Neonatal Foreskin Fibroblasts Research Articles

Spontaneous Efficient Differentiation of Human Pluripotent Stem Cells (hPSC) Upon Co-culture of hPSCs with Human Neonatal Foreskin Fibroblasts in 3D.

Pluripotent stem cells (PSCs) form well-formed embryoid bodies (EBs) in 3D culture. These EBs are formed in culture media lacking leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) in mouse and human PSCs, respectively. EBs are excellent technical tools for understanding developmental biology and inducing controlled differentiation in succeeding experimental steps. Technically speaking, EBs are spontaneously differentiated PSCs in 3D and exhibit all three lineages in a time-point/sequential manner. For example, ectoderm will form first, followed by mesoderm and endoderm. We have attempted to co-culture human neonatal foreskin-derived fibroblast cells in our laboratory with the PSCs first in 2D conditions followed by the induction of EBs (PSC+fibroblasts co-cultured) in low attachment dishes. We also performed spontaneous differentiation of such EBs (co-cultured with fibroblasts). We checked the presence of markers of various lineages, namely, ectoderm, mesoderm, and endoderm in days 6, 10, and 12day EBs. We have also compared the fibroblast co-cultured EBs, along with control EBs (derived from only PSCs). This co-culture system mimics the natural conditions of uterine implantation and the role of the endometrial fibroblasts in the induction of further embryonic development. The fibroblast co-cultured iPSC EBs had better roundness scores than the normal iPSC EBs and had a higher expression of lineage-specific markers.

WITHDRAWN: Transdifferentiation of Human Neonatal Foreskin Fibroblasts into Keratinocyte-like Cells via Regulating the AKT/P53/WNT/LEF1 Axis

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Malaysian and Chinese King Cobra Venom Cytotoxicity in Melanoma and Neonatal Foreskin Fibroblasts Is Mediated by Age and Geography.

Snake venoms constitute a complex, rapidly evolving trait, whose composition varies between and within populations depending on geographical location, age and preys (diets). These factors have determined the adaptive evolution for predatory success and link venom heterogeneity with prey specificity. Moreover, understanding the evolutionary drivers of animal venoms has streamlined the biodiscovery of venom-derived compounds as drug candidates in biomedicine and biotechnology. The king cobra (Ophiophagus hannah; Cantor, 1836) is distributed in diverse habitats, forming independent populations, which confer differing scale markings, including between hatchlings and adults. Furthermore, king cobra venoms possess unique cytotoxic properties that are used as a defensive trait, but their toxins may also have utility as promising anticancer-agent candidates. However, the impact of geographical distribution and age on these potential venom applications has been typically neglected. In this study, we hypothesised that ontogenetic venom variation accompanies the morphological distinction between hatchlings and adults. We used non-transformed neonatal foreskin (NFF) fibroblasts to examine and compare the variability of venom cytotoxicity between adult captive breeding pairs from Malaysian and Chinese lineages, along with that of their progeny upon hatching. In parallel, we assessed the anticancer potential of these venoms in human-melanoma-patient-derived cells (MM96L). We found that in a geographical distribution and gender-independent manner, venoms from hatchlings were significantly less cytotoxic than those from adults (NFF; ~Log EC50: 0.5-0.6 vs. 0.2-0.35 mg/mL). This is consistent with neonates occupying a semifossorial habitat, while adults inhabit more above-ground habitats and are therefore more conspicuous to potential predators. We also observed that Malaysian venoms exhibited a slightly higher cytotoxicity than those from the Chinese cobra cohorts (NFF; Log EC50: 0.1-0.3 vs. 0.3-0.4 mg/mL), which is consistent with Malaysian king cobras being more strongly aposematically marked. These variations are therefore suggestive of differential anti-predator strategies associated with the occupation of distinct niches. However, all cobra venoms were similarly cytotoxic in both melanoma cells and fibroblasts, limiting their potential medical applications in their native forms.

Colloidal silver against macrophage infections and biofilms of atypical mycobacteria.

Skin and soft tissue infection (SSTI) caused by atypical mycobacteria such as Mycobacterium abscessus and Mycobacterium avium intracellulare complex (MAIC) have increased in recent years. Current therapeutic options are limited, and hence new and better therapies are urgently required. Colloidal Silver (CS) has been identified for its widespread antibacterial properties and silver-impregnated dressings have been used for SSTIs caused by various pathogens. The efficacy of Green Synthesized Colloidal Silver (GSCS) was investigated for bacterial growth inhibition (BGI) using a microdilution method and minimum biofilm eradication concentration (MBEC) using resazurin assay and confocal scanning laser microscopy (CSLM) of M. abscessus (n = 5) and MAIC (n = 5). The antibacterial effect of GSCS against M. abscessus infected macrophages was also evaluated. The in vitro cytotoxicity of GSCS on a human keratinocyte cell line (HaCaT) and neonatal foreskin fibroblasts was analyzed by the crystal violet proliferation assay. Average BGI and MBEC of GSCS varied between 0.7 and 22 ppm for M. abscessus and MAIC. The concentration of 3 ppm reduced M. abscessus-infection in macrophages significantly. GSCS was not cytotoxic to HaCaT and neonatal foreskin fibroblast cells at concentrations < 3 ppm up to 2h exposure time. GSCS therefore, has the potential for topical application against atypical mycobacterial SSTI.

Anti-Fibrotic Potential of Tomentosenol A, a Constituent of Cerumen from the Australian Native Stingless Bee, Tetragonula carbonaria.

Bioactivity-guided fractionation was used to isolate two compounds, tomentosenol A (1) and torellianone A (2), from a cerumen extract from Tetragonula carbonaria. The anti-fibrotic activity of these compounds was examined using human cultured neonatal foreskin fibroblasts (NFF) and immortalised keratinocytes (HaCaTs). Tomentosenol A (1), inhibited NFF and HaCaT cell proliferation and prevented NFF and HaCaT scratch wound repopulation at 12.5–25 µM concentrations. These inhibitory effects were associated with reduced cell viability, determined by tetrazolium dye (MTT) and sulforhodamine B (SRB) assays. Compound 1 further inhibited transforming growth factor-β1 (TGF-β1)-stimulated, NFF-myofibroblast differentiation and soluble collagen production; and was an effective scavenger of the model oxidant, 2,2-diphenyl-1-picrylhydrazyl (DPPH·), with an EC50 value of 44.7 ± 3.1 µM. These findings reveal significant anti-fibrotic potential for cerumen-derived tomentosenol A (1).

Silibinin relieves UVB-induced apoptosis of human skin cells by inhibiting the YAP-p73 pathway.

Excessive exposure to UVB induces skin diseases. Silibinin, a flavonolignan used for treating liver diseases, is found to be effective against UVB-caused skin epidermal and dermal cell damage. In this study we investigated the molecular mechanisms underlying. Human nonmalignant immortalized keratinocyte HaCaT cells and neonatal human foreskin fibroblasts HFFs were exposed to UVB irradiation. We showed that pre-treatment with silibinin dose-dependently decreased UVB-induced apoptosis of HaCaT cells. Furthermore, we showed that silibinin treatment inhibited nuclear translocation of YAP after UVB irradiation. Molecular docking analysis and DARTS assay confirmed the direct interaction of silibinin with YAP. Silencing YAP by siRNA had no influence on the survival of HaCaT cells, whereas inhibiting classical YAP-TEAD signaling pathway by siRNA targeting TEAD1 or its pharmaceutical inhibitor verteporfin further augmented UVB-induced apoptosis, suggesting that YAP-TEAD pathway was prosurvival, which did not participate in the protective effect of silibinin. We then explored the pro-apoptotic YAP-p73 pathway. p73 was upregulated in UVB-irradiated cells, but reduced by silibinin cotreatment. The mRNA and protein levels of p73 target genes (PML, p21 and Bax) were all increased by UVB but decreased by silibinin co-treatment. Inhibiting p73 by using siRNA reduced UVB-induced apoptosis, suggesting that downregulation of p73 was responsible for the cytoprotective effect of silibinin. In HFFs, the upregulated YAP-p73 pathway by UVB irradiation was also suppressed by silibinin. Collectively, YAP-p73 pathway is a major cause of the death of UVB-exposed epidermal HaCaT cells and dermal HFFs. Silibinin directly inhibits YAP-p73 pathway, exerting the protective action on UVB-irradiated skin cells.

Pharmacological Characterisation of Pseudocerastes and Eristicophis Viper Venoms Reveal Anticancer (Melanoma) Properties and a Potentially Novel Mode of Fibrinogenolysis.

Venoms are a rich source of potential lead compounds for drug discovery, and descriptive studies of venom form the first phase of the biodiscovery process. In this study, we investigated the pharmacological potential of crude Pseudocerastes and Eristicophis snake venoms in haematological disorders and cancer treatment. We assessed their antithrombotic potential using fibrinogen thromboelastography, fibrinogen gels with and without protease inhibitors, and colourimetric fibrinolysis assays. These assays indicated that the anticoagulant properties of the venoms are likely induced by the hydrolysis of phospholipids and by selective fibrinogenolysis. Furthermore, while most fibrinogenolysis occurred by the direct activity of snake venom metalloproteases and serine proteases, modest evidence indicated that fibrinogenolytic activity may also be mediated by selective venom phospholipases and an inhibitory venom-derived serine protease. We also found that the Pseudocerastes venoms significantly reduced the viability of human melanoma (MM96L) cells by more than 80%, while it had almost no effect on the healthy neonatal foreskin fibroblasts (NFF) as determined by viability assays. The bioactive properties of these venoms suggest that they contain a number of toxins suitable for downstream pharmacological development as candidates for antithrombotic or anticancer agents.

Selective eradication of human non-small cell lung cancer cells using aptamer-decorated nanoparticles harboring a cytotoxic drug cargo

Targeted cancer therapy is currently the leading modality to enhance treatment selectivity and efficacy, as well as to minimize untoward toxicity to healthy tissues. Herein, we devised and studied nanoparticles (NPs) composed of the biocompatible block-copolymer PEG-PCL entrapping the hydrophobic chemotherapeutic drug paclitaxel (PTX), which are targeted to human non-small cell lung cancer (NSCLC) cells. To achieve selective NSCLC targeting, these NPs were decorated with single-stranded oligonucleotide-based S15 aptamers (S15-APTs), which we have recently shown to serve as efficient tumor cell targeting ligands. Prepared without using surfactants, these 15 nm PEG-PCL/PTX NPs entered NSCLC cells via clathrin-mediated endocytosis. These NPs demonstrated efficient encapsulation of PTX, high selectivity to- and potent eradication of human A549 NSCLC cells, with a remarkable half maximal inhibitory concentration (IC50) of 0.03 μM PTX. In contrast, very high IC50 values of 1.7, 4.2, 43, 87, and 980 µM PTX were obtained towards normal human bronchial epithelial BEAS2B, cervical carcinoma HeLa, colon adenocarcinoma CaCo-2, neonatal foreskin fibroblast FSE, and human embryonic kidney HEK-293 cells, respectively. These results demonstrate 2–5 orders of magnitude difference in the selective cytotoxicity towards NSCLCs, reflecting a potentially outstanding therapeutic window. Moreover, the dual utility of aptamer-decorated NPs for both drug stabilization and selective tumor targeting was studied by increasing APT concentrations during NP “decoration”. The optimal aptamer density on the surface of NPs for selective targeting, for high fluorescence diagnostic signal and for maintaining small particle size to enable endocytosis, was achieved by using 30 nM APTs during NP decoration. Collectively, our findings suggest that these APT-decorated NPs hold great preclinical promise in selective targeting and eradication of human NSCLC cells without harming normal tissues.

Generation of integration free human iPSC line SORMi002-A from neonatal foreskin fibroblasts.

Circumcision is a common surgical procedure usually performed on neonates worldwide. Skin removed after circumcision is abundant and often discarded. Neonatal foreskin fibroblasts (NFFs) can be easily isolated from it for application in biomedical research including induced pluripotent stem cell (iPSC) generation. In this study NFFs were reprogrammed to iPSCs using non-integrating episomal reprogramming plasmids. The iPSCs generated express pluripotency markers. They form embryoid bodies and differentiate in vitro into cells of endoderm, mesoderm and ectoderm. They have a normal male karyotype, are genetically identical to the parent fibroblasts and are transgene free.

Linear Peptide Analogues from Jatropha’s Orbitides Promote Migration of Human Neonatal Foreskin Fibroblasts in vitro and Collagen Deposition

Orbitides are a class of small naturally occurring cyclic peptides with structural and functional diversities. Their chemical properties make this class feasible to be obtained by solid phase peptide synthesis (SPPS). Therefore, this synthetic accessibility enables useful application and facilitates the identification of analogues, bioactivity studies, and thus, enables them to be applied to obtain peptide libraries. The aim of this work was to investigate the effects of orbitides and their linear synthetic analogues on the migration of neonatal human foreskin fibroblasts. The screening of linear peptide analogues, originally designed from natural orbitides isolated from Jatropha species, demonstrated that some molecules (linear pohlianin B and linear ribifolin) have the potential to induce fibroblast migration and collagen deposition and may thus contribute to accelerating the processes of wound healing and tissue repair. These results also demonstrate the significance of using peptides as an important tool for the discovery of simple and novel drug scaffolds.

Lupeol, a Pentacyclic Triterpene, Promotes Migration, Wound Closure, and Contractile Effect In Vitro: Possible Involvement of PI3K/Akt and p38/ERK/MAPK Pathways.

Skin wound healing is a dynamic and complex process involving several mediators at the cellular and molecular levels. Lupeol, a phytoconstituent belonging to the triterpenes class, is found in several fruit plants and medicinal plants that have been the object of study in the treatment of various diseases, including skin wounds. Various medicinal properties of lupeol have been reported in the literature, including anti-inflammatory, antioxidant, anti-diabetic, and anti-mutagenic effects. We investigated the effects of lupeol (0.1, 1, 10, and 20 μg/mL) on in vitro wound healing assays and signaling mechanisms in human neonatal foreskin keratinocytes and fibroblasts. Results showed that, at high concentrations, Lupeol reduced cell proliferation of both keratinocytes and fibroblasts, but increased in vitro wound healing in keratinocytes and promoted the contraction of dermal fibroblasts in the collagen gel matrix. This triterpene positively regulated matrix metalloproteinase (MMP)-2 and inhibited the NF-κB expression in keratinocytes, suggesting an anti-inflammatory effect. Lupeol also modulated the expression of keratin 16 according to the concentration tested. Additionally, in keratinocytes, lupeol treatment resulted in the activation of Akt, p38, and Tie-2, which are signaling proteins involved in cell proliferation and migration, angiogenesis, and tissue repair. These findings suggest that lupeol has therapeutic potential for accelerating wound healing.

Gomesin inhibits melanoma growth by manipulating key signaling cascades that control cell death and proliferation

Consistent with their diverse pharmacology, peptides derived from venomous animals have been developed as drugs to treat disorders as diverse as hypertension, diabetes and chronic pain. Melanoma has a poor prognosis due in part to its metastatic capacity, warranting further development of novel targeted therapies. This prompted us to examine the anti-melanoma activity of the spider peptides gomesin (AgGom) and a gomesin-like homolog (HiGom). AgGom and HiGom dose-dependently reduced the viability and proliferation of melanoma cells whereas it had no deleterious effects on non-transformed neonatal foreskin fibroblasts. Concordantly, gomesin-treated melanoma cells showed a reduced G0/G1 cell population. AgGom and HiGom compromised proliferation of melanoma cells via activation of the p53/p21 cell cycle check-point axis and the Hippo signaling cascade, together with attenuation of the MAP kinase pathway. We show that both gomesin peptides exhibit antitumoral activity in melanoma AVATAR-zebrafish xenograft tumors and that HiGom also reduces tumour progression in a melanoma xenograft mouse model. Taken together, our data highlight the potential of gomesin for development as a novel melanoma-targeted therapy.

GRP78-targeted in-silico virtual screening of novel anticancer agents.

Overexpression of GRP78 in a variety of cancers such as glioblastoma, leukemia, lung, prostate, breast, gastric, and colon makes it a prime target for anticancer drug development. Present study reports GRP78-based design of novel anticancer agents using in-silico methods. As a first step toward the work, the interactions between GRP78 and 15 known ligands were modeled by docking simulation. The docked complex, GRP78-13, superior to other compounds with respect to its experimental activity and energy descriptors, was deduced into a structure-based pharmacophore. This hypothesis was applied as a screening filter to Asinex and Chemdiv databases. Finally, 23 hits were tested in vitro. Among these, VH1019 and VH1011 induced a concentration-dependent strong broad antiproliferative effect in glioma (U87-MG), breast cancer (MCF-7), and prostate cancer (DU-145) cell lines as compared to nontumorigenic control, neonatal foreskin fibroblast (HFF-1). These compounds showed preferential growth inhibition of cancer cells over normal cells. The acetohydrazide derivative VH1019 was identified as a potential new chemotype for GRP78 inhibitors with an IC50 of 12.7μM in MCF-7.

Fiber-modified hexon-chimeric oncolytic adenovirus targeting cancer associated fibroblasts inhibits tumor growth in gastric carcinoma.

ObjectiveTo evaluate the effects of fiber-modified hexon-chimeric recombinant oncolytic adenovirus targeting cancer associated fibroblasts (CAFs) on the gastric CAFs and the transplantation tumor mice model of gastric carcinoma (GC).ResultsCompared with BJ cells and GPFs, the reproduction and infectivity of P9, P9-4C or GP adenoviruses were markedly higher in gastric CAFs. In addition, P9, P9-4C or GP had a significantly relatively more killing effect on gastric CAFs compared with GPFs, and have less oncolytic effect in BJ cells. Furthermore, in transplantation tumor mice model of GC we found significantly higher hexon protein expression in tumor tissues, more decreasing tumor growth and increasing inhibitory rates after treatment of P9, P9-4C or GP adenoviruses compared with Ad adenovirus.Materials and MethodsBased on the construction of the recombinant oncolytic adenoviruses pRCAdHVR48-SDF1p-Ad/EGFP (Ad, as control) with the E1A gene transcription regulated by stromal-derived factor 1 (SDF1) promoter and the hexon replaced by hexon-chimeric (H5HVR48) gene, three fiber-modified hexon-chimeric oncolytic adenovirus through the modification fiber protein by insertion of different short peptides specifically binding to fibroblast activation protein (FAP), including pRCAdHVR48-SDF1p-FAP-P9/EGFP (P9), pRCAdHVR48-SDF1p-FAP-P9-4C/EGFP (P9-4C), pRCAdHVR48-SDF1p-FAP-GP/EGFP (GP), and their corresponding replication-defective adenovirus in parallel were reconstructed. Then the reproduction, infectivity and killing ability of the four above recombinant adenoviruses were evaluated in gastric CAFs compared with gastric para-mucosa fibroblasts (GPFs) and neonatal human foreskin fibroblasts (BJ). Furthermore, transplantation tumor mice model of GC was established, and then treated by the four above recombinant adenoviruses. Tumor size and tumor growth inhibitory rates were calculated, and histomorphology by HE staining and hexon expressions by immunohistochemistry were evaluated in tumor tissues.ConclusionsThe fiber-modified hexon-chimeric recombinant oncolytic adenovirus targeting CAFs can relatively specifically kill gastric CAFs and inhibit GC cells growth in vivo.

Methanolic Extract of Costus pictus D. DON Induces Cytotoxicity in Liver Hepatocellular Carcinoma Cells Mediated by Histone Deacetylase Inhibition.

Background:Leaves of Costus pictus D. Don, (insulin plant) are used as dietary supplement for the treatment of diabetes.Objective:The antidiabetic activity of this plant is well documented, but its activity on different cell types and mechanism remains unknown. Thus, the present study evaluates the cytotoxicity of C. pictus methanolic extract (CPME) against various cancer and normal cells.Materials and Methods:Dried leaves of C. pictus were extracted using methanol and were subjected to histone deacetylase (HDAC) inhibition and toxicity studies.Results:The CPME displayed a selective toxicity toward tested cancer cells in a dose- and time-dependent manner. CPME exhibited significant cytotoxicity on Liver hepatocellular carcinoma cells (Hep G2) (half maximal inhibitory concentration IC50 = 6.7 mg/ml). Since CPME demonstrates both antidiabetic, anticancer activity, and HDAC enzyme play a detrimental role in both the complications, we have evaluated the CPME-induced HDAC regulation on Hep G2 cell lines. CPME showed a notable HDAC inhibition (55%). Furthermore, CPME did not show any genotoxicity or membrane instability at the tested concentrations.Conclusion:CPME demonstrates selective cytotoxicity toward tumor cells at a lower concentration through HDAC inhibition.SUMMARY C. pictus is used as munching supplementary food for the treatment of diabetesCPME selectively induces cytotoxicity in cancer cells leaving normal cells healthySelective toxicity to cancer cells are attributed by the inhibition of HDAC enzymeCPME did not show any genotoxicity and membrane instability in blood cellsCPME could be potential source of HDAC inhibitor. Abbreviations used: A549: Human lung carcinoma cells, CPME: Costus pictus methanolic extract, DMEM: Dulbecco's modified eagle's medium, DMSO: Dimethyl sulfoxide, ELISA: Enzyme-linked immunosorbent assay, 5-FU: 5-Fluorouracil, Hep G2: Liver hepatocellular carcinoma cells, HEK-293: Human embryonic kidney cells, Hela: Human cervical carcinoma cells, HT-29: Human colorectal adenocarcinoma cells, HDAC: Histone deacetylase, IC50: Half maximal inhibitory concentration, MCF-7: Human breast adenocarcinoma cells, MDA-MB-435S: Human breast cancer cells, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, NFF: Neonatal foreskin fibroblasts, PHA: Phytohemagglutinin, PBS: Phosphate buffer saline, RPMI-1640: Roswell Park Memorial Institute Medium.

Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth

Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9–370 nM), with belinostat, panobinostat and vorinostat having 8–45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 μM) or S. mansoni schistosomula (IC50 > 10 μM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 ∼10 μM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4–7 and 4–10 after infection (P < 0.05), respectively.

Generation of human iPS cell line SKiPSc1 from healthy Human Neonatal Foreskin Fibroblast cells

The SKiPSc1 induced pluripotent stem (iPS) cell line was generated from Human Neonatal Foreskin Fibroblasts (HNFFs) obtained from a healthy donor infant that were reprogrammed using non-integrating Sendai viral vectors expressing Oct3/4, Sox2, c-Myc, and Klf4.

Nucleophilic substitution reactions of [(η5-Cp*)Ru(η6-C6H5CO2H)]+: Synthesis, characterization and cytotoxicity of organoruthenium ester and amide complexes

This article outlines the synthesis of an electrophilic organoruthenium carboxylic acid of the structure [(η5-Cp*)Ru(η6-C6H5CO2H)]+ and explores the behavior of this molecule under a variety of nucleophilic substitution conditions using a range of oxygen and nitrogen based nucleophiles including alcohols, primary and secondary amines and aromatic sulfonamides. The resulting organoruthenium ester [(η5-Cp*)Ru(η6-C6H5COOR)]+ and amide [(η5-Cp*)Ru(η6-C6H5CONHR)]+ or [(η5-Cp*)Ru(η6-C6H5CONR)]+ complexes are additionally reported. All prepared complexes have been fully characterized using Fourier-transform IR and NMR spectroscopy and electrospray mass spectrometry with single-crystal X-ray structural determinations reported for three complexes: 3[(η5-Cp*)Ru(η6-C6H5CO2H)]B(C6H5)4.[(η5-Cp*)Ru(η6-C6H5CO2)].H2O, [(η5-Cp*)Ru(η6-C6H5CONHCH2Ph)]PF6 and [(η5-Cp*)Ru(η6-C6H5CONHSO2C6H4COMe)]PF6. Complexes were also evaluated for in vitro cytotoxic activity against the MCF7 (hormone-dependant breast cancer), MDA-MD-231 (hormone-independent breast cancer), MM96L (human melanoma) tumourigenic cell lines and the normal NFF (neonatal foreskin fibroblasts) human cell line.

344. En Route to Non-Viral Genetic Engineering: Kinetics of DNA Uptake and Transgene Expression Following Repeated Transfection with Multiple Episomal Plasmids in Human Primary Fibroblast

Non-viral approach to cellular reprogramming or genome editing of mammalian cells often involve co-delivery of multiple types of nucleic acid molecules. Whether the method involves co-transfection with multiple episomal plasmid DNAs, a mixture of mRNA/gRNA oligomers or a combination of both DNA and RNA molecules, the efficacy of these modular approaches hinges upon the efficiency at which all the molecular factors are co-delivered and co-expressed at their optimal stoichiometric ratios. A significant rate-limiting step thus lies in the lack of an efficient co-transfection, in which a subset of the transfected population may be devoid of one, two, or more of the factors required, but the proportion at which these event occur is not clear. Further, because non-viral transfection is a transient process, repeated transfection is often employed to sustain transgene expression, as is often the case in non-viral episomal based cellular reprogramming. However, the effectiveness of these subsequent rounds of transfection in maintaining transgene expression among the transfected cells is presently unclear. In this study, we examined the kinetics of DNA uptake and transgene expression following cationic reagent-mediated non-viral transfection of primary human neonatal foreskin fibroblast with multiple episomal plasmid DNAs. To measure the level of DNA uptake and transgene expression following co-transfection, we employed two fluorescent reporter gene plasmids (eGFP and mtagBFP2) and covalently labeled them with either FITC or Cy5. Cells were transfected using XtremeGENE HP with either one or both of the labeled plasmids. More than 90% of the cells transfected were positive for either FITC or Cy5 plasmid DNA. When the labeled plasmids were mixed at 1:1 ratio or diluted with unlabeled DNA, there was a proportional decrease in the level of fluorescence in the respective fluorescent channel consistent with the relative input ratios between the two labeled DNAs. We also saw a strong correlation in the co-expression of both reporter genes following co-transfection with the majority of the transfected cells dually expressing both GFP and BFP (64%), however, there were subsets of singly transfected cells that express only GFP (8%) or BFP (27%). We next looked at the effectiveness of repeated transfection in enhancing/sustaining transgene expression in transfected cells. In order to distinguish the population of repeatedly transfected ones from new transfection events in subsequent rounds of transfection, we employed the same two reporter gene set-up (eGFP/mTagBFP2) in which cells were transfected with GFP first, followed by a second transfection with BFP a few days later; cells that were repeatedly transfected would then express BFP in addition to GFP. Our result showed that, while the overall transfection efficiency was higher with repeated transfection, to our surprise, the majority of the transfected cells remained GFP+; only a subset of the 40% of the transfected cells were positive for both GFP and BFP (~9% total), with the remaining attributed to newly transfected cells expressing only BFP. Taken together, these data suggest that while cationic reagent can efficiently co-deliver and co-transfect multiple episomal factors, the effectiveness of this modular approach in non-viral genetic engineering may be limited in cases where sustained expression is required due to the majority of the transfected cells being refractory to subsequent rounds of transfection. Addressing these rate-limiting steps should help increase the utility and efficiency of the system.

Apoptotic transition of senescent cells accompanied with mitochondrial hyper-function.

Defined as stable cell-cycle arrest, cellular senescence plays an important role in diverse biological processes including tumorigenesis, organismal aging, and embryonic development. Although increasing evidence has documented the metabolic changes in senescent cells, mitochondrial function and its potential contribution to the fate of senescent cells remain largely unknown. Here, using two in vitro models of cellular senescence induced by doxorubicin treatment and prolonged passaging of neonatal human foreskin fibroblasts, we report that senescent cells exhibited high ROS level and augmented glucose metabolic rate concomitant with both morphological and quantitative changes of mitochondria. Furthermore, mitochondrial membrane potential depolarized at late stage of senescent cells which eventually led to apoptosis. Our study reveals that mitochondrial hyper-function contributes to the implementation of cellular senescence and we propose a model in which the mitochondrion acts as the key player in promoting fate-determination in senescent cells.