Two-Step Generation of Oligodendrocyte Progenitor Cells From Mouse Fibroblasts for Spinal Cord Injury

Oligodendrocyte progenitor cells (OPCs) are attracting attention as the ideal cell therapy for spinal cord injury (SCI). Recently, advanced reprogramming and differentiation techniques have made it possible to generate therapeutic cells for treating SCI. In the present study, we used directly-induced neural stem cells (DNSCs) from fibroblasts to establish OPCs (DN-OPCs) capable of proliferation and confirmed their OPC-specific characteristics. Also, we evaluated the effect of transplanted DN-OPCs on SCI in rats. The DN-OPCs exhibited an OPC-specific phenotype and electrophysiological function and could be differentiated into oligodendrocytes. In the SCI model, transplanted DN-OPCs improved behavior recovery, and showed engraftment into the host spinal cord with expression of myelin basic protein. These results suggest that DN-OPCs could be a new source of potentially useful cells for treating SCI

Internal evaluation of a physically-based distributed model using data from a Mediterranean mountain catchment

An evaluation of the performance of a physically-based distributed model of a small Mediterennean mountain catchment is presented. This was carried out using hydrological response data, including measurements of runoff, soil moisture, phreactic surface level and actual evapotranspiration. A-priori model parameterisation was based as far as possible on property data measured in the catchment. Limited model calibration was required to identify an appropriate value for terms controlling water loss to a deeper regional aquifer. The model provided good results for an initial calibration period, when judge in terms of catchment discharge. However, model performance for runoff declined substantially when evaluated againts a consecutive, rather drier, period of data. Evaluation against other catchment responses allowed identification of the problems responsible for the observed lack of model robustness in flow simulation. In particular, it was shown that an incorrect parameterisation of the soil water was preventing adequate representation of drainage from soils during hydrogeraph recessions. This excess moisture was then being removed via an overestimation of evapotranspiration. It also appeared that the model underestimated canopy interception. The results presented here suggest that model evaluation against catchment scale variables summarising its water balance can be of great use in identifying problems with model parameterisation, even for distributed models. Evaluation using spatially distributed data yielded less useful information on model performance, owing to the relative sparseness of data points, and problems of mismatch of scale between the measurement and the model grid.This work was carried out as part of project VAHMPIRE (Validating Hydrological Models using Process Studies and Internal Data from Research Basins: tools for assessing the hydrological impacts of environmental change), which was funded by the European Commission Framework IV Environment and Climate Program (Contract No. ENV4- CT95-0134). Simulations were carried out on a UNIX workstation funded jointly by UK Nirex Ltd. and NERC grant GR3/ E0009.Peer Reviewe

Epiblast Stem Cell Subpopulations Represent Mouse Embryos of Distinct Pregastrulation Stages

SummaryEmbryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast

Induced neural stem cells from distinct genetic backgrounds exhibit different reprogramming status

Somatic cells could be directly converted into induced neural stem cells (iNSCs) by ectopic expression of defined transcription factors. However, the underlying mechanism of direct lineage transition into iNSCs is largely unknown. In this study, we examined the effect of genetic background on the direct conversion process into an iNSC state. The iNSCs from two different mouse strains exhibited the distinct efficiency of lineage conversion as well as clonal expansion. Furthermore, the expression levels of endogenous NSC markers, silencing of transgenes, and in vitro differentiation potential were also different between iNSC lines from different strains. Therefore, our data suggest that the genetic background of starting cells influences the conversion efficiency as well as reprogramming status of directly converted iNSCs.ope

A Plant-Derived Antigen–Antibody Complex Induces Anti-Cancer Immune Responses by Forming a Large Quaternary Structure

The antigen–antibody complex (AAC) has novel functions for immunomodulation, encouraging the application of diverse quaternary protein structures for vaccination. In this study, GA733 antigen and anti-GA733 antibody proteins were both co-expressed to obtain the AAC protein structures in a F1 plant obtained by crossing the plants expressing each protein. In F1 plant, the antigen and antibody assembled to form a large quaternary circular ACC structure (~30 nm). The large quaternary protein structures induced immune response to produce anticancer immunoglobulins G (IgGs) that are specific to the corresponding antigens in mouse. The serum containing the anticancer IgGs inhibited the human colorectal cancer cell growth in the xenograft nude mouse. Taken together, antigens and antibodies can be assembled to form AAC protein structures in plants. Plant crossing represents an alternative strategy for the formation of AAC vaccines that efficiently increases anticancer antibody production

Optimization of Matrigel-based culture for expansion of neural stem cells

<div><p></p><p>Neural stem cells (NSCs) are capable of self-renewal and can differentiate into neurons and two types of glial cells. NSCs have great potential for basic studies of neurogenesis and neurodegenerative diseases, and for therapeutic transplantation applications. NSCs can be derived from the fetal or adult brain. <i>In vitro</i> expansion of NSCs may help to elucidate their properties involved in neurogenesis and is crucial to obtain on-demand the large numbers of these cells needed for clinical transplantation trials. Laminin is an extracellular matrix molecule commonly used to culture NSCs as an attached monolayer. However, laminin is costly if NSCs need to be cultured in large quantities. Matrigel is a soluble basement membrane biomaterial, which has been widely used for feeder-free cultures of human embryonic stem cells. In the present study, we evaluated the ability of Matrigel to support the attachment and long-term proliferation of NSCs. We investigated the growth and adherence of NSCs derived from the fetal mouse brain on Matrigel at various concentrations (0.01–1%). We found that 0.02% Matrigel is sufficient for NSCs to be expanded <i>in vitro</i> in long-term culture. The NSCs cultured on Matrigel-coated plates showed typical cellular and molecular characteristics and had multipotency to be differentiated into neurons and glial cells. The Matrigel-based monolayer culture system established in this study provides a cost-effective approach to maintain NSCs <i>in vitro</i> in long-term cultures.</p></div

Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts

Generation of induced pluripotent stem cells (iPSCs) by defined factors (OCT4, SOX2, C-MYC, and KLF4) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (OCT4, SOX2, C-MYC, and KLF4) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (OCT4/p53, SOX2/KLF4, L-MYC/LIN28A) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors