Human Neonatal Foreskin Fibroblasts Research Articles

WITHDRAWN: Transdifferentiation of Human Neonatal Foreskin Fibroblasts into Keratinocyte-like Cells via Regulating the AKT/P53/WNT/LEF1 Axis

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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.

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.

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.

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.

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.

Therapeutic transdifferentiation of human fibroblasts into endothelial cells using forced expression of lineage-specific transcription factors

Transdifferentiation is the direct conversion from one somatic cell type into another desired somatic cell type. This reprogramming method offers an attractive approach for regenerative medicine. Here, we demonstrate that neonatal fibroblasts can be transdifferentiated into endothelial cells using only four endothelial transcription factors, namely, ETV2, FLI1, GATA2, and KLF4. We observed a significant up-regulation of endothelial genes including KDR, CD31, CD144, and vWF in human neonatal foreskin (BJ) fibroblasts infected with the lentiviral construct encoding the open reading frame of the four transcription factors. We observed morphological changes in BJ fibroblasts from the fibroblastic spindle shape into a more endothelial-like cobblestone structures. Fluorescence-activated cell sorting analysis revealed that ~16% of the infected cells with the lentiviral constructs encoding 4F expressed CD31. The sorted cells were allowed to expand for 2 weeks and these cells were immunostained and found to express endothelial markers CD31. The induced endothelial cells also incorporated fluorescence-labeled acetylated low-density lipoprotein and efficiently formed capillary-like networks when seeded on Matrigel. These results suggested that the induced endothelial cells were functional in vitro. Taken together, we successfully demonstrated the direct conversion of human neonatal fibroblasts into endothelial cells by transduction of lentiviral constructs encoding endothelial lineage-specific transcription factors ETV2, FLI1, GATA2, and KLF4. The directed differentiation of fibroblasts into endothelial cells may have significant utility in diseases characterized by fibrosis and loss of microvasculature.

Synthesis and Antiplasmodial Evaluation of Analogues Based on the Tricyclic Core of Thiaplakortones A-D.

Six regioisomers associated with the tricyclic core of thiaplakortones A–D have been synthesized. Reaction of 1H-indole-4,7-dione and 1-tosyl-1H-indole-4,7-dione with 2-aminoethanesulfinic acid afforded a regioisomeric series, which was subsequently deprotected and oxidized to yield the tricyclic core scaffolds present in the thiaplakortones. All compounds were fully characterized using NMR and MS data. A single crystal X-ray structure was obtained on one of the N-tosyl derivatives. All compounds were screened for in vitro antiplasmodial activity against chloroquine-sensitive (3D7) and multidrug-resistant (Dd2) Plasmodium falciparum parasite lines. Several analogues displayed potent inhibition of P. falciparum growth (IC50 < 500 nM) but only moderate selectivity for P. falciparum versus human neonatal foreskin fibroblast cells.

Gene expression of OCT4, SOX2, KLF4 and MYC (OSKM) induced pluripotent stem cells: identification for potential mechanisms.

BackgroundSomatic cells could be reprogrammed to induced pluripotent stem cells (iPS) by ectopic expression of OCT4, SOX2, KLF4 and MYC (OSKM). We aimed to gain insights into the early mechanisms underlying the induction of pluripotency.MethodsGSE28688 containing 14 gene expression profiles were downloaded from GEO, including untreated human neonatal foreskin fibroblasts (HFF1) as control, OSKM-induced HFF1 (at 24, 48, 72 h post-transduction of OSKM encoding viruses), two iPS cell lines, and two embryonic stem (ES) cell lines. Differentially expressed genes (DEGs) were screened between different cell lines and the control by Limma package in Bioconductor. KEGG pathway enrichment analysis was performed by DAVID. The STRING database was used to construct protein-protein interaction (PPI) network. Activities and regulatory networks of transcription factors (TFs) were calculated and constructed by Fast Network Component Analysis (FastNCA).ResultsCompared with untreated HFF1, 117, 347, 557, 2263 and 2307 DEGs were obtained from three point post-transduction HFF1, iPS and ES cells. Meanwhile, up-regulated DEGs in first two days of HFF1 were mainly enriched in RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling pathways. Down-regulated DEGs at 72 h were significantly enriched in focal adhesion pathway which was similar to iPS cells. Moreover, ISG15, IRF7, STAT1 and DDX58 were with higher degree in PPI networks during time series. Furthermore, the targets of six selected TFs were mainly enriched in screened DEGs.ConclusionIn this study, screened DEGs including ISG15, IRF7 and CCL5 participated in OSKM-induced pluripotency might attenuate immune response post-transduction through RLR and TLR signaling pathways.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2503890341543007.

Synthesis and antimalarial evaluation of amide and urea derivatives based on the thiaplakortone A natural product scaffold.

A series of amide (8–32, 40–45) and urea (33, 34, 36–39) analogues based on the thiaplakortone A natural product scaffold were synthesised and screened for in vitro antimalarial activity against chloroquine-sensitive (3D7) and chloroquine- and mefloquine-resistant (Dd2) Plasmodium falciparum parasite lines. Several analogues displayed potent inhibition of P. falciparum growth (IC50 <500 nM) and good selectivity for P. falciparum versus human neonatal foreskin fibroblast cells (selectivity index >100). Two of these compounds, 8 and 33, exhibited good aqueous solubility and metabolic stability, and when administered subcutaneously to mice (32 mg kg(-1)), plasma concentrations remained above 0.2 μM for at least 8 h. Both 8 and 33 were well tolerated in mice after subcutaneous administration of 32 mg kg(-1) twice daily for 4 days. Using this regimen blood stage P. berghei was suppressed by 52% for 8 and 26% for 33, relative to the vehicle control.

Structure Determination of Pentacyclic Pyridoacridine Alkaloids from the Australian Marine Organisms Ancorina geodides and Cnemidocarpa stolonifera

AbstractChemical investigation of two marine samples A. geodides and C. stolonifera led to the isolation of six pentacyclic pyridoacridine alkaloids, in which two compounds, ancorine A and cnemidine A, are new pyridoacridines. Their structures were determined by NMR spectroscopic analysis supported by quantum mechanical calculations of 13C NMR chemical shifts. This is the first study demonstrating that the pyridoacridine structure class can be assigned on the basis of quantum mechanical calculations. The isolated pyridoacridine alkaloids showed selective cytotoxicity against human prostate cancer cells (PC3) compared with human neonatal foreskin fibroblast noncancer cells (NFF).

Potent cytotoxic peptides from the Australian marine sponge Pipestela candelabra.

Two consecutive prefractionated fractions of the Australian marine sponge extract, Pipestela candelabra, were identified to be selectively active on the human prostate cancer cells (PC3) compared to the human neonatal foreskin fibroblast non-cancer cells (NFF). Twelve secondary metabolites were isolated in which four compounds are new small peptides. Their structures were characterized by spectroscopic and chemical analysis. These compounds inhibited selectively the growth of prostate cancer cells with IC50 values in the picomolar to sub-micromolar range. Structure-activity relationship of these compounds is discussed.

Paracrine regulation of fetal lung morphogenesis using human placenta-derived mesenchymal stromal cells

BackgroundRecent experimental work suggests the therapeutic role of mesenchymal stromal cells (MSC) during perinatal lung morphogenesis. The purpose of this study was to investigate the potential paracrine effects of human placenta-derived mesenchymal stromal cells (PL-MSCs) on pulmonary development. MethodsHuman MSCs were isolated from preterm placental chorion. Normal E14.5–15.5 fetal rat lungs were subsequently harvested and cultured ex vivo in the presence of conditioned media from PL-MSCs for 72 h. The lungs were analyzed morphometrically and by quantitative DNA, protein, and gene expression. Postnatal human bone marrow-derived mesenchymal stromal cells and neonatal foreskin fibroblasts (FF) were used as controls. ResultsThe MSC phenotype of the isolated placental cells was confirmed. Compared with lungs cultured in the absence of PL-MSCs, fetal lung growth was markedly accelerated on exposure to PL-MSC conditioned media as demonstrated by increases in Δlung surface area, terminal bud formation, and Δterminal bud formation. Pulmonary growth was predominantly impacted by enhanced branching morphogenesis, as shown by 73.5 ± 6.1 terminal buds after stimulation with PL-MSCs compared with 46.7 ± 5.7 terminal buds in control unconditioned media (P < 0.05). Significant differences were noted favoring PL-MSCs over FFs based on terminal bud formation and Δterminal bud formation (P < 0.05). There was significant upregulation of club cell secretory protein in lungs exposed to PL-MSCs compared with all other groups. ConclusionsThese data suggest that human PL-MSCs are potent paracrine stimulators of pulmonary morphogenesis in a fetal organ culture model. Cell therapies based on autologous or donor-derived PL-MSCs may represent a novel strategy for enhancing perinatal lung growth.

Abstract 362: Fibronectin facilitates extracellular matrix assembly and vascular morphogenesis .

Abstract Tumor growth is facilitated by oxygen and nutrients supplied by tumor blood vessels. Recently, we reported that de-cellularized extracellular matrix (ECM) deposited by co-cultures of fibroblasts with breast cancer cell lines supported vasculogenesis of endothelial cells (ECs). The most robust changes in vasculogenesis and phenotype were attributed to ECM deposited by co-cultures with metastatic MDA-MB-231 breast cancer cells. We have utilized this co-culture set up to investigate how fibronectin, a matrix protein involved in angiogenesis and breast tumor progression, influences vasculogenesis of ECs grown on de-cellularized ECM. Based on our previous study, MDA-MB-231 and human neonatal foreskin fibroblasts were grown at a 1:1 ratio for 9 days followed by de-cellularization using 50mM NH4OH and 0.4% triton-X. Following ECM de-cellularization, we found that ECM proteins collagens I and IV, laminin and tenascin-C were highly co-localized with a fibrillar fibronectin matrix and absent in regions where the fibrillar fibronectin matrix was lacking. Through use of a fibronectin inhibitor peptide, pUR4B (Dr. Jane Sottile, University of Rochester), reported to prevent fibronectin accumulation in the matrix, we were able to eliminate fibronectin assembly in the matrix. Interestingly, the deposition of collagen I and tenascin-C in the matrix could not be detected with the addition of the fibronectin inhibitor pUR4B. Examining vasculogenesis of ECs on de-cellularized ECM, we found that the presence of fibronectin was increased both intra-and extra-cellularly following vascular morphogenesis. Moreover, the vascular network appeared to degrade laminin and collagens I and IV in the de-cellularized ECM. Up-regulated expression of the gelatinases and collagenases MMPs 1,2 and 9 and MT1-MMP was observed in ECs which had formed a vascular network on ECM, a mechanism likely contributing to decreased laminin and collagens I and IV after vasculogenesis. Blocking fibronectin integrins α5β1 and αvβ3prevented vascular formation of ECs on intact, de-cellularized ECM, suggesting that fibronectin is important for EC attachment and subsequent vasculogenesis. Inhibition of fibronectin assembly into the matrix during vasculogenesis of ECs on intact, de-cellularized ECM reduced vascular formation, but did not abrogate EC attachment to the ECM, implicating a contribution from EC-derived fibronectin during vasculogenesis. In vivo experiments are being performed to elucidate the role of fibronectin in the assembly of the tumor ECM and in the growth of tumor vessels during the progression of human xenograft breast tumors. Together, these results suggest that fibronectin is a critical component of the ECM, facilitating the assembly of a number of matrix proteins and promoting vasculogenesis on de-cellularized ECM. Citation Format: Abigail Hielscher, Connie Qiu, Sharon Gerecht. Fibronectin facilitates extracellular matrix assembly and vascular morphogenesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 362. doi:10.1158/1538-7445.AM2013-362

Neuronal Differentiation of a Human Induced Pluripotent Stem Cell Line (FS-1) Derived from Newborn Foreskin Fibroblasts

Isolation of induced pluripotent stem cells (iPSCs) from fully differentiated somatic cells has revolutionized existing concepts of cell differentiation and stem cells. Importantly, iPSCs generated from somatic cells of patients can be used to model different types of human diseases. They may also serve as autologous cell sources that can be used in transplantation therapy. In this study, we investigated the neuronal properties of an iPSC line that is derived from human neonatal foreskin fibroblasts (FS-1). We initially examined the morphology and marker expression of FS-1 cells at undifferentiated stage. We then spontaneously differentiated FS-1 cells in suspension culture and examined the expression of markers representing three germ layers. We finally differentiated FS-1 cells into neuronal lineages by co-culturing them with PA6 stromal cells, and found that, under the conditions we used, they have a tendency to differentiate into more forebrain-type neurons, suggesting that FS-1 iPSC-derived neural cells will be useful to be used in cell therapy of stroke or Huntington's disease, among others. Taken together, FS-1 cells derived from human neonatal fibroblasts exhibit very similar properties with human ES cells, and can provide useful sources for cell therapy and various other applications.

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells.

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates.Electronic Supplementary MaterialSupplementary material is available for this article at 10.2478/s11658-010-0039-8 and is accessible for authorized users.

A Cyclic AMP Analog, 8-Br-cAMP, Enhances the Induction of Pluripotency in Human Fibroblast Cells

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by ectopic expression of four transcription factors. However, the efficiency of human iPS cell generation is extremely low and therefore elucidating the mechanisms underlying cellular reprogramming is of prime importance. We demonstrate that 8-Bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP) improves the reprogramming efficiency of human neonatal foreskin fibroblast (HFF1) cells transduced with the four transcription factors by 2-fold. The combination of 8-Br-cAMP and VPA synergistically increases the efficiency to 6.5-fold. The effect of 8-Br-cAMP or VPA may in part be due to the up-regulation of cytokine-related and inflammatory pathways. Remarkably, the synergistic effect of 8-Br-cAMP and VPA on cellular reprogramming may be due to the transient decrease of p53 protein during the early stages of reprogramming. However, it could also be due to additional differentially regulated genes and pathways such as the up-regulation of cytokine-related, inflammatory pathways and self-renewal supporting gene, namely cyclin-encoding CCND2, and the associated genes CCNA1 and CCNE1. Conversely, we also see the down-regulation of the p53 (CCNB2, GTSE1, SERPINE1) and cell cycle (PLK1, CCNB2) pathways. Our data demonstrates that a cyclic AMP analog, 8-Br-cAMP, enhances the efficiency of cellular reprogramming. In addition, 8-Br-cAMP and VPA have a synergistic effect on cellular reprogramming, which may be in part due to the transient down-regulation of the p53 signaling pathway during the early stages of reprogramming.

Differential regulation on human skin fibroblast by α 1 adrenergic receptor subtypes

Alpha 1 adrenoceptor (α 1-AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for α 1 adrenergic [ 3H]-prazosin revealed two saturated and specific binding sites with high or low affinity. Competitive binding assay with different antagonist subtypes, defined pharmacologically three major types of α 1-AR. The α 1-AR agonists (from 1 × 10 −10 to 1 × 10 −4 M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1 × 10 −9 M and maximal inhibition at 1 × 10 −6 M. Prazosin, abolished the stimulatory (pA 2: 9.24) and inhibitory (pA 2: 8.80) actions of α 1-AR agonists. The α 1-AR stimulation resulted in the activation of phosphoinositide turnover (InsP) via phospholipase C (PLC) involving calcium/calmodulin (CaM) and nitric oxide synthase (NOS) that correlates with the DNA synthesis increment; whereas the inhibition resulted in a decrease of cyclic AMP (cAMP) accumulation via adenylate cyclase inhibition. The potency displayed by the specific antagonists tested in binding, DNA synthesis, InsP and NOS at low agonist concentration suggests that they can be elicited by the activation of the same receptor (α 1B-AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of α 1D-AR coupled to G i protein. Non-functional α 1A-AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of α 1-AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.