For Fibroblast Growth Factor 2 Research Articles

Effects of Growth Factors on the Differentiation of Dental Stem Cells: A Systematic Review and Meta-analysis (Part I).

To evaluate the biological interaction between dental stem cells (DSCs) and different growth factors in the field of regenerative endodontics. A systematic search was conducted in the electronic databases up to October 2021. This study followed the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. Ex vivo studies evaluating the biological interactions of DSCs and growth factors were included. The meta-analysis was performed according to the type of growth factor. The outcomes were cell viability/ proliferation and mineralization. Standardized mean differences (SMDs) were estimated using the random-effect maximum-likelihood method (P < .05). Additional analysis was performed to find any potential source of heterogeneity. Twenty articles were included in the systematic review; meta-analysis was performed for fibroblast growth factor-2 (FGF-2) and Transforming growth factor-ß1 (TGF-β1) (n = 5). Results showed that use of FGF-2 significantly increased cell proliferation on day 1-(SMD = 3.56, P = 0.00), 3-(SMD = 9.04, P = 0.00), 5-(SMD = 8.37, P = 0.01), and 7 (SMD=8.51, P=0.00) than the control group. TGF-ß1 increased alkaline phosphatase (ALP) activity more than control only on day 3 (SMD = 3.68, P = 0.02). TGF-β1 had no significant effect on cell proliferation on days 1 and 3 (P > 0.05) and on ALP activity on days 5 and 7 (P > 0.05). Meta-regression analysis showed that different covariates (i.e., cell type, passage number, and growth factors' concentration) could significantly influence the effect sizes at different follow- ups (P < 0.05). Specific growth factors might enhance the proliferation and mineralization of DSCs; however, the obtained evidence was weak. Due to the high heterogeneity among the included studies, other growth factors' inhibitory/stimulatory effects on DSCs could not be evaluated.

L-glutamine supplementation reduced morphological damage in the renal glomerulus of rats with Walker-256 tumor.

To evaluate the effects of the experimental subcutaneous Walker-256 tumor and L-glutamine supplementation, an antioxidant, on the glomerular morphology of rats. Twenty Wistar rats were distributed into four groups (n = 5): control (C); control treated with 2% L-glutamine (CG); rats with Walker-256 tumor (WT); and rats with Walker-256 tumor treated with 2% L-glutamine (WTG). Renal histological samples were submitted to periodic acid-Schiff and Masson's Trichrome staining to analyze glomerular density, morphometry of glomerular components and glomerulosclerosis; and to immunohistochemistry for fibroblast growth factor-2 (FGF-2). WT showed 50% reduction in body mass gain and cachexia index > 10%, while WTG demonstrated reduction in cachexia (p < 0.05). WT revealed reduction of glomerular density, increase in the glomerular tuft area, mesangial area, matrix in the glomerular tuft, decrease in the urinary space and synechia, and consequently higher glomerulosclerosis (p < 0.05). L-glutamine supplementation in the WTG improved glomerular density, and reduced glomerular tuft area, urinary space, mesangial area, and glomerulosclerosis compared to WT(p < 0.05). WT showed higher collagen area and FGF-2 expression compared to C (p < 0.05). WTG presented lower collagen fibers and FGF-2 expression compared to WT (p < 0.05). L-glutamine supplementation reduced cachexia and was beneficial for glomerular morphology of the rats, as well as it reduced kidney damage and improved the remaining glomeruli morphology.

Affibody-mediated controlled release of fibroblast growth factor 2

Protein therapeutics possess high target affinity and specificity, yet short residence times, which limit their broad utility. To overcome this challenge, we used affinity interactions to modulate protein release from a hydrogel delivery vehicle thereby prolonging therapeutic availability. Specifically, we designed an affibody-modified hyaluronan (HA)-based hydrogel as a delivery platform for fibroblast growth factor 2 (FGF2), a neuroprotective and neuroregenerative factor in the central nervous system (CNS). We identified a highly specific affibody binding partner with moderate affinity for FGF2 using yeast surface display and flow cytometry-based screening. Importantly, we demonstrated controlled release of bioactive FGF2 from the hydrogel by varying the ratio of affibody to protein and showed increased thermal stability of FGF2 in the presence of affibody. This versatile delivery platform will allow the distinct, simultaneous release of multiple proteins based on specific affinity interactions.

Fibroblast growth factor 2: Role in prenatal alcohol-induced stimulation of hypothalamic peptide neurons

Prenatal alcohol exposure (PAE) increases alcohol consumption and risk for alcohol use disorder. This phenomenon in rodents is suggested to involve a stimulatory effect of PAE, in female more than male offspring, on neurogenesis and density of neurons expressing neuropeptides in lateral hypothalamus (LH), including melanin-concentrating hormone (MCH), known to promote alcohol intake. With evidence suggesting a role for fibroblast growth factor 2 (FGF2) and its receptor FGFR1 in stimulating neurogenesis and alcohol drinking, we investigated here whether the FGF2-FGFR1 system is involved in the PAE-induced increase in MCH neurons, in postnatal offspring of pregnant rats given ethanol orally (embryonic day 10–15) at a low-moderate (2 g/kg/day) or high (5 g/kg/day) dose. Our results demonstrate that PAE at the low-moderate but not high dose stimulates FGF2 and FGFR1 gene expression and increases the density of MCH neurons co-expressing FGF2, only in females, but FGFR1 in both sexes. PAE induces this effect in the dorsal but not ventral area of the LH. Further analysis of FGF2 and FGFR1 transcripts within individual MCH neurons reveals an intracellular, sex-dependent effect, with PAE increasing FGF2 transcripts positively related to FGFR1 in the nucleus as well as cytoplasm of females but transcripts only in the cytoplasm of males. Peripheral injection of FGF2 itself (80 μg/kg, s.c.) in pregnant rats mimics these effects of PAE. Together, these results support the involvement of the FGF2-FGFR1 system in mediating the PAE-induced, sex dependent increase in density of MCH neurons, possibly contributing to increased alcohol consumption in the offspring.

Ubiquitin-conjugating enzyme V variant 1 enables cellular responses toward fibroblast growth factor signaling in endothelium.

Ubiquitination has been shown to provide an essential regulatory role in modulating the duration and amplitude of the signaling activity in angiogenesis. While successive enzymatic reactions mediated by three distinct types of enzymes commonly known as E1, E2, and E3 are required for ubiquitination, the role of E3s which govern the final step of ubiquitination has been extensively analyzed in angiogenesis. In contrast, the role of E2s, which determine the context and functional consequences of ubiquitination, remains largely unknown with respect to angiogenesis. To better elucidate the role of E2s in modulating endothelial behaviors during angiogenesis, we first systematically analyze the expression pattern of E2s in endothelial cells (ECs) using previously published scRNA-seq data and identify ubiquitin-conjugating enzyme variant 1 (UBE2V1), an unconventional E2 without innate catalytic activity, as one of the most abundantly expressed E2s in ECs. While ubiquitously expressed in diverse cell types, abrogation of UBE2V1significantly impairs proliferation and viability of human umbilical vein endothelial cells (HUVECs) without affecting other cell types, suggesting that UBE2V1 is likely to possess nonredundant functions in ECs. Consistent with this idea, UBE2V1 appears to be critical for morphogenesis and migration of ECs during angiogenesis. Interestingly, we find that UBE2V1 is essential for fibroblast growth factor 2 (FGF2)-induced angiogenesis, but appears to have minor effects on vascular endothelial growth factor-A-induced angiogenesis in vitro as well as in vivo. Therefore, it seems that UBE2V1 could enable ECs to distinguish two related yet distinct angiogenic cues. Mechanistically, we show that UBE2V1 promotes ubiquitination of MEK kinase 1, a key mediator of FGF2signaling, to enhance phosphorylation of extracellular signal-regulated kinase 1/2 in HUVECs. Taken together, our results illustrate the unique role of UBE2V1 as a key modulator for angiogenic behaviors in ECs.

FGF-2 release and bonding/physical properties of 4-META/MMA-based adhesive resins incorporating small FGF-2-loaded polymer particles

ObjectivesNon-biodegradable particles comprising hydroxyethyl methacrylate (HEMA) and trimethylolpropane trimethacrylate (TMPT) have been reported as useful carriers for fibroblast growth factor-2 (FGF-2). They have also been successfully incorporated into the 4-[2-(methacryloyloxy)ethoxycarbonyl]phthalic anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin to promote tissue regeneration. However, smaller particles are required to obtain restorative materials acceptable for clinical use. The aim of this study was to investigate the ability of the 4-META/MMA-TBB resin that comprises small FGF-2-loaded particles to release FGF-2 and promote cell proliferation. In addition, the bonding and physical properties of the experimental resin were evaluated. MethodsThe small particles loaded with FGF-2 were newly fabricated and incorporated into the commercial 4-META/MMA-TBB resin. Release profiles of FGF-2 from the experimental resins were assessed, and the cell proliferation cultured with the eluate was evaluated. The bonding and physical properties of the resins were evaluated using shear bond strength and three-point bending tests, and by measuring the curing time, water absorption, and water dissolution. ResultsSustained release of FGF-2 from the experimental resins for two weeks was observed, and the released FGF-2 was demonstrated to promote cell proliferation. All bonding and physical properties of the 4-META/MMA-TBB resins were found acceptable for clinical use. SignificanceThe small FGF-2-loaded particles incorporated into the 4-META/MMA-TBB resin had the same abilities to release FGF-2 and proliferate cells, as those exhibited by the conventionally sized particles. In addition, there were no adverse influences on bonding and physical properties, suggesting that the bioactive adhesive resin was acceptable for clinical use.

Fully Synthetic Heparan Sulfate-Based Neural Tissue Construct That Maintains the Undifferentiated State of Neural Stem Cells

Heparin and heparan sulfate (HS) are attractive components for constructing biomaterials due to their ability to recruit and regulate the activity of growth factors. The structural and functional heterogeneity of naturally derived heparin and HS is, however, an impediment for the preparation of biomaterials for regenerative medicine. To address this problem, we have prepared hydrogels modified by well-defined synthetic HS-derived disaccharides. Human induced pluripotent cell-derived neural stem cells (HIP-NSCs) encapsulated in a polyethylene glycol-based hydrogel modified by a pendent HS disaccharide that is a known ligand for fibroblast growth factor-2 (FGF-2) exhibited a significant increase in proliferation and self-renewal. This observation is important because evidence is emerging that undifferentiated stems cells can yield significant therapeutic benefits via their paracrine signaling mechanisms. Our data indicate that the HS disaccharide protects FGF-2, which has a very short biological half-live, from degradation. It is anticipated that, by careful selection of a synthetic HS oligosaccharide, it will be possible to control retention and release of specific growth factor, which in turn will provide control over cell fate.

Development of a novel dental resin cement incorporating FGF-2-loaded polymer particles with the ability to promote tissue regeneration

ObjectiveAiming to achieve bioactive dental resins that promote healing of surrounding tissues, we developed novel poly(2-hydroxyethyl methacrylate/trimethylolpropane trimethacrylate) (polyHEMA/TMPT) particles. These particles have been reported to be useful as a non-biodegradable carrier for fibroblast growth factor-2 (FGF-2) in vitro. The aim of this study was to evaluate the ability of an adhesive resin incorporating FGF-2-loaded polymer particles to promote tissue regeneration in vitro and in vivo. MethodsExperimental adhesive resins were prepared by incorporating FGF-2-loaded polyHEMA/TMPT particles into a 4-META/MMA-based adhesive resin, and the release profiles of FGF-2 were evaluated. The proliferation of osteoblast-like cells in the eluate from cured experimental resin was assessed. When the experimental resin was implanted into rat calvaria defects, bone regeneration was evaluated by microcomputed tomography and histological observations. ResultsSustained release of FGF-2 from the experimental resin was observed for 14 days. Eluate from the cured experimental resin significantly promoted the proliferation of osteoblast-like cells. Significantly greater bone regeneration was observed using the experimental resin compared with the control resin without FGF-2. Significance4-META/MMA-based adhesive resin incorporating FGF-2-loaded polymer particles is useful to promote tissue regeneration, suggesting that its application would be beneficial for root-end filling or the repair of fractured roots in cases with severely damaged periodontal tissue.

Application of Laser Capture Microdissection to Craniofacial Biology: Characterization of Anatomically Relevant Gene Expression in Normal and Craniosynostotic Rabbit Sutures.

Fusion of the cranial sutures is thought to depend on signaling among perisutural tissues. Mapping regional variations in gene expression would improve current models of craniosynostosis. Laser capture microdissection (LCM) isolates discrete cell populations for gene expression analysis. LCM has rarely been used in the study of mineralized tissue. This study sought to evaluate the potential use of LCM for mapping of regional gene expression within the cranial suture. Coronal sutures were isolated from 10-day-old wild-type and craniosynostotic (CS) New Zealand White rabbits, and LCM was used to isolate RNA from the sutural ligament (SL), osteogenic fronts (OF), dura mater, and periosteum. Relative expression levels for Fibroblast Growth Factor 2 (FGF2), Fibroblast Growth Factor Receptor 2 (FGFR2), Transforming Growth Factor Beta 2 (TGFβ-2), Transforming Growth Factor Beta 3 (TGFβ-3), Bone Morphogenetic Protein 2 (BMP-2), Bone Morphogenetic Protein 4 (BMP-4), and Noggin were determined using quantitative real-time PCR. A fivefold increase in TGFβ2 expression was detected in the CS SL relative to wild type, whereas 152-fold less TGFβ-3 was detected within the OF of CS animals. Noggin expression was increased by 10-fold within the CS SL, but reduced by 13-fold within the CS dura. Reduced expression of FGF2 was observed within the CS SL and dura, whereas increased expression of FGFR2 was observed within the CS SL. Reduced expression of BMP-2 was observed in the CS periosteum, and elevated expression of BMP-4 was observed in the CS SL and dura. LCM provides an effective tool for measuring regional variations in cranial suture gene expression. More precise measurements of regional gene expression with LCM may facilitate efforts to correlate gene expression with suture morphogenesis and pathophysiology.

ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation.

The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis.

Co-localization of LTBP-2 with FGF-2 in fibrotic human keloid and hypertrophic scar

We have recently shown that Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) has a single high-affinity binding site for fibroblast growth factor-2 (FGF-2) and that LTBP-2 blocks FGF-2 induced cell proliferation. Both proteins showed strong co-localisation within keloid skin from a single patient. In the current study, using confocal microscopy, we have investigated the distribution of the two proteins in normal and fibrotic skin samples including normal scar tissue, hypertrophic scars and keloids from multiple patients. Consistently, little staining for either protein was detected in normal adult skin and normal scar samples but extensive co-localisation of the two proteins was observed in multiple examples of hypertrophic scars and keloids. LTBP-2 and FGF-2 were co-localised to fine fibrous elements within the extracellular matrix identified as elastic fibres by immunostaining with anti-fibrillin-1 and anti-elastin antibodies. Furthermore, qPCR analysis of RNA samples from multiple patients confirmed dramatically increased expression of LTBP-2 and FGF-2, similar TGF-beta 1, in hypertrophic scar compared to normal skin and scar tissue. Overall the results suggest that elevated LTBP-2 may bind and sequester FGF-2 on elastic fibres in fibrotic tissues and modulate FGF-2's influence on the repair and healing processes.

HIV-Tat Protein Forms Phosphoinositide-dependent Membrane Pores Implicated in Unconventional Protein Secretion

HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel β sheet content. Intriguingly, oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2 but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was critically required for efficient membrane pore formation by HIV-Tat oligomers. These properties are strikingly similar to what has been reported previously for fibroblast growth factor 2 (FGF2), providing strong evidence of a common core mechanism of unconventional secretion shared by HIV-Tat and fibroblast growth factor 2.

FUNCTIONAL HUMAN ES-DERIVED OPCS AT LOW OXYGEN

We have established an efficient system to specify NG2/PDGF-Rα/OLIG2+ oligodendrocyte precursor cells (OPCs) from human embryonic stem cells (hESCs) at low, physiological (3%) oxygen levels. This was achieved via both forebrain and spinal cord origins, with up to 98% of cells expressing NG2. Developmental insights reveal a critical role for fibroblast growth factor 2 (FGF-2) in OLIG2 induction via ventral forebrain pathways. The OPCs mature in vitro to express O4 (46%) and subsequently become galactocerebroside (GALC), O1, and myelin basic protein-positive (MBP+) multibranching oligodendrocytes. These were cultured alongside hESC-derived neurons. The electrophysiological properties of human OPCs are similar to those of rat OPCs, with large voltage-gated sodium currents and the ability to fire action potentials. Exposure to a selective retinoid X receptor agonist increased the proportion of O4+ oligodendrocytes that express MBP from 5% to 30%. Thus, we have established a developmentally engineered system to investigate the biological properties of human OPCs and test the effects of putative remyelinating agents prior to clinical application.

1α,25(OH)₂D₃ inhibits FGF-2 release from oral squamous cell carcinoma cells through down-regulation of HBp17/FGFBP-1.

Heparin-binding protein 17/fibroblast growth factor binding protein-1 (HBp17/FGFBP-1, GenBank accession no. NP-005121) is prominent for its role as the chaperone for fibroblast growth factor-2 (FGF-2), which plays a crucial role in angiogenesis as well as promoting tumor growth. HBp17/FGFBP-1 has been proposed as a candidate biomarker for a number of cancers since it is frequently found to be elevated in many cancer types including in the tissue and cell lines of oral squamous cell carcinomas (OSCC). Previously, we reported that 1α,25(OH)2D3 suppressed the HBp17/FGFBP-1 expression in OSCC by inhibiting nuclear factor-kappaB (NF-κB) expression via vitamin D3 receptor (VDR). In this paper, to further characterize the inhibitory effect of 1α,25(OH)2D3 on HBp17/FGFBP-1, we examined the cellular localization of HBp17/FGFBP-1 protein and FGF-2 protein in the UE OSCC cell line. We found that the treatment of OSCC cells with 40-nM 1α,25(OH)2D3 suppressed HBp17/FGFBP-1 expression both in the nucleus and cytosol and reduced FGF-2 release into the culture medium. The expression of HBp17/FGFBP-1 and FGF-2 was analyzed by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). In summary, the ability of 1α,25(OH)2D3 to suppress the expression of HBp17/FGFBP-1 and FGF-2 strongly suggests a therapeutic potential as a molecular-targeted anticancer drug for FGF-dependent cancers.

Prolyl Isomerase Pin1-mediated Conformational Change and Subnuclear Focal Accumulation of Runx2 Are Crucial for Fibroblast Growth Factor 2 (FGF2)-induced Osteoblast Differentiation

Fibroblast growth factor 2 (FGF2) signaling plays a pivotal role in bone growth/differentiation through the activation of osteogenic master transcription factor Runx2, which is mediated by the ERK/MAPK-dependent phosphorylation and the p300-dependent acetylation of Runx2. In this study, we found that Pin1-dependent isomerization of Runx2 is the critical step for FGF2-induced Runx2 transactivation function. We identified four serine or threonine residues in the C-terminal domain of Runx2 that are responsible for Pin1 binding and structural modification. Confocal imaging studies indicated that FGF2 treatment strongly stimulated the focal accumulation of Pin1 in the subnuclear area, which recruited Runx2. In addition, active forms of RNA polymerase-II also colocalized in the same subnuclear compartment. Dipentamethylene thiuram monosulfide, a Pin1 inhibitor, strongly attenuated their focal accumulation as well as Runx2 transactivation activity. The Pin1-mediated structural modification of Runx2 is an indispensable step connecting phosphorylation and acetylation and, consequently, transcriptional activation of Runx2 by FGF signaling. Thus, the modulation of Pin1 activity may be a target for the regulation of bone formation.

High Yields of Oligodendrocyte Lineage Cells from Human Embryonic Stem Cells at Physiological Oxygen Tensions for Evaluation of Translational Biology

SummaryWe have established and efficient system to specify NG2/PDGF-Rα/OLIG2+ oligodendrocyte precursor cells (OPCs) from human embryonic stem cells (hESCs) at low, physiological (3%) oxygen levels. This was achieved via both forebrain and spinal cord origins, with up to 98% of cells expressing NG2. Developmental insights reveal a critical role for fibroblast growth factor 2 (FGF-2) in OLIG2 induction via ventral forebrain pathways. The OPCs mature in vitro to express O4 (46%) and subsequently become galactocerebroside (GALC), O1, and myelin basic protein-positive (MBP+) multibranching oligodendrocytes. These were cultured alongside hESC-derived neurons. The electrophysiological properties of human OPCs are similar to those of rat OPCs, with large voltage-gated sodium currents and the ability to fire action potentials. Exposure to a selective retinoid X receptor agonist increased the proportion of O4+ oligodendrocytes that express MBP from 5% to 30%. Thus, we have established a developmentally engineered system to investigate the biological properties of human OPCs and test the effects of putative remyelinating agents prior to clinical application.

Dermatan sulfotransferase Chst14/D4st1, but not chondroitin sulfotransferase Chst11/C4st1, regulates proliferation and neurogenesis of neural progenitor cells

Chondroitin sulfates (CSs) and dermatan sulfates (DSs) are enriched in the microenvironment of neural stem cells (NSCs) during development and in the adult neurogenic niche, and have been implicated in mechanisms governing neural precursor migration, proliferation and differentiation. In contrast to previous studies, in which a chondroitinaseABC-dependent unselective deglycosylation of both CSs and DSs was performed, we used chondroitin 4-O-sulfotransferase-1 (Chst11/C4st1)- and dermatan 4-O-sulfotransferase-1 (Chst14/D4st1)-deficient NSCs specific for CSs and DSs, respectively, to investigate the involvement of specific sulfation profiles of CS and DS chains, and thus the potentially distinct roles of CSs and DSs in NSC biology. In comparison to wild-type controls, deficiency for Chst14 resulted in decreased neurogenesis and diminished proliferation of NSCs accompanied by increased expression of GLAST and decreased expression of Mash-1, and an upregulation of the expression of the receptors for fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF). By contrast, deficiency in Chst11 did not influence NSC proliferation, migration or differentiation. These observations indicate for the first time that CSs and DSs play distinct roles in the self-renewal and differentiation of NSCs.

Renal collecting system growth and function depend upon embryonic γ1 laminin expression

In order to understand the functions of laminins in the renal collecting system, the Lamc1 gene was inactivated in the developing mouse ureteric bud (UB). Embryos bearing null alleles exhibited laminin deficiency prior to mesenchymal tubular induction and either failed to develop a UB with involution of the mesenchyme, or developed small kidneys with decreased proliferation and branching, delayed renal vesicle formation and postnatal emergence of a water transport deficit. Embryonic day 12.5 kidneys revealed an almost complete absence of basement membrane proteins and reduced levels of α6 integrin and FGF2. mRNA levels for fibroblast growth factor 2 (FGF2) and mediators of the GDNF/RET and WNT11 signaling pathway were also decreased. Furthermore, collecting duct cells derived from laminin-deficient kidneys and grown in collagen gels were found to proliferate and branch slowly. The laminin-deficient cells exhibited decreased activation of growth factor- and integrin-dependent pathways, whereas heparin lyase-treated and β1 integrin-null cells exhibited more selective decreases. Collectively, these data support a requirement of γ1 laminins for assembly of the collecting duct system basement membrane, in which immobilized ligands act as solid-phase agonists to promote branching morphogenesis, growth and water transport functions.

Human Cytokine Expression Profile in Various Conditioned Media for In Vitro Expansion Bone Marrow and Umbilical Cord Blood Immunophenotyped Mesenchymal Stem Cells

Introduction Bone marrow (BM) represents the major source of mesenchymal stem cells (MSCs); however, umbilical cord blood (UCB) MSCs have some advantages over BM, such as a higher differentiation capability and noninvasive collection methods. Objectives We compared antigen expression and cytokine-secretion by MSC from BM and UCB expanded with media supplemented with fetal bovine serum (FBS) or human platelet lysate (HPL). Materials and Methods We compared protocols for the expansion of hMSC starting from samples of BM or UCB by morphological analysis, calculation of population doubling numbers, and cytometry techniques using monoclonal antibodies (BD Biosciences). Using the last technique, cytokines were detected in brain homogenate supernatant fluids of MSC cultured in various media, using the Bio-Plex cytokine assay system (BD Biosciences). Results Calculating the number population doubling (PD) and colony-forming unit- 1fibroblast (CFU-F) assays showed significantly better expansion with HPL compared with a selected batch of FBS and within fewer days: PD about 5 for 10%HPL versus 25 for fibroblast growth factor2 (FGF2) medium. By flow cytometry, we observed a greater number of BM MSCs compared with UCB MSCs, as well as differences in the expression of some MSC antigens, particularly CD105, CD90, and CD31. Analysis of cytokines: FGFb, RANTES, VEGF, IL-6, IL-8, G-CSF, and GM-CSF showed only some of them to be expressed: namely, IL-6, IL-8, and VEGF. MSCs derived from UCB showed low concentrations of these cytokines compared with MSCs derived from BM.

Toward a Novel Endogenous Anxiolytic Factor, Fibroblast Growth Factor 2

Toward a Novel Endogenous Anxiolytic Factor, Fibroblast Growth Factor 2