Cytokines in neural regeneration.

Growth factors with already established multiple effects on non-neural cells continue to be of considerable interest to researchers with regard to the nervous system, where regulation of cell maintenance and plasticity in relation to lesion and regeneration is part of their functional repertoire. Fibroblast growth factors, interleukins, and type beta transforming growth factors are prominent representatives of such proteins. Ciliary neurotrophic factor is another multifunctional neurokine. The proposed role of this molecule as a 'lesion factor', however, is still not firmly settled.     

Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes

Background aimsMucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) in which an absence of sulfamidase results in incomplete degradation and subsequent accumulation of its substrate, heparan sulfate. Most neurodegenerative LSD remain untreatable. However, therapy options, such as gene, enzyme end cell therapy, are under investigation. Previously, we have constructed an embryonic stem (ES) cell line (NS21) that over-expresses human sulphamidase as a potential treatment for murine MPS IIIA.MethodsIn the present study the sulfatase-modifying factor I (SUMF1) and enhanced green fluorescence protein (eGFP) genes were co-introduced under a cytomegalovirus (CMV) promoter into NS21 cells, to enhance further sulfamidase activity and provide a marker for in vivo cell tracking, respectively. eGFP was also introduced under the control of the human elongation factor-1α (hEF-1α) promoter to compare the stability of transgene expression.ResultsDuring differentiation of ES cells into glial precursors, SUMF1 was down-regulated and was hardly detectable by day 18 of differentiation. Likewise, eGFP expression was heterogeneous and highly unstable. Use of a human EF-1α promoter resulted in more homogeneous eGFP expression, with ～ 50% of cells eGFP positive following differentiation into glial precursors. Compared with NS21 cells, the outgrowth of eGFP-expressing cells was not as confluent when differentiated into glial precursors.ConclusionsOur data suggest that SUMF1 enhances sulfamidase activity in ES cells, hEF-1α is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression.     

Single-cell analysis of tumors: Creating new value for molecular biomarker discovery of cancer stem cells and tumor-infiltrating immune cells

Biomarker-driven individualized treatment in oncology has made tremendous progress through technological developments, new therapeutic modalities and a deeper understanding of the molecular biology for tumors, cancer stem cells and tumor-infiltrating immune cells. Recent technical developments have led to the establishment of a variety of cancer-related diagnostic, prognostic and predictive biomarkers. In this regard, different modern OMICs approaches were assessed in order to categorize and classify prognostically different forms of neoplasia. Despite those technical advancements, the extent of molecular heterogeneity at the individual cell level in human tumors remains largely uncharacterized. Each tumor consists of a mixture of heterogeneous cell types. Therefore, it is important to quantify the dynamic cellular variations in order to predict clinical parameters, such as a response to treatment and or potential for disease recurrence. Recently, single-cell based methods have been developed to characterize the heterogeneity in seemingly homogenous cancer cell populations prior to and during treatment. In this review, we highlight the recent advances for single-cell analysis and discuss the challenges and prospects for molecular characterization of cancer cells, cancer stem cells and tumor-infiltrating immune cells.     

筛选抑制酿酒酵母生长的人类基因

【目的】基于人类基因文库,构建一个筛选抑制酿酒酵母生长的人类基因的方法,并运用此方法筛选含有生长抑制性人源蛋白质的酿酒酵母,用于分析人类基因的生理功能及其抑制剂的寻找。【方法】利用Gateway~(TM)重组技术将人类蛋白质编码基因构建到酿酒酵母表达质粒中。得到的质粒分别转化酿酒酵母细胞中,分析哪些基因的表达会抑制酿酒酵母的生长,并用绿色荧光蛋白标签对典型候选基因在酿酒酵母中的定位进行观察。【结果与结论】本研究建立了抑制酿酒酵母生长的人类基因的筛选方法,并运用此方法成功地从2991个人类蛋白质编码基因中筛选到29个显著抑制酿酒酵母生长的基因。其中一些是引起人类疾病的致病基因。例如,PDLIM4参与到骨质疏松症和前列腺癌的形成和发展,但其生理功能尚不清楚。我们的研究可能为揭示这些候选基因的功能和调节机制提供新的途径。     

Reproducible preparation of spheroids of pancreatic hormone positive cells from human iPS cells: An in vitro study

BackgroundTransplantation of islets of Langerhans is regarded as a promising therapy for type 1 diabetes. A large number of β-cells are required for the treatment of human type 1 diabetes. Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, have been considered as new sources for cell replacement therapy.MethodsCell aggregates were prepared from human iPS cells using agarose microwell plates and differentiated into pancreatic endocrine cells by changing the culture media with different additives.ResultsAfter 20 days of culture, approximately 30% of cells in aggregates were positive for C-peptide. After another 14 days in culture, the cells gained an ability to alter C-peptide release in response to changes in the glucose concentration.ConclusionsUniform aggregates of human iPSCs were easily prepared on agarose microwell plates and efficiently differentiated into the pancreatic endocrine lineage. Thus, aggregate culture is a suitable method for preparing islet-like aggregates from human iPSCs.General significanceOur results indicate that the microwell plate is suitable for scaling up the preparation of pancreatic endocrine cells from human iPS cells in a robotic system.     

Global transcriptome analysis of human bone marrow stromal cells (BMSC) reveals proliferative, mobile and interactive cells that produce abundant extracellular matrix proteins, some of which may affect BMSC potency

Background aimsBone marrow stromal cells (BMSC) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSC were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality.MethodsEarly passage BMSC prepared from marrow aspirates of seven healthy subjects were compared with three human embryonic stem cell (hESC) samples, CD34⁺ cells from three healthy subjects and three fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35 000 probes.ResultsBMSC gene expression signatures of BMSC differed from those of hematopoietic stem cells (HSC), hESC and fibroblasts. Genes upregulated in BMSC were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The upregulated genes most probably belonged to pathways for integrin signaling, integrin-linked kinase (ILK) signaling, NF-E2-related factor-2 (NFR2)-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wingless-type MMTV integration site (Wnt/β catenin signaling. Among the most highly upregulated genes were structural extracellular matrix (ECM) proteins (α5 and β5 integrin chains, fibronectin and collagen type IIIα1 and Vα1) and functional EMC proteins [connective tissue growth factor (CTGF), transforming growth factor beta-induced protein (TGFBI) and A disintegrin and metalloproteinase (ADAM12)].ConclusionsGlobal analysis of human BMSC suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects.     

A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide

The active form of fibronectin is its extracellular matrix form, which allows for the attachment of cells and influences both the growth and migration of cells. The matrix form is assembled by cells; however, many cells are defective in this regard. Several regions within fibronectin have been shown to play a role in matrix assembly by cells. One such region has been localized into the first type III repeat of fibronectin (Chernousov, M. A., F. J. Fogerty, V. E. Koteliansky, and D. F. Mosher. J. Biol. Chem. 266:10851-10858). We have identified this site as a fibronectin-fibronectin binding site and reproduced it as a synthetic peptide. This site is contained in a 14-kD fragment that corresponds to portions of the first two type III repeats. The 14-kD fragment was found to bind to cell monolayers and to inhibit fibronectin matrix assembly. The 14-kD fragment only slightly reduced the binding of fibronectin to cell surfaces but it significantly inhibited the subsequent incorporation of fibronectin into the extracellular matrix. The 14-kD fragment also bound to purified fibronectin and inhibited fibronectin-fibronectin binding. A synthetic 31-amino acid peptide (P1) representing a segment of the 14-kD fragment retained the ability to inhibit fibronectin-fibronectin binding. Peptide P1 specifically bound fibronectin from plasma in affinity chromatography, whereas a column containing another peptide from the 14-kD fragment did not. These results define a fibronectin-fibronectin binding site that appears to promote matrix assembly by allowing the assembly of fibronectin molecules into nascent fibrils. The 14-kD fragment and the P1 peptide that contain this site inhibit matrix assembly by competing for the fibronectin-fibronectin binding.     

Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells

Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-l-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein-protein interactions in cells.     

Human adipose stromal vascular cell delivery in a fibrin spray

Background aimsAdipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system.MethodsSVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVage system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel fibrin spray system. SVF cell growth and differentiation were documented by immunofluorescence staining and photomicrography.ResultsSVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SVF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD146+ and α-smooth muscle actin+ perivascular/stromal cells.ConclusionsHuman adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.     

Growth factors induce early pre-replicative changes in senescent human fibroblasts.

As human fibroblasts in culture senesce their response to platelet-derived growth factor (PDGF) becomes attenuated. To clarify at which level such cells are blocked in the pre-replicative part of the cell cycle, we have analysed PDGF-induced pre-replicative events in senescent (phase III) cultures. We found that phase III cells retain a normal number of PDGF receptors and that these are functional with regard to PDGF-induced receptor autophosphorylation. Phase III cells also respond to PDGF by rapid actin reorganization and increased levels of c-fos and c-myc mRNA, similar to growth-arrested phase II fibroblasts. However, the expression of the nuclear antigen K-67, which in phase II cell is induced in S-phase and continues to be expressed throughout the cell cycle, is not induced in phase III cells in response to PDGF. We conclude that phase III human fibroblasts, although blocked with regard to proliferation, still retain a functional growth factor receptor system, and display early responses when exposed to growth factors, such as changes in the cytoskeleton and the expression of proto-oncogenes.     

Upregulation of transforming growth factor-beta type I receptor by interferon consensus sequence-binding protein in osteosarcoma cells

Transforming growth factor-beta (TGF-β) is a known tumor suppressor, which also exerts a tumor promoting activity at an advanced stage of cancer. Previously, we reported that expression of interferon consensus sequence-binding protein (ICSBP), also known as interferon regulatory factor-8, is positively correlated with TGF-β type I receptor (TGF-β RI) expression in osteosarcoma patient tissues. In this study, we demonstrated that ICSBP upregulated TGF-β RI and induced epithelial-to-mesenchymal transition-like phenomena in human osteosarcoma cell lines. As determined by soft agar growth of osteosarcoma cells and xenografted mouse models, ICSBP increased tumorigenicity, which was reversed by ICSBP knock-down or a TGF-β RI inhibitor. To test whether ICSBP directly regulates the promoter activity of TGF-β RI, we performed a TGF-β RI promoter assay, an electro mobility shift assay, and a chromatin immunoprecipitation assay. We observed that TGF-β RI promoter was activated in ICSBP-overexpressing osteosarcoma cells. Exploiting serial deletions and mutations of the TGF-β RI promoter, we found a putative ICSBP-binding site at nucleotides −216/−211 (GGXXTC) in the TGF-β RI promoter. Our data suggest that ICSBP upregulates TGF-β RI expression by binding to this site, causing ICSBP-mediated tumor progression in osteosarcoma cells. In addition, we found a positive correlation between ICSBP and TGF-β RI expression in several types of tumors using the cBioportal database.SummaryWe demonstrated that interferon consensus sequence-binding protein upregulates transforming growth factor-beta type I receptor (TGF-β RI) expression by binding to nucleotides −216/−211 (GGXXTC) in the TGF-β RI promoter, which resulted in increased tumorigenicity and tumor progression in human osteosarcoma cells.     

Direct lineage conversions: unnatural but useful?

Classic experiments such as somatic cell nuclear transfer into oocytes and cell fusion demonstrated that differentiated cells are not irreversibly committed to their fate. More recent work has built on these conclusions and discovered defined factors that directly induce one specific cell type from another, which may be as distantly related as cells from different germ layers. This suggests the possibility that any specific cell type may be directly converted into any other if the appropriate reprogramming factors are known. Direct lineage conversion could provide important new sources of human cells for modeling disease processes or for cellular-replacement therapies. For future applications, it will be critical to carefully determine the fidelity of reprogramming and to develop methods for robustly and efficiently generating human cell types of interest.     

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.     

The human LT system. II. Immunological relationships of LT molecules released by mitogen activated human lymphocytes in vitro.

Materials with LT activity present in supernatants from PHA stimulated human lymphocytes in vitro are very heterogeneous and can be separated into multiple molecular weight classes, termed complex, α, β, and γ. Several of these classes can be further resolved into subclasses by other physical and chemical methods. The immunologic relationships of these materials one to another were examined employing various rabbit anti-humn LT sera which will neutralize LT activity on L-929 cells in vitro. These studies reveal: (a) LT activities are due to a distinct group of substances which are immunologically related one to another and can exist in several molecular weight forms; (b) a high MW class of molecules, termed complex, appears to contain all currently known LT classes and subclasses; (c) LT classes and subclasses both have common (public) and discrete (private) antigenic specificities; (d) human LT classes and subclasses do not appear to share Ag determinants with materials with LT activity released by lectin stimulated lymphoid cells from rabbit, rat, hamster, guinea pig, or mouse; and (e) human LT molecules are not immunologically related to cell toxins released by glass adherent human peripheral blood monocytes or PMN cells. These data indicate human LT molecules form a “discrete system” of lymphocyte derived cell toxins, which can associate together into various related but different MW forms in the supernatant.     

Cellular succession in the epithelium lining the pharyngeal cavity of planarians

Regardin cell renewal of planarian epithelial tissue, little is known at present except for the epidermis (Tyler, 1984). According to Skaer (1965), the definitive epidermal cells in planarian embryos originate in the parenchyma where they develop as precursor cells which then migrate into the epidermis. This process of development is also reported in regenerating planarians (Spiegelman & Dudley, 1973; Morita & Best, 1974; Hori, 1978). Recently the validity of Skaer's conclusion has clearly been confirmed by Ishii & Sakurai (1998) using SEM and TEM.The present study deals with the origin and renewal of cells of another epithelium — that lining the pharyngeal cavity of a freshwater planarian Dugesia japonica. This epithelium is known to consist of cells with secretory granduls of fingerprint-like structure (Ishii, 1966; Bowen & Ryder, 1973; Gamo & Garcia-Corrales, 1988; Asai, 1991). The characteristic feature of these specific granules, as well as epitheliosomes (Tyler, 1984) of the epidermis, is very useful as a cell marker for distinguishing cell types and following cell differentiation. Gamo & Garcia-Corrales (1988) claimed the cell populations of the pharyngeal epithelium of Dugesia gonocephala is morphologically and functionally heterogeneous. The sequence of morphological alteration offers the oppurtunity to distinguish at least three functional stages. These represent different adaptive forms of a single cell type, and indicate transformation from one to the next induced by cell renewal. It suggests a beta cell (noeblast) origin of the epithelial cells.In the present study SEM observations revealed theat there are morphologically three distinct cell types which are considered to be juvenile, mature, and senile cells respectively, judging from the differences in cells and the many transitional forms among them (Fig. 1). When observed by TEM, the cytoplasmic patterns of these cell types are correspondingly different from one another, similarly the surface specializations of the plasma membranes of each cell type which facilitate their identification by TEM also vary. Apparently alterations of cell morphology are involved in ageing in a single cell type. Morphological features of each cell type are summarized. Juveniles (R) rarely occur, are small cells provided with prominent apical ruffles and considered to be phagocytic because the cells often contain newly formed phagosomes. Mature cells (F) predominate, are large, polygonal, flat cells, presumably with an intense secretory activity. The cells contain many profiles of granular ER and a small number of specific (secretory) granules. Senile cells (M) are intermittently distributed, irregularly contoured cells with heavy surface microvilli. The specific granules are dense in the apical cytoplasm, and profiles of granular ER decease in number.The precursor epithial cells that are seen in the parenchyma exhibit a considerable degree of differentiation before their translocation into the lining. The primordial cells observed are neoblast-like, with several cohromatoid bodies and many free ribosomes (Morita et al., 1969). These results obtain thus support the evidence and prediction of Gamo & Garcia-Corrales (1988). The observed mode of cell replacement appears to be the same as found in the epidermis (Lentz, 1967).     

Role of Type 2C Protein Phosphatases in Growth Regulation and in Cellular Stress Signaling

ABSTRACT

A number of interesting features, phenotypes, and potential clinical applications have recently been ascribed to the type 2C family of protein phosphatases. Thus far, 16 different PP2C genes have been identified in the human genome, encoding (by means of alternative splicing) for at least 22 different isozymes. Virtually ever since their discovery, type 2C phosphatases have been predominantly linked to cell growth and to cellular stress signaling. Here, we provide an overview of the involvement of type 2C phosphatases in these two processes, and we show that four of them (PP2Cα, PP2Cβ, ILKAP, and PHLPP) can be expected to function as tumor suppressor proteins, and one as an oncoprotein (PP2Cδ /Wip1). In addition, we demonstrate that in virtually all cases in which they have been linked to the stress response, PP2Cs act as inhibitors of cellular stress signaling. Based on the vast amount of experimental evidence obtained thus far, it therefore seems justified to conclude that type 2C protein phosphatases are important physiological regulators of cell growth and of cellular stress signaling.     

Neuronal organization of the accessory olfactory bulb of the lizard Podarcis hispanica: Golgi study

The neuronal organization of the accessory olfactory bulb (AOB), which receives sensory information from the vomeronasal organ, was described in a squamate reptile (Podarcis hispanica) by means of light microscopy. Using the Golgi-impregnation method, seven neuronal types could be distinguished: Periglomerular cells constitute a morphologically heterogeneous population of small neurons located between and around the glomeruli. The mitral cells are diffusely distributed in the AOB. Their cell bodies are usually located within the mitral cell layer, but some of them could be also observed in the plexiform layers. Mitral cells were classified into three subgroups on the basis of their sizes and dendritic tree morphologies. Thus, the “outer mitral cells” have the biggest cell bodies, and their distal secondary dendrites are mainly distributed rostrocaudally in the external plexiform layer. The “inner mitral cells” have large cell bodies, and their secondary dendrites are distributed dorsoventrally and are located deeper than those of the other two subgroups. The third type, the “small mitral cells,” is the smallest one among mitral cells in the AOB, and from their cell bodies, only two main dendritic trunks arise. The granule cells are composed of several categories based on their different cell body locations and dendritic tree morphologies. Thus, the “superficial granule cells” are located exclusively in the external plexiform layer and have small dendritic fields. The “middle granule cells” have fusiform cell bodies—situated in the internal plexiform layer—and present a wide dendritic projection area. Finally, the “deep granule cells” are distributed throughout the granule cell layer and include a great variety of dendritic tree morphologies. The distribution and morphological features of all neuronal types constituting the AOB of Podarcis were compared with those reported on other vertebrates. The results suggest that the lamination pattern and neuronal organization of the AOB in lizards are more similar to that of mammals than to that of the remaining vertebrates.