Fibroblast Growth Factor

A prerequisite for successful embryo implantation is adequate preparation of receptive endometrium and the establishment and maintenance of a viable embryo. The success of implantation further relies upon a two-way dialogue between the embryo and uterus. However, molecular bases of these preimplantation and implantation processes in humans are not well known. We performed genome expression analyses of human embryos (n ϭ 128) and human endometria (n ϭ 8). We integrated these data with protein-protein interactions in order to identify molecular networks within the endometrium and the embryo, and potential embryo-endometrium interactions at the time of implantation. For that, we applied a novel network profiling algorithm HyperModules, which combines topological module identification and functional enrichment analysis. We found a major wave of transcriptional down-regulation in preimplantation embryos. In receptive-stage endometrium, several genes and signaling pathways were identified, including JAK-STAT signaling and inflammatory pathways. The main curated embryo-endometrium interaction network highlighted the importance of cell adhesion molecules in the implantation process. We also identified cytokine-cytokine receptor interactions involved in implantation, where osteopontin (SPP1), leukemia inhibitory factor (LIF) and leptin (LEP) pathways were intertwining. Further, we identified a number of novel players in human embryo-endometrium interactions, such as apolipoprotein D (APOD), endothelin 1 (END1), fibroblast growth factor 7 (FGF7), gastrin (GAST), kringle containing trnasmembrane protein 1 (KREMEN1), neuropilin 1 (NRP1), serpin peptidase inhibitor clade A member 3 (SERPINA3), versican (VCAN), and others. Our findings provide a fundamental resource for better understanding of the genetic network that leads to successful embryo implantation. We demonstrate the first systems biology approach into the complex molecular network of the implantation process in humans. (Molecular Endocrinology 26: 203-217, 2012) S uccessful embryo implantation is an absolute requirement for the reproduction of mammalian species. In humans, implantation involves complex interactions between the embryo and the maternal endometrium, all of which must be performed within an optimal time frame.

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118. © 2010 Wiley Periodicals, Inc.

Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) are actively pursued for stimulation of bone formation. To assess their promise for systemic therapy of osteoporosis, we ascertained the effects of bFGF and BMP-2 on bone marrow cells in vitro. Bone marrow cells were obtained from young (8 weeks) and adult (32 weeks) rats by femoral aspiration and were exposed to osteogenic medium (ie, basal medium with 10 mM ␤-glycerolphosphate and 100 nM dexamethasone) containing the growth factors. The cell viability in osteogenic medium was reduced after 3 weeks but not if the concentration of ␤-glycerolphosphate/dexamethasone was reduced to 3 mM/30 nM. Unlike BMP-2, bFGF at 2-50 ng/mL was capable of enhancing longterm cell viability. Continuous treatment of bone marrow cells for 3 weeks resulted in dose-dependent stimulation of mineralization by BMP-2, but not by bFGF, whose activity was optimal at 2-10 ng/mL. To explore the effect of shortterm exposure, bone marrow cells were treated with growth factors for 1 week and subsequent mineralization was investigated. BMP-2 exposure increased the extent of mineralization, but bFGF was not effective after the short exposure. We concluded bFGF was more potent (ie, required lower concentration) for stimulating osteogenic parameters, but BMP-2 effects were lasting on the bone marrow cells.

In a novel assay of angiogenesis in the quail chorioal-(relative to 1.49 { 0.02 and 583 { 60/cm 2 for conlantoic membrane (CAM), we measured vascular pattrols), and of angiostatin-treated samples, 1.43 { 0.02 tern and angiogenic rate after homogeneous exposure and 424 { 74/cm 2 (relative to 1.50 { 0.02 and 616 { of the entire vascular tree to recognized modulators of 59/cm 2 for controls). To establish normalization valvessel growth. In comparison to phosphate-buffered ues for rates of angiogenesis, we analyzed untreated saline (PBS)-treated controls, the vascular stimulator, CAMs of E6 to E12. From E7 to E10 in skeletonized basic fibroblast growth factor (bFGF or FGF-2), inimages, D f increased linearly from 1.37 { 0.01 to 1.54 creased the rate of angiogenesis by a maximum of { 0.01 and r v from 311 { 67 to 746 { 124/cm 2 (in 72%, whereas a recently discovered angiogenic inhibiboth cases, r 2 Å 1.000). Thus, the rates of normal tor, angiostatin, decreased the rate of vascular growth angiogenic growth as measured by D f and r v were by a maximum of 68%. The perturbants were applied 0.06/day and 138/cm 2-day, respectively. From E10 to in PBS to the CAM of 7-day-old embryos (E7) cultured E12, D f and r v declined slightly. Differences between in petri dishes, and the embryos were cultured further the vasculature of untreated and PBS-treated CAMs until fixation at E8 or E9. For morphometry of the were statistically insignificant. In conclusion, vascuquasi-two-dimensional CAM vasculature, digital imlar branching pattern and density in the quail CAM ages of arterial endpoints from the middle region of were stimulated by bFGF and inhibited by angiothe CAM were acquired in grayscale at a magnification statin. We quantified these changes with statistical of 101, binarized to black/white, and skeletonized. significance by D f and r v , which are expressed relative The pattern of vessel branching was assessed by meato the rates of normal developmental angiogenesis surement of the fractal dimension (D f), and vessel measured for the two parameters in untreated quail density (r v), with the method of grid intersection. embryos. ᭧ 1998 Academic Press Correlations between these two statistical techniques Key Words: angiogenesis; fractal dimension; grid interwere linear (r 2 ranged from 0.967 to 0.985). For skelesection; chorioallantoic membrane; quail; basic fibrotonized images at E9, D f and r v of bFGF-treated samblast growth factor (bFGF); angiostatin. ples were 1.55 { 0.01 and 782 { 26/cm 2 , respectively

Salmosin is a snake venom-derived novel disintegrin that antagonizes platelet aggregation. In this study, we investigated its functional specificity in tumor angiogenesis. Salmosin significantly inhibited bovine capillary endothelial cell proliferation induced by basic fibroblast growth factor but had no effect on normal growth of the cell. The basic fibroblast growth factor-induced in vivo angiogenesis in the chorioallantoic membrane was disrupted by salmosin treatment without affecting normal embryonic angiogenesis. Adhesion of the bovine capillary endothelial cells to vitronectin was also inhibited by the binding of salmosin to the alpha(v)beta3 integrin. Both the metastatic-tumor growth and the solid-tumor growth that developed in mice were effectively suppressed by salmosin treatment. Several lines of experimental evidence strongly suggest that the tumor-specific antiangiogenic activity of salmosin disrupts tumor growth by blocking the alpha(v)beta3 integrin that is expressed o...

The pancreas is an endodermally derived organ that initially appears as a dorsal and ventral protrusion of the primitive gut epithelium. The pancreatic progenitor cells present in these early pancreatic anlagen proliferate and eventually give rise to all pancreatic cell types. The fibroblast growth factor receptor (FGFR) 2b high-affinity ligand FGF10 has been linked to pancreatic epithelial cell proliferation, and we have shown previously that Notch signalling controls pancreatic cell differentiation by means of lateral inhibition. In the developing pancreas, activated intracellular Notch appears to be required for maintaining cells in the progenitor state, in part by blocking the expression of the pro-endocrine gene neurogenin 3 (ngn3), and hence endocrine cell differentiation. Here, we show that persistent expression of Fgf10 in the embryonic pancreas of transgenic mice also inhibits pancreatic cell differentiation, while stimulating pancreatic epithelial cell proliferation. We provide evidence that one of the effects of the persistent expression of Fgf10 in the developing pancreas is maintained Notch activation, which results in impaired expression of ngn3 within the pancreatic epithelium. Together, our data suggest a role for FGF10/FGFR2b signalling in regulation of pancreatic cell proliferation and differentiation and that FGF10/FGFR2b signalling affects the Notch-mediated lateral inhibition pathway. Developmental Dynamics 228: 185-193, 2003.

Glutamine synthetase (GS) has long been known to be expressed exclusively in pericentral hepatocytes most proximal to the central veins of liver lobuli. This enzyme as well as its peculiar distribution complementary to the periportal compartment for ureogenesis plays an important role in nitrogen metabolism, particularly in homeostasis of blood levels of ammonium ions and glutamine. Despite this fact and intensive studies in vivo and in vitro, many aspects of the regulation of its activity on the protein and on the genetic level remained enigmatic. Recent experimental advances using transgenic mice and new analytic tools have revealed the fundamental role of morphogens such as wingless-type MMTV integration site family member signals (Wnt), beta-catenin, and adenomatous polyposis coli in the regulation of this particular enzyme. In addition, novel information concerning the structure of transcription factor binding sites within regulatory regions of the GS gene and their interactions with signalling pathways could be collected. In this review we focus on all aspects of the regulation of GS in the liver and demonstrate how the new findings have changed our view of the determinants of liver zonation. What appeared as a simple response of hepatocytes to blood-derived factors and local cellular interactions must now be perceived as a fundamental mechanism of adult tissue patterning by morphogens that were considered mainly as regulators of developmental processes. Though GS may be the most obvious indicator of morphogen action among many other targets, elucidation of the complex regulation of the expression of the GS gene could pave the road for a better understanding of the mechanisms involved in patterning of liver parenchyma. Based on current knowledge we propose a new concept of how morphogens, hormones and other factors may act in concert, in order to restrict gene expression to small subpopulations of one differentiated cell type, the hepatocyte, in different anatomical locations. Although many details of this regulatory network are still missing, and an era of exciting new discoveries is still about to come, it can already be envisioned that similar mechanisms may well be active in other organs contributing to the fine-tuning of organ-specific functions.

FGF receptor 2 isoform IIIb (FGFR2b), originally discovered as a receptor for FGF7, is known to be an important receptor in vertebrate morphogenesis, because FGFR2b null mice exhibit agenesis or dysgenesis of various organs, which undergo budding and branching morphogenesis. Since FGF7 null mice do not exhibit marked defects in organogenesis, it has been considered that other FGF(s) than FGF7 might function as a major ligand for FGFR2b during organogenesis. One of the candidate ligands is FGF10, because FGF10 binds to FGFR2b with high affinity and the formation of the limb and lung is arrested in FGF10 null mice as found in FGFR2b-deficient mice. Previous analyses of FGF10 null mice revealed that FGF10 is required for limb and lung development. To elucidate the role of FGF10 in wide-range organogenesis, we further analyzed the phenotypes of the FGF10 knockout mice. We found diverse phenotypes closely related to those for FGFR2b-deficient mice, which includes the absence of thyroid, pituitary, and salivary glands, while minor defects were observed in the formation of teeth, kidneys, hair follicles, and digestive organs. These results suggest that FGF10 acts as a major ligand for FGFR2b in mouse multi-organ development.

The fate of cortical progenitors, which progressively generate neurons and glial cells during development, is determined by temporally and spatially regulated signaling mechanisms. We found that the transcription factor Sip1 (Zfhx1b), which is produced at high levels in postmitotic neocortical neurons, regulates progenitor fate non-cell autonomously. Conditional deletion of Sip1 in young neurons induced premature production of upper-layer neurons at the expense of deep layers, precocious and increased generation of glial precursors, and enhanced postnatal astrocytogenesis. The premature upper-layer generation coincided with overexpression of the neurotrophin-3 (Ntf3) gene and upregulation of fibroblast growth factor 9 (Fgf9) gene expression preceded precocious gliogenesis. Exogenous application of Fgf9 to mouse cortical slices induced excessive generation of glial precursors in the germinal zone. Our data suggest that Sip1 restrains the production of signaling factors in postmitotic neurons that feed back to progenitors to regulate the timing of cell fate switch and the number of neurons and glial cells throughout corticogenesis.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allelespecific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

FGF23 dosage with an automated assay: a single-center experience in patients with renal chronic disease A key pathogenic role in the development of secondary hyperparathyroidism in renal patients is played by the fibroblast growth factor 23 (FGF23), a 32 kDa protein secreted by osteocytes in response to dietary phosphorus load. FGF23 elicits a phosphaturic response and inhibits calcitriol synthesis. Measurement of FGF23 levels in patients with chronic renal insufficiency can be useful to better define abnormalities of calcium-phosphate metabolism as well as to select those patients in need of dietary interventions targeted to reduce phosphorus intake. Unfortunately, up to now some limits in its application as well as economic factors have prevented the routine use of FGF23 quantification in clinical practice. The aim of our study was to evaluate the circulating levels of FGF23 in patients with chronic kidney disease using the LIAISON® FGF23 (DiaSorin) assay, a new automated chemiluminescence method that measures the intact molecule and has been approved for clinical use. Twenty-three consecutive patients (14 males and 9 females, with mean age 72.3±10.9 years) with stage 3a-5ND CKD were enrolled. The FGF23 assay was also tested in 10 random patients with end-stage renal disease on hemodialysis. Serum levels of FGF23, PTH, 25(OH)VitD, calcium, phosphorus, creatinine and albumin were measured under fasting conditions. Within the CKD group, FGF23 serum levels were higher than normal in 16 cases. Serum FGF23 levels correlated significantly with serum phosphorus (r = 0.600, p<0.01), calcium (r =-0.546, p<0.05) and eGFR (r =-0.495, p<0.05). In particular, the negative relationship between residual renal function and circulating FGF23 was significant in patients with eGFR<30 mL/min/1.73mq (r =-0.636, p<0.05) but not in those with eGFR>30 mL/ min/1.73 mq. Although from a small cohort of patients, our data confirm that FGF23 levels increase already in the early stages of CKD; they also confirm that when eGFR levels are <30 mL/min/1.73 mq FGF23 increases rapidly and linearly, reaching extremely high levels in the dialysis population. The availability of an automated assay quantifying the amounts of intact FGF23 molecule, and approved for clinical use by the European Community, represents a valuable tool for obtaining an optimal pharmacological and dietary management of patients with CKD-MBD since the earliest stages of renal disease.

Therapeutic angiogenesis is a novel approach to the treatment of myocardial ischemia based on the use of proangiogenic growth factors to induce the growth of new blood vessels to supply the myocardium at risk. This study was designed to assess the safety and efficacy of a single intrapericardial injection of basic fibroblast growth factor (FGF-2) in a porcine model of

An ideal scaffold that provides a combination of suitable mechanical properties along with biological signals is required for successful ligament/tendon regeneration in mesenchymal stem cell-based tissue engineering strategies. Among the various fibre-based scaffolds that have been used, hybrid fibrous scaffolds comprising both microfibres and nanofibres have been recently shown to be particularly promising. This study developed a biohybrid fibrous scaffold system by coating bioactive bFGF-releasing ultrafine PLGA fibres over mechanically robust slowly-degrading degummed knitted microfibrous silk scaffolds. On the ECM-like biomimetic architecture of ultrafine fibres, sustained release of bFGF mimicked the ECM in function, initially stimulating mesenchymal progenitor cell (MPC) proliferation, and subsequently, their tenogeneic differentiation. The biohybrid scaffold system not only facilitated MPC attachment and promoted cell proliferation, with cells growing both on ultrafine PLGA fibres and silk microfibres, but also stimulated tenogeneic differentiation of seeded MPCs. Upregulated gene expression of ligament/tendon-specific ECM proteins and increased collagen production likely contributed to enhancing mechanical properties of the constructs, generating a ligament/tendon analogue that has the potential to be used to repair injured ligaments/tendons.

Objectives: The fibroblast growth factor receptor 3 gene (FGFR3) plays a critical role in cartilage growth-plate differentiation and bony development. It has been shown that 97% of patients with achondroplasia have a G to A transition mutation at position 1138 (c.1138 GNA) of codon 380 of the FGFR3 gene.

Objective. Recent data have shown that abnormal subchondral bone remodeling plays an important role in osteoarthritis (OA) onset and progression, and it was suggested that abnormal mechanical pressure applied to the articulation was responsible for these metabolic changes. This study was undertaken to evaluate the effects of cyclic compression on osteoblasts from OA subchondral bone.

the BM of MM patients, and the MVD correlated with the grade of myeloma cell invasion of the BM in the untreated MM cases. It was recently reported that thalidomide might be effective for the treatment of MM. We assessed the effect of thalidomide on angiogenesis in BM treatment of 11 patients with refractory MM. The concentration of M-protein in the serum or urine of seven of the 11 patients was reduced by at least 30% after thalidomide treatment, and MVD in the BM decreased in three of these seven cases in response to thalidomide. Increased plasma concentrations of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) were observed in all 11 cases before thalidomide administration and both levels were reduced after treatment with thalidomide. Augmented angiogenesis in the bone marrow of MM patients was confirmed in the present study. It seems that thalidomide is effective in the treatment of MM through the impairment of angiogenesis by decreasing FGF-2 and VEGF production. This is the first report on pathological evidence in the bone marrow of MM before and after thalidomide treatment, in Japan.

Triton X-100 extracted ciliary membrane protein from isolated cilia, prepared from the protozoon Tetrahymena thermophila, were fractionated by affinity chromatography on columns with covalently bound fibroblast growth factor (FGF), insulin, or concanavalin A (ConA), respectively. The eluted proteins were further analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels, isoelectric focusing, and by immunoblotting techniques using antibodies against the FGF receptor, platetelet derived growth factor (PDGF) receptor ␣-subunit, and insulin receptor ␤-subunit. The particular antibodies were chosen because the peptides PDGF, FGF, insulin, and ConA are chemoattractants in this organism and corresponding binding (receptor) proteins could be expected to be identified.

This study has assessed the amounts of insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), transforming growth factor b (TGF-b), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) and their binding to extracellular matrix components of Wharton's jelly. Studies were performed on the umbilical cords taken from human newborns delivered by healthy mothers. Wharton's jelly was separated and submitted to homogenisation and extraction with acetic acid and TriseHCl buffer. The assays of growth factors were carried out with the use of ELISA commercial kits, together with SDS/polyacrylamide gel electrophoresis of tissue extracts followed by Western immunoblotting. Several growth factors, viz. acidic FGF, basic FGF, EGF, IGF-I, PDGF and TGF-b were detected in Wharton's jelly. The amounts of these factors per gram of tissue vary from about 40 pg (EGF, PDGF) to about 200 ng (IGF-I). The amounts of peptide growth factors calculated per microgram of DNA are distinctly higher in Wharton's jelly in comparison to the umbilical cord artery. Western blot analysis demonstrated that almost the entire amount of these factors is bound to high molecular weight components. Since the number of cells in Wharton's jelly is very low and the amounts of extracellular matrix components are very high, it is concluded that the cells are strongly stimulated by peptide growth factors to produce large amounts of collagen and glycosaminoglycans. Placenta (2005), 26, 747e752

Ethnopharmacological relevance: Eclipta alba is traditionally known to potentiate hair growth promotion. Aim of the study: The study was aimed to investigate the efficacy of methanol extract of Eclipta alba as hair growth promoter. Materials and methods: Pigmented C57/BL6 mice, preselected for their telogen phase of hair growth were used. In these species, the truncal epidermis lacks melanin-producing melanocytes and melanin production is strictly coupled to anagen phase of hair growth. The extract was applied topically to assess telogen to anagen transition. Immunohistochemical investigation was performed to analyze antigen specificity. Animals in anagen phase of hair growth were positive for FGF-7 and Shh and negative for BMP4, whereas the animals in telogen phase were positive only for BMP4 antigen. Results: The methanol extract of whole plant when tested for hair growth promoting potential, exhibited dose dependent activity in C57BL6 mice. The activity was assessed by studying the melanogenesis in resected skin, follicle count in the subcutis, skin thickness and surrogate markers in vehicle control and extract treated animals. Conclusion: These findings suggest that methanol extract of Eclipta alba may have potential as a hair growth promoter.

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally active, extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

including steroid hormones, aging, environmental enrichment, genetic background, stresses, and running (Gould et al.studies from single cell cultures show that EGF and FGF-2 are mitogens for mouse-derived neu-Summary rospheres (Reynolds and Weiss, 1996; Gritti et al., 1996) and that other unknown factors, present in conditioned We have purified and characterized a factor, from the medium (CM), are required to support neural stem cell conditioned medium of neural stem cell cultures, proliferation (Temple, 1989; Kilpatrick and Bartlett, 1993; which is required for fibroblast growth factor 2's Davis and Temple, 1994; Qian et al., 1997; Palmer et al., (FGF-2) mitogenic activity on neural stem cells. This 1997; Johansson et al., 1999).

The A391E mutation in the transmembrane domain of FGFR3 leads to aberrant development of the cranium. It has been hypothesized that the mutant glutamic acid in the membrane enhances FGFR3 ligand-independent activation by over-stabilizing the FGFR3 dimer due to hydrogen bonding. We previously tested this hypothesis in lipid bilayers and showed that the mutation stabilizes the isolated FGFR3 TM domain dimer by −1.3 kcal/mole. Here we further test the hypothesis, by investigating the effect of the A391E mutation on the activation of full-length FGFR3 in HEK293T cells in the absence of ligand. We find that the mutation enhances the ligandindependent activation propensity of FGFR3 by −1.7 kcal/mole. This value is similar to the stabilization of the isolated A391E mutant TM domain dimer in lipid bilayers, and is consistent with the observed strength of hydrogen bonds in membranes, thus supporting the above hypothesis.

Background: There is an ongoing controversy on the benefits of treatment protocols, including dental lasers and photodynamic therapy (PDT). The purpose of this study is to compare the local biologic effects of PDT, diode soft laser (DSL) therapy, and conventional deep scaling and root planing (SRP) in residual pockets.

Nanofibrous double-layer matrices were prepared by electrospinning technique with the bottom layer formed from PCL (poly-e-caprolactone)/PLLA (poly-l-lactic acid) nanofibers and the upper layer from PCL/Gelatin nanofibers. Bottom layer was designed to give mechanical strength to the system, whereas upper layer containing gelatin was optimized to improve the cell adhesion. Gelatin microspheres were incorporated in the middle of two layers for controlled growth factor delivery. Successful fabrication of the blend nanofibers were shown by spectroscopy. Scanning electron microscopy results demonstrated that bead-free nanofibers with uniform morphology could be obtained by 10% w/v concentrations of PCL/PLLA and PCL/Gelatin solutions. Microspheres prepared by 15% gelatin concentration and crosslinked with 7.5% glutaraldehyde solution were chosen after in vitro release studies for the incorporation to the double-layer matrices. The optimized conditions were used to prepare fibroblast growth factor-2 (FGF-2) loaded microspheres. Preliminary cell culture studies showed that the FGF-2 could be actively loaded into the microspheres and enhanced the cell attachment and proliferation. The complete system had a slow degradation rate in saline (18% weight loss in 2 months) and it could meanwhile preserve its integrity. This sandwich system prevented microsphere leakage from the scaffold, and the hydrophilic and bioactive nature of the fibers at the upper layer promoted cell attachment to the surface. PLLA/ PCL layer, on the other hand, improved the mechanical properties of the system and enabled better handling. V C 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000-000, 2013.

Previous work in our laboratory has shown that scopoletin, one of the main bioactive constituents of Erycibe obtusifolia Benth stems, exerts anti-arthritic activity in vivo partly by preventing synovial angiogenesis. The present study was performed to further investigate the anti-angiogenic potential of scopoletin, focusing on the mechanisms of action in vitro. In the aortic ring sprouting assay, scopoletin (10, 30 and 100 μM) significantly inhibited the growth of endothelial sprouts in a concentration-dependent manner. As to human umbilical vein endothelial cells (HUVECs), scopoletin could inhibit their proliferation, migration and tubule formation induced by FGF-2, especially the proliferation. It also remarkably decreased the expression of VEGF at mRNA and protein levels, and the phosphorylations of IKKα and IκB but not Akt, as well as the degradation of IκB caused by FGF-2 in HUVECs. These findings suggest that scopoletin is substantially able to attenuate FGF-2-induced angiogenesis, and it might act by directly preventing the stimulation action of FGF-2 and by indirectly decreasing the production of VEGF. Scopoletin down-regulated the VEGF expression through NF-κB rather than PI-3K/Akt signaling pathway.

It has become increasingly evident that growth cone guidance depends on the concerted actions of cytoskeletal proteins, molecular motors and cell adhesion molecules. Recent studies suggest that modulation of coupling between extracellular substrates and intracellular cytoskeletal networks via cell surface receptors is an important mechanism for regulating directed neuronal growth.

The canonical Wnt/β-catenin pathway is an essential component of multiple developmental processes. To investigate the role of this pathway in the ectoderm during facial morphogenesis, we generated conditional βcatenin mouse mutants using a novel ectoderm-specific Cre recombinase transgenic line. Our results demonstrate that ablating or stabilizing β-catenin in the embryonic ectoderm causes dramatic changes in facial morphology. There are accompanying alterations in the expression of Fgf8 and Shh, key molecules that establish a signaling center critical for facial patterning, the frontonasal ectodermal zone (FEZ). These data indicate that Wnt/β-catenin signaling within the ectoderm is critical for facial development and further suggest that this pathway is an important mechanism for generating the diverse facial shapes of vertebrates during evolution.

There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patientused transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (.75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by fibroblast growth factor (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within 15 min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale 'genetic modification' of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions.

Chemotherapy-induced alopecia represents one of the major unresolved problems of clinical oncology. The underlying molecular pathogenesis in humans is virtually unknown because of the lack of adequate research models. Therefore, we have explored whether microdissected, organ-cultured, human scalp hair follicles (HFs) in anagen VI can be exploited for dissecting and manipulating the impact of chemotherapy on human HFs. Here, we show that these organ-cultured HFs respond to a key cyclophosphamide metabolite, 4-hydroperoxycyclophosphamide (4-HC), in a manner that resembles chemotherapy-induced HF dystrophy as it occurs in vivo: namely, 4-HC induced melanin clumping and melanin incontinence, down-regulated keratinocyte proliferation, massively up-regulated apoptosis of hair matrix keratinocytes, prematurely induced catagen, and up-regulated p53. In addition, 4-HC induced DNA oxidation and the mitochondrial DNA common deletion. The organ culture system facilitated the identification of new molecular targets for chemotherapyinduced HF damage by microarray technology (eg, interleukin-8, fibroblast growth factor-18, and glypican 6). It was also used to explore candidate chemotherapy protectants, for which we used the cytoprotective cytokine keratinocyte growth factor as exemplary pilot agent. Thus, this novel system serves as a powerful yet pragmatic tool for dissecting and manipulating the im-pact of chemotherapy on the human HF. (Am J Pathol

Stem cell lines that provide a renewable and scaleable supply of central nervous system cell types would constitute an invaluable resource for basic and applied neurobiology. Here we describe the generation and long-term expansion of multiple human foetal neural stem (NS) cell lines in monolayer culture without genetic immortalization. Adherent human NS cells are propagated in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. However, they produce astrocytes, oligodendrocytes, and neurons upon exposure to appropriate differentiation factors. Single cell cloning demonstrates that human NS cells are tripotent. They retain a diploid karyotype and constant neurogenic capacity after over 100 generations. In contrast to human neurospheres, we observe no requirement for the cytokine leukaemia inhibitory factor (LIF) for continued expansion of adherent human NS cells. Human NS cells can be stably transfected to provide reporter lines and readily imaged in live monolayer cultures, creating the potential for high content genetic and chemical screens.

The development and evolution of mechanosensory cells and the vertebrate ear is reviewed with an emphasis on delineating the cellular, molecular and developmental basis of these changes. Outgroup comparisons suggests that mechanosensory cells are ancient features of multicellular organisms. Molecular evidence suggests that key genes involved in mechanosensory cell function and development are also conserved among metazoans.

Fgf10 has a prominent role in organogenesis. In the developing lung, Fgf10 is dynamically expressed in the distal mesenchyme from where it diffuses and activates its epithelial receptor, Fgfr2b, to trigger budding. Little is known about how Fgf10 expression is regulated. Here we have identified a mouse lung-specific mesenchymal cell line, MLg, which expresses endogenous Fgf10 and responds to known regulators of Fgf10 in a way that is reminiscent of the early lung. To gain insights into the mechanisms involved in the transcriptional regulation of Fgf10 in these cells, we have cloned and analyzed approximately a 4.5 kb region of the mouse Fgf10 promoter. Promoter deletion analysis and Luciferase reporter assays revealed an upstream region of the Fgf10 promoter with selective enhancer activity in the MLg, but not in the non-lung-derived cell line NIH3T3. Our data suggest that a potential lung mesenchyme-specific enhancer may exist within this region of the Fgf10 promoter.

Objective. – Bone marrow cells implantation (BMI) has been reported to efficiently improve ischemic heart disease. However, BMI strategies are generally invasive. To establish a BMI strategy for ischemic heart disease, we performed implantation of autologous cryopreserved mononuclear cells (MNCs) from bone marrow (BM) retrogradely into the myocardium via the coronary vein in pigs with acute myocardial infarction (AMI) and

The retinal pigmented epithelium (RPE) is known to be the site of the primary lesion in inherited retinal dystrophy in the Royal College of Surgeons (RCS) rat, a model for retinitis pigmentosa. Although the only functional defect so far detected in these cells is their failure to efficiently phagocytose shed photoreceptor outer segment debris, the actual cause of photoreceptor cell death is still unknown. Recently the possibility of "trophic factors" important in photoreceptor survival produced by normal RPE but not by dystrophic RPE has been suggested. Hence we decided to investigate the presence and abundance of two candidate diffusible factors, the acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), as well as their high affinity cell surface receptors (FGF-R). mRNA was isolated from primary cultures of purified normal and dystrophic RPE and analyzed by PCR amplification using specific oligonucleotide primers for aFGF and bFGF: the size and abundance of amplified fragments was similar for both cell types. Also, aFGF protein, detected by imrnunocytochemistry using specific antisera, appeared to be present in approximately equal amounts and distributed in a similar pattern. However, scatchard analysis of radio-labelled bFGF binding to primary cultures of normal and dystrophic rat RPE revealed that dystrophic RPE possess only 29% the number of surface receptors compared to congenic normal cells. Furthermore, the level of expression of FGF-R2 mRNA, but not that of FGF-R1, was significantly different. Other parameters measured (receptor affinity, profile of ligand internalization and degradation, receptor molecular weight and mitogenic activity) did not show any significant differences between normal and dystrophic RPE. The precise role of FGF-R deficiency in the etiology of the disease hence remains to be determined. but it indicates the importance of trophic factors in the normal functioning of the retina.

The aim of the study was to analyze molecular mechanisms involved in FSH and basic fibroblast growth factor (bFGF) regulation of lactate production in rat Sertoli cells. The regulation of the availability of pyruvate, which is converted to lactate, could be a mechanism utilized by hormones to ensure lactate supply to germ cells. On one hand, the regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) expression could result in increased glycolysis, while an increase in pyruvate availability may also result from a lower conversion to acetyl-CoA by negative regulation of pyruvate dehydrogenase complex (PDC) activity by phosphorylation. Sertoli cell cultures obtained from 20-day-old rats were used. Stimulation of the cultures with FSH or bFGF showed that FSH increases Pfkfb1 and Pfkfb3 expression while bFGF increases Pfkfb1 mRNA levels. Additionally, we observed that FSH-stimulated lactate production was inhibited in the presence of a PFKFB3 inhibitor, revealing the p...