Gold nanoparticle-based surface-enhanced Raman scattering for noninvasive molecular probing of embryonic stem cell differentiation

This study reports the use of gold nanoparticle-based surface-enhanced Raman scattering (SERS) for probing the differentiation of mouse embryonic stem (mES) cells, including undifferentiated single cells, embryoid bodies (EBs), and terminally differentiated cardiomyocytes. Gold nanoparticles (GNPs) were successfully delivered into all 3 mES cell differentiation stages without affecting cell viability or proliferation. Transmission electron microscopy (TEM) confirmed the localization of GNPs inside the following cell organelles: mitochondria, secondary lysosome, and endoplasmic reticulum. Using bright- and dark-field imaging, the bright scattering of GNPs and nanoaggregates in all 3 ES cell differentiation stages could be visualized. EB (an early differentiation stage) and terminally differentiated cardiomyocytes both showed SERS peaks specific to metabolic activity in the mitochondria and to protein translation (amide I, amide II, and amide III peaks). These peaks have been rarely identified in undifferentiated single ES cells. Spatiotemporal changes observed in the SERS spectra from terminally differentiated cardiomyocyte tissues revealed local and dynamic molecular interactions as well as transformations during ES cell differentiation.     

The influence of RAMP1 overexpression on CGRP‐induced osteogenic differentiation in MG‐63 cells in vitro: An experimental study

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor‐like receptor (CRLR) in MG‐63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG‐63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG‐63 cells. Real‐time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post‐induction of differentiation. The CGRP‐induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP‐mediated differentiation of MG‐63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP‐induced MG‐63 differentiation. J. Cell. Biochem. 114: 314–322, 2013. © 2012 Wiley Periodicals, Inc.     

Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA₁ to LPA₆) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA₄ and LPA₆ knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA₂ knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2 S)-OMPT (LPA₃ agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA₄ and LPA₆ knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

     

Peripheral blood progenitor cell cycle kinetics following priming with pIXY321 in patients treated with the "ICE" regimen.

PURPOSE: Treatment with hematopoietic growth factors increases the percentage of hematopoietic progenitor cells in cell cycle. Following withdrawal of certain growth factors, preclinical data suggest that there is a transient fall in the percentage of progenitor cells in cycle below the baseline, thus providing a window to administer chemotherapy with reduced risk of myelotoxicity. PATIENTS AND METHODS: Patients with histologically confirmed, previously untreated neoplasia, were treated with pIXY321 by subcutaneous injection at a dose of 375 microg/m2 twice daily (total dose 750 microg/m2/day) for seven days (days -8 to -2), followed by a two-day rest (days -1 to 0). Patients received ICE (ifosfamide, carboplatin and etoposide) on days 1 to 3. On day 4, pIXY321 was resumed until hematologic recovery. Peripheral blood was collected on days -8, -2, -1, 1, and cell cycle distribution was determined using flow cytometry. RESULTS: Twenty patients were treated in this study and received a total of 54 cycles. Partial responses were observed in three of 13 patients with non-small cell lung cancer (23 percent) and two of five patients with small cell lung cancer (40 percent). Six of 15 patients had an increased number of cells in S+G2/M on day 1 of ICE following seven days of pIXY321 and two days off (days -1 to 0). The average increase was 63 percent (range 6-253). Seven patients had a decreased number of cells in S+G2/M. The average decrease was 55 percent (range 6.3-78). There were no significant differences among the fifteen patients with regards to the observed toxicity of the chemotherapy. DISCUSSION: pIXY321 in this schedule did not consistently decrease the percentage of cycling progenitor cells in the peripheral blood. Future studies should define whether other growth factors and/or schedules can synchronize progenitor cell cycling and protect the marrow compartment from cycle specific chemotherapy.     

Reciprocal osteoblastic and osteoclastic modulation in co-cultured MG63 osteosarcoma cells and human osteoclast precursors

Osteosarcoma is usually associated with a disturbed bone metabolism. The aim of this work was to characterize the reciprocal interactions between MG63 osteosarcoma cells and osteoclasts, in a co-culture system. Co-cultures were characterized throughout 21 days for the osteoclastogenic response and the expression of osteoblastic markers. Monocultures of MG63 cells and peripheral blood mononuclear cell (PBMC) and co-cultures of PBMC + human bone marrow cells (hBMC) were also performed. Compared to PBMC cultures, co-cultures yielded significantly increased gene expression of osteoclast-related markers, tartarate-acid resistant phosphatase (TRAP) activity, TRAP-positive multinucleated cells, cells with actin rings and vitronectin receptors (VNR) and calcitonin receptors (CTR) and calcium phosphate resorbing ability. Results showed that the development of functional osteoclasts required a very low number of MG63 cells, suggesting a high osteoclastogenic-triggering capacity of this cell line. Subjacent mechanisms involved the pathways MEK and NF-kB, although with a lower relevance than that observed on PBMC monocultures or co-cultures of hBMC + PBMC; PGE2 production also had a contribution. Compared to MG63 cell monocultures, the co-culture expressed lower levels of COL1 and ALP, and higher levels of BMP-2, suggesting that PBMC also modulated the osteoblastic behavior. While M-CSF appeared to be involved in the osteoclastogenic response on the MG63 + PBMC co-cultures, RANKL does not seem to be a key player in the process. On the other hand, sphingosine-1-phosphate production might contribute to the modulation of the osteoblastic behavior. Results suggest that the reciprocal modulation between osteosarcoma and osteoclastic cells might contribute to the disturbed bone metabolism associated with bone tumors.     

RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, ∼63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.     

Expression of apoptosis and cell cycle related genes in proliferating and colcemid arrested cells of divergent lineage.

Progression through the cell cycle and redirection of cells towards programmed cell death (apoptosis) are tightly inter-related processes. However the requirement for tissue and cell type specificity suggests that a wide variety of mechanisms are used to achieve the same purpose. To examine this issue, we investigated cell cycle (c-myc, p53, p21/WAF) and apoptosis related (bcl-2, bcl-X(L), bax-alpha) gene expression in two cell lines of very different origin under proliferating and apoptosis-inducing conditions. Transformed human osteosarcoma cells (MG63) and non-transformed human kidney embryonal fibroblasts (293-0) were kept in culture in medium containing 10% FCS and growth arrest was induced by the addition of 50 ng/ml colcemid. Colcemid treatment caused growth arrest and elevated expression of cyclin B1 protein in both cell lines. Apoptosis was significantly elevated in both cell lines after colcemid exposure for at least one cell cycle. However the pattern of expression of cell cycle and apoptosis related genes, determined by RT-PCR, was quite different between the two cell lines during exponential growth and cell cycle arrest. Colcemid treatment did not markedly influence c-myc, p53 and p21/WAF expression in MG63 cells but did suppress c-myc and increase p21/WAF in 293-0 cells. Furthermore colcemid treated MG63 cells exhibited elevated bcl-2 and bax-alpha expression while similar treatment of 293-0 cells resulted in decreased bcl-X(L) and slightly increased bax-alpha expression. While growth arrest and apoptosis were induced in both MG63 and 293 cells following colcemid treatment, the differences in gene expression suggest that the mechanism by which these cells determine cell fate is quite different and may determine the sensitivity of different cell populations to anti-neoplastic drug therapy. The distinct patterns of gene expression should be carefully defined before mechanisms of apoptotic cell death are studied.     

Insulin stimulates osteoblast proliferation and differentiation through ERK and PI3K in MG‐63 cells

Insulin has been proposed to be an anabolic agent in bone, but the mechanisms underlying insulin effects on osteoblast differentiation are still not clear. To explore the mechanisms of action of insulin on osteoblast growth and differentiation, human osteoblastic cell line‐MG‐63 was used and stimulated by insulin in the presence or absence of ERK inhibitor PD98059, PI3‐K inhibitor LY294002, or inhibitor PD98059 + LY294002. The results showed that insulin positively regulated the expression of its receptor. Insulin stimulated the proliferation of MG‐63 cells in a time‐ and dose‐dependent manner and blockade of both MAPK and PI3K pathways could inhibit the cell proliferation. In addition, ALP activity, the secretion of type I collagen, OC gene expression, and mineralized nodule formation were increased in the insulin treated group, whereas these indicators were decreased after treatment with blocking agents. However, treatment with PI3‐K inhibitor LY294002 significantly reversed the down‐regulation of Runx2 expression and treatment with ERK inhibitor PD98059 remarkably decreased up‐regulation of Osx and IGF‐1 expression after insulin treatment. Therefore, the data obtained from this study suggested that insulin promoted osteoblast proliferation and differentiation through MAPK and PI3K pathway in MG‐63 cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Mechanobiology of MG63 osteoblast-like cells adaptation to static magnetic forces

The aim of this study was to explore the biophysical effects of static magnetic field on osteoblastic cells. MG63 cells were exposed to 0.25 and 0.4-T static magnetic fields (SMF). The cell cycle effects were tested by flow cytometry. The differentiation of the cells was assessed by detecting the changes in prostaglandin E2, osteocalcin, and extracellular matrix expression. Membrane fluidity was used to evaluate the alterations in the biophysical properties of cellular membranes after the SMF simulations. Our results show that SMF exposure increases prostaglandin E2 level and extracellular matrix express in MG63 cells. On the other hand, MG63 cells exposed to 0.4-T SMF exhibited a significant decrease in membrane fluidity at 8 h. Based on these findings, it appears reasonable to suggest that SMF affect osteoblastic maturation by increasing membrane rigidity and then inducing differentiation pathway.     

Aaptamine, a spongean alkaloid, activates p21 promoter in a p53-independent manner

Aaptamine, a benzonaphthyridine alkaloid was isolated from a marine sponge on the guidance of a bioassay using the transfected human osteosarcoma MG63 cells (MG63luc(+)). Aaptamine activated p21 promoter stably transfected in MG63 cells dose-dependently at the concentrations of 20-50microM. Expression of p21 and its mRNA in the wild-type MG63 cells also increased by aaptamine-treatment. Furthermore, the cell cycle of MG63 cells was arrested at the G2/M phase within 48h by the aaptamine-treatment. To analyze a responsive element of p21 promoter in the up-regulation of p21 by aaptamine, MG63 cells were transiently transfected with a series of the deleted or mutated promoter segments, and induction of luciferase with aaptamine treatment was examined by using these corresponding transfected cells. The activation of p21 promoter by aaptamine was led through acting Sp1 sites between -82 and -50bp in a p53-independent manner.     

MODULATION OF OSTEOGENIC DIFFERENTIATION IN HUMAN SKELETAL CELLS IN VITRO BY 5-AZACYTIDINE

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.     

Death receptor-3 mediates apoptosis in human osteoblasts under narrowly regulated conditions

We previously reported that a soluble form of the TNF-family receptor death receptor-3 (DR3) is expressed in osteoblasts. DR3 regulates death or differentiation in other tissues, and DR3 ligands occur in bone, but the function of DR3 in the osteoblast was unknown. We studied the expression of DR3 and the effects crosslinking antibodies to DR3 or of natural DR3 ligands in human osteoblasts. Western analysis showed that nontransformed osteoblasts and the MG63 osteosarcoma cell line produce both soluble decoy receptor and transmembrane isoforms of DR3. Cell surface labeling showed that low and high DR3-expressing osteoblast populations occur. Verification of by cloning showed a point mutation in DR3 from MG63 cells. Activation of DR3 by antibody crosslinking or with DR3 ligands caused apoptosis in osteoblasts and in MG63 cells, but only in low-density cell cultures. In dense cultures apoptosis did not occur, but nuclear factor-kappaB nuclear translocation was observed under some conditions. Crosslinking of DR3 in high-density MG63 cultures blocked expression of bone matrix elements. DR3 activation in high-density nontransformed osteoblasts had only minor effects on cell maturation. We conclude that DR3 activation can mediate apoptosis in osteoblasts. Its activity is, however, highly restricted by its soluble ligand-binding isoform and possibly also by alternate survival signals. In the presence of survival signals, DR3 may affect cell maturation although effects on differentiation were clearly seen only in the MG63 transformed cell line.     

Influence of collagen and chondroitin sulfate (CS) coatings on poly-(lactide-co-glycolide) (PLGA) on MG 63 osteoblast-like cells

Poly-(lactide-co-glycolide) (PLGA) is an FDA-approved biodegradable polymer which has been widely used as a scaffold for tissue engineering applications. Collagen has been used as a coating material for bone contact materials, but relatively little interest has focused on biomimetic coating of PLGA with extracellular matrix components such as collagen and the glycosaminoglycan chondroitin sulfate (CS). In this study, PLGA films were coated with collagen type I or collagen I with CS (collagen I/CS) to investigate the effect of CS on the behaviour of the osteoblastic cell line MG 63. Collagen I/CS coatings promoted a significant increase in cell number after 3 days (in comparison to PLGA) and after 7 days (in comparison to PLGA and collagen-coated PLGA). No influence of collagen I or collagen I/CS coatings on the spreading area after 1 day of culture was observed. However, the cells on collagen I/CS formed numerous filopodia and displayed well developed vinculin-containing focal adhesion plaques. Moreover, these cells contained a significantly higher concentration of osteocalcin, measured per mg of protein, than the cells on the pure collagen coating. Thus, it can be concluded that collagen I/CS coatings promote MG 63 cell proliferation, improve cell adhesion and enhance osteogenic cell differentiation.     

Vascular prostheses with controlled release of antibiotics Part 2. In vitro biological evaluation of vascular prostheses treated by cyclodextrins

Viability tests by the colony forming method show no toxicity for all CDs (beta-CD, gamma-CD, HPbeta-CD and HPgamma-CD) and their associated polymer. A survival rate of 100% is observed for all CDs at high concentration 400 ppm. Proliferation tests revealed a low proliferation of L132 cells on grafted vascular prostheses and untreated prostheses and good proliferation on Melinex (film form of PET). A proliferation of 17% is observed after 3 days of incubation and decrease at 4% after 6 days on prostheses. Melinex exhibits a proliferation rate as the controls. Vitality tests confirm proliferation tests and show a good vitality of cells even for low cell amounts. From these experiments it becomes obvious that the decreasing proliferation rate is not a cytotoxic effect but is due to the chemical and/or physical surface characteristics. A similar result is obtained for cell adhesion kinetics between grafted vascular prostheses and control. After 2 h adhesion, a lower adhesion is observed on untreated prostheses. Theses results were confirmed by immunochemistry and morphology tests. This cell adhesion inhibiting effect of the PET prostheses contributes to a better "survival" of vascular prostheses without secondary obstruction or stenosis.     

Response of MG63 Osteoblast Cells to Surface Modification of Ti-6Al-4V Implant Alloy by Laser Interference Lithography

The response of human osteoblast-like osteosarcoma cells (MG63) to surface modification of Ti-6Al-4V implant alloy was investigated by Laser Interference Lithography (LIL).In this work,laser interference lithography was employed to fabricate the microstructures of grooves,dots and dimples onto the surfaces of Ti-6Al-4V samples.Two and three beam LIL systems were developed to carry out the experiments.The laser treatment resulted in the increases of the roughness and the contact angle of water on the implant alloy surfaces.The proliferation of osteoblasts was analyzed by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay for the time periods of 4 hours,2 days,3 days,and 6 days.The MTT test results demonstrated that the laser treatment surfaces had a positive impact on the proliferation of osteoblast cells after 24 hours.The alloy surface morphology and the morphological changes of MG63 cells cultured on the laser textured Ti-6Al-4V surface were observed by Scanning Electron Microscope (SEM).The SEM results indicated that the osteoblast cells were aligned on grooved surfaces and they were prolonged with the structures.Enzymatic detachment results showed that the 20 μm grooved structures provided the better cell adhesion to the textured Ti-6Al-4V surfaces.     

The responses of hemopoietic precursor cells in mice to bacterial cell-wall components

The influence upon differnt cellular and humoral parameters of hemopoiesis of three structurally unrelated, highly purified bacterial cell-wall components (BCWC) was investigated. The spleens of C57BL/6 mice assayed 6 days after the injection of either lipid A or outer-membrane lipoportein, but not murein, showed a marked increase in granulocyte-macrophage, eosinophil, and megakaryocyte progenitor cell levels. The number of pluripotent hemopoietic stem cells (CFU-S) also increased in the spleens of mice treated with either lipid A or lipoprotein. Similar results were obtained following the injection of lipoprotein or lipid A into CBA or C57BL/6.nu mice. Genetically anemic W^f/W^f mice were found to have spontaneously elevated numbers of splenic progenitor cells, which increased further after the injection of lipid A. The proportions of the different splenic progenitor cell types were similar in both untreated and lipid A treated W^f/W^f mice, and in normal littermate controls. When tested in vitro, unfractionated or partially purified post-lipid A serum was found to stimulate the growth of granulocyte-macrophage progenitor cells (GM-CFC), but no detectable stimulation of eosinohphil, megakryocyte, or erythroid progenitor cells was observed. The data suggest that the rise in splenic levels of the different progenitor cells is not mediated by the corresponding types of CSF, but more likely by proliferation and differentiation of CFU-S.     

Dietary iron and the integrity of the developing rat brain: a study with the artificially-reared rat pup.

Inadequate iron nutrition is thought to affect many aspects of brain development. Iron is a component of enzyme systems in DNA synthesis, the respiratory chain, neurotransmitter and lipid metabolism. The iron content of the striatum increases post-natally, with neuronal differentiation, myelin lipid and receptor formation: Seventy percent of the iron in the brain is associated with myelin. In an attempt to dissociate the global effects of under-and/or malnutrition and to produce exclusively an iron deficiency, we have used the gastrostomy-reared rat pup fed milk substitutes which vary only in their iron content. To ensure the pups did not have adequate iron reserves at birth, dams were fed a meal diet of low iron content (3 ppm) throughout gestation. The pups were then artificially reared on milk with (43 ppm), and without added iron (2.5 ppm) from 6 up to 21 days after birth. At 21 days of age, body weights of iron deficient pups were about 90% those of control animals. At 21 days of age, the pups were weaned, then fed standard laboratory rat chow. Brain was examined at 42 days of age (for young adults) and up to 6 months of age (180 days as mature adults). Morphometric analysis of sagittal sections of the cerebellum at 21 and 63 days of age revealed a deficit in white matter formation in pups fed low-iron at 21 days of age when compared to controls. This deficit was partially recouped by age 63 days. By contrast, animals fed milk supplemented with iron showed greater definition in white matter formation than controls at 21 days of age; indicative of precocious maturation of the white matter tracts. Our findings indicate that iron deficiency, without under/mal-nutrition and other variables, does not result in extensive growth deficits in body and brain weight. However, the iron status profoundly influences the development of myelination in that the process is delayed in iron deficiency.     

Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells

Background  

Novel approaches to treat human cancer that are effective with minimal toxicity profiles are needed. We evaluated gold nanoparticles (GNPs) in human hepatocellular and pancreatic cancer cells to determine: 1) absence of intrinsic cytotoxicity of the GNPs and 2) external radiofrequency (RF) field-induced heating of intracellular GNPs to produce thermal destruction of malignant cells. GNPs (5 nm diameter) were added to 2 human cancer cell lines (Panc-1, Hep3B). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and propidium iodide-fluorescence associated cell sorting (PI-FACS) assessed cell proliferation and GNP-related cytotoxicity. Other GNP-treated cells were exposed to a 13.56 MHz RF field for 1, 2, or 5 minutes, and then incubated for 24 hours. PI-FACS measured RF-induced cytotoxicity.     

Reconstitution of a developmental clock in vitro: a critical role for astrocytes in the timing of oligodendrocyte differentiation

The rat optic nerve contains three types of macroglial cells: type 1 astrocytes first appear at embryonic day 16 (E16), oligodendrocytes at birth (E21), and type 2 astrocytes between postnatal days 7 and 10. The oligodendrocytes and type 2 astrocytes develop from a common, bipotential O-2A progenitor cell. We show here that although O-2A progenitor cells in E17 optic nerve prematurely stop dividing and differentiate into oligodendrocytes within 2 days in culture, when cultured on a monolayer of type 1 astrocytes, they continue to proliferate; moreover, the first cells differentiate into oligodendrocytes after 4 days in vitro, which is equivalent to the time that oligodendrocytes first appear in vivo. Our findings suggest that the timing of oligodendrocyte differentiation depends on an intrinsic clock in the O-2A progenitor cell that counts cell divisions that are driven by a growth factor (or factors) produced by type 1 astrocytes.