and day 3 (primitive streak). Scale bars = 200 µm.</p

Evaluation of transcriptional activation of other inducible lineages markers in hESC-derived NPCs.

<p>Transcriptional expression of (a) <i>FOXD1</i> and <i>HOXB11</i> (metanephric stroma/ureteric bud), (b) bone (<i>RUNX2</i> and <i>COL1A1</i>), (c) vascular endothelium (<i>PECAM1</i> and <i>TIE2</i>), (d) smooth muscle (<i>MYOSIN11</i> and <i>CALPONIN</i>), (e) liver (<i>ALB</i> and <i>AAT</i>), and (f) neuron (<i>TUJ1</i> and <i>MAP2</i>) were analyzed by q-PCR in hESC-derived NPCs. Relative expression values were normalized to GAPDH, and fold-changes are shown by mean ± S.E.M (n = 3).</p