Adult Human Dermal Fibroblasts Research Articles

Synthesis and structure-property relationship of lipoic acid-containing porphyrin derivatives for mitochondria-targeting applications

In this research, synthesis and characterization of a novel unsymmetric water-soluble porphyrin having three N-methylpyridinium and one lipoic acid-based meso-substituents were described. Effects of incorporation between a lipoic acid unit and a pyridinium-substituted porphyrin macrocycle, and the presence of Mn(II) as a metal center of the porphyrin ring on cytotoxicity of the compounds in Human keratinocyte (HaCaT) and Human dermal fibroblast, adult (HDFa) cells, and on mitochondria-targeting activity in the HaCaT cells were investigated. The biological studies revealed that the target lipoic acid-containing Mn(II)-porphyrin had low cytotoxicity with IC50 values bigger than 115 µM and of 87 µM in the HaCaT and HDFa cells, respectively. Moreover, this target compound also exhibited satisfactory mitochondria-targeting activity in the HaCaT cells.

Differential modulation of Bax/Bcl-2 ratio and onset of caspase-3/7 activation induced by derivatives of Justicidin B in human melanoma cells A375.

Diphyllin and its derivatives are well known cytotoxic natural products structurally related to the anti-cancer drug podophyllotoxin. We here study their structure-activity relationship upon human melanoma cells for first time. To this end, human melanoma A375 cells were incubated with Justicidin B and its 4-methoxylated or 4-glycosylated derivatives to evaluate their selective cytotoxicity and study their effects on cell cycle distribution, caspase activation, apoptosis induction using Annexin V-FITC/PI staining, cell morphology and western blot analysis. Diphyllin methyl ether (GI50 = 3.66 μM) and Justicidin B (GI50 = 1.70 μM) caused an elevation of both early and late apoptosis populations whereas Diphyllin apioside (GI50 = 0.84 μM) and its acetate (GI50= 0.39 μM) enhanced late apoptosis population only (Annexin V-positive/PI-positive). All induced cell cycle arrest at S phase and classic morphological indicators of apoptosis (blebbing, apoptotic bodies, and nuclear fragmentation) accompanied with an elevation of cells with low DNA content (sub-G1). All compounds increased the Bax/Bcl-2 ratio by enhancing Bax expression which evidences the involvement of the mitochondria (intrinsic pathway) in the apoptotic process. These caspase-3/7 results evidence that 4-methoxylation or 4-O-glycosylation of Justicidin B -a caspase independent mitochondrial apoptosis-inducer- triggers caspase-3/7 activation at different times (24h vs. 48h, respectively). Interestingly, the methoxylation causes attenuation of Bcl-2 protein expression contrarily to Diphyllin methyl ether or the O-glycosylated derivatives. Finally, the compounds exhibited significantly less toxicity when tested in adult human dermal fibroblasts and their GI50 in melanoma Sk-Mel-5 cells was not influenced by MDR1/Pgp inhibitors. This study may inform the synthesis of future Diphyllin derivatives with different apoptosis mechanism of action towards human melanoma cells.

Tissue-protective effects of French maritime pine bark (Pycnogenol) on glutamate-induced cytotoxicity in adult human dermal fibroblasts

Tissue-protective effects of French maritime pine bark (Pycnogenol) on glutamate-induced cytotoxicity in adult human dermal fibroblasts

Synthesis and Evaluation of a Sodium Alginate-4-Aminosalicylic Acid Based Microporous Hydrogel for Potential Viscosupplementation for Joint Injuries and Arthritis-Induced Conditions.

A microporous hydrogel was developed using sodium alginate (alg) and 4-aminosalicylic acid (4-ASA). The synthesized hydrogel was characterized using various analytical techniques such as Fourier transform infrared spectroscopy (FTIR), Carbon-13 nuclear magnetic resonance (13C-NMR), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). Additonal carboxyl and hydroxyl functional groups of 4-ASA provided significant lubrication and stress-triggered sol-gel transition to the conjugated hydrogel. In addition, cytotoxicity analysis was undertaken on the conjugated hydrogel using human dermal fibroblast-adult (HDFa) cells, displaying non-toxic characteristics. Drug release profiles displaying 49.6% in the first 8 h and 97.5% within 72 h, similar to the native polymer (42.8% in first 8 h and 90.1% within 72 h). Under applied external stimuli, the modified hydrogel displayed significant gelling properties and structure deformation/recovery behaviour, confirmed using rheological evaluation (viscosity and thixotropic area of 8095.3 mPas and 26.23%, respectively). The modified hydrogel, thus, offers great possibility for designing smart synovial fluids as a biomimetic aqueous lubricant for joint-related injuries and arthritis-induced conditions. In addtion, the combination of thixotropy, non-toxicity, and drug release capabilities enables potential viscosupplementation for clinical application.

Inhibiting function of human fetal dermal mesenchymal stem cells on bioactivities of keloid fibroblasts

BackgroundKeloid is one kind of benign skin disease caused by hyperplasia of fibroblasts and collagen fibrils. It is refractory due to the lack of an effective treatment at present, which puts pressure on seeking a new therapeutic regimen. Mesenchymal stem cells (MSCs) from fetal skin are considered to play a crucial role in scarless healing. Nevertheless, the efficacy of them in keloid disorders remains poorly understood.MethodsKeloid fibroblasts (KFs), human adult dermal fibroblasts (ADFs), and human fetal dermal mesenchymal stem cells (FDMSCs) were isolated to single cells and cultured in Dulbecco’s modified Eagle’s medium (DMEM). ADFs and FDMSCs were used to generate ADF-conditioned medium (A-CM) and FDMSC-conditioned medium (F-CM). The effects of A-CM and F-CM on KFs were tested using MTT assay, BrdU assay, TUNEL assay, quantitative polymerase chain reaction, Western blot, and annexin V-FITC/PI binding assay,.ResultsFDMSCs inhibited the bioactivity of KFs, downregulated the expression of the antiapoptotic protein BCL-2, and upregulated the expression of the proapoptotic protein BAX of KFs by secreting some soluble substances, thus accelerating the apoptosis of KFs.ConclusionF-CM induces apoptosis of KFs, providing a novel treatment strategy for keloid disorders.

Effects of hypoxia on the phenotype transformation of human dermal fibroblasts to myofibroblasts and the mechanism

Objective: To investigate the effects of hypoxia on the phenotype transformation of human dermal fibroblasts to myofibroblasts and the mechanism. Methods: The third passage of healthy adult human dermal fibroblasts in logarithmic phase were cultured in DMEM medium containing 10% fetal bovine serum for the following five experiments. (1) In experiments 1, 2, and 3, cells were divided into normoxia group and hypoxia group according to the random number table, with 10 dishes in each group. Cells of normoxia group were cultured in incubator containing 21% oxygen, while those of hypoxia group with 1% oxygen. At post culture hour (PCH) 0 and 48, 5 dishes of cells were collected from each group, respectively. mRNA expressions of markers of myofibroblasts including alpha smooth muscle actin (α-SMA), type Ⅰ collagen, and type Ⅲ collagen of cells were determined with real time fluorescent quantitative reverse transcription polymerase chain reaction in experiment 1. Protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen of cells were determined with Western blotting in experiment 2. The protein expression of nuclear factor-kappa B (NF-κB) of cells was determined with Western blotting in experiment 3. (2) In experiment 4, cells were divided into normoxia group, hypoxia group, and hypoxia+ pyrrolidine dithiocarbamate (PDTC) group according to the random number table, with 5 dishes in each group. Cells in the former two groups were treated the same as those in experiment 1. Cells in hypoxia+ PDTC group were treated the same as those in hypoxia group plus adding 4 mL PDTC with a final molarity of 10 μmol/L in the culture medium. At PCH 48, the protein expression of NF-κB of cells was determined with Western blotting. (3) In experiment 5, cells were divided into normoxia group, hypoxia group, hypoxia+ PDTC group, and normoxia+ PDTC group according to the random number table, with 5 dishes in each group. Cells in the former three groups were treated the same as those in experiment 4. Cells in normoxia+ PDTC group were treated the same as those in normoxia group plus adding 4 mL PDTC with a final molarity of 10 μmol/L in the culture medium. At PCH 48, protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen of cells were determined with Western blotting. Data were processed with analysis of variance of factorial design, one-way analysis of variance, and LSD-t test. Results: (1) Compared with those of normoxia group at corresponding time point, mRNA expressions and protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen and the protein expression of NF-κB in fibroblasts of hypoxia group were not changed obviously at PCH 0 (with t values from -1.21 to 2.04, P values above 0.05), while mRNA expressions and protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen and the protein expression of NF-κB significantly increased at PCH 48 (with t values from -12.57 to -3.44, P values below 0.01). (2) At PCH 48, the protein expression of NF-κB in fibroblasts of hypoxia group was 0.83±0.12, significantly higher than that of normoxia group (0.17±0.06, t=-16.96, P<0.001). The protein expression of NF-κB in fibroblasts of hypoxia+ PDTC group was 0.31±0.08, significantly lower than that of hypoxia group (t=12.73, P<0.001). (3) At PCH 48, protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in fibroblasts of hypoxia group were 0.73±0.09, 1.25±0.10, and 1.16±0.07, respectively, significantly higher than those of normoxia group (0.14±0.06, 0.87±0.08, and 0.77±0.13, respectively, with t values from 9.24 to 11.24, P values below 0.001). The protein expression of α-SMA in fibroblasts of normoxia+ PDTC group was 0.24±0.07, significantly higher than that of normoxia group (t=4.22, P<0.01). Protein expressions of type Ⅰ collagen and type Ⅲ collagen in fibroblasts of normoxia+ PDTC group were 0.25±0.06 and 0.32±0.11, respectively, significantly lower than those of normoxia group (with t values respectively -4.31 and -3.88, P values below 0.01). Protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in fibroblasts of hypoxia+ PDTC group were 0.09±0.08, 0.38±0.12, and 0.47±0.08, respectively, significantly lower than those of hypoxia group (with t values from 11.78 to 22.98, P values below 0.001). Conclusions: Hypoxia can significantly up-regulate the expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in human dermal fibroblasts, which may promote the phenotype transformation of fibroblasts to myofibroblasts, and this is likely to be associated with the activation of NF-κB signal pathway.

SOX17 Regulates Conversion of Human Fibroblasts Into Endothelial Cells and Erythroblasts by Dedifferentiation Into CD34+ Progenitor Cells.

The mechanisms underlying the dedifferentiation and lineage conversion of adult human fibroblasts into functional endothelial cells have not yet been fully defined. Furthermore, it is not known whether fibroblast dedifferentiation recapitulates the generation of multipotent progenitors during embryonic development, which give rise to endothelial and hematopoietic cell lineages. Here we established the role of the developmental transcription factor SOX17 in regulating the bilineage conversion of fibroblasts by the generation of intermediate progenitors. CD34+ progenitors were generated after the dedifferentiation of human adult dermal fibroblasts by overexpression of pluripotency transcription factors. Sorted CD34+ cells were transdifferentiated into induced endothelial cells and induced erythroblasts using lineage-specific growth factors. The therapeutic potential of the generated cells was assessed in an experimental model of myocardial infarction. Induced endothelial cells expressed specific endothelial cell surface markers and also exhibited the capacity for cell proliferation and neovascularization. Induced erythroblasts expressed erythroid surface markers and formed erythroid colonies. Endothelial lineage conversion was dependent on the upregulation of the developmental transcription factor SOX17, whereas suppression of SOX17 instead directed the cells toward an erythroid fate. Implantation of these human bipotential CD34+ progenitors into nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice resulted in the formation of microvessels derived from human fibroblasts perfused with mouse and human erythrocytes. Endothelial cells generated from human fibroblasts also showed upregulation of telomerase. Cell implantation markedly improved vascularity and cardiac function after myocardial infarction without any evidence of teratoma formation. Dedifferentiation of fibroblasts to intermediate CD34+ progenitors gives rise to endothelial cells and erythroblasts in a SOX17-dependent manner. These findings identify the intermediate CD34+ progenitor state as a critical bifurcation point, which can be tuned to generate functional blood vessels or erythrocytes and salvage ischemic tissue.

An ultra-sensitive biophysical risk assessment of light effect on skin cells.

The aim of this study was to analyze photo-dynamic and photo-pathology changes of different color light radiations on human adult skin cells. We used a real-time biophysical and biomechanics monitoring system for light-induced cellular changes in an in vitro model to find mechanisms of the initial and continuous degenerative process. Cells were exposed to intermittent, mild and intense (1-180 min) light with On/Off cycles, using blue, green, red and white light. Cellular ultra-structural changes, damages, and ECM impair function were evaluated by up/down-regulation of biophysical, biomechanical and biochemical properties. All cells exposed to different color light radiation showed significant changes in a time-dependent manner. Particularly, cell growth, stiffness, roughness, cytoskeletal integrity and ECM proteins of the human dermal fibroblasts-adult (HDF-a) cells showed highest alteration, followed by human epidermal keratinocytes-adult (HEK-a) cells and human epidermal melanocytes-adult (HEM-a) cells. Such changes might impede the normal cellular functions. Overall, the obtained results identify a new insight that may contribute to premature aging, and causes it to look aged in younger people. Moreover, these results advance our understanding of the different color light-induced degenerative process and help the development of new therapeutic strategies.

Chromatin-modifying agents convert fibroblasts to OCT4+ and VEGFR-2+ capillary tube-forming cells.

RationaleThe human epigenome is plastic. The goal of this study was to address if fibroblast cells can be epigenetically modified to promote neovessel formation.Methods and resultsHere, we used highly abundant human adult dermal fibroblast cells (hADFCs) that were treated with the chromatin-modifying agents 5-aza-2'-deoxycytidine and trichostatin A, and subsequently subjected to differentiation by activating Wnt signaling. Our results show that these epigenetically modified hADFCs increasingly expressed β-catenin, pluripotency factor octamer-binding transcription factor-4 (OCT4, also known as POU5F1), and endothelial cell (EC) marker called vascular endothelial growth factor receptor-2 (VEGFR-2, also known as Fetal Liver Kinase-1). In microscopic analysis, β-catenin localized to cell-cell contact points, while OCT4 was found to be localized primarily to the nucleus of these cells. Furthermore, in a chromatin immunoprecipitation experiment, OCT4 bound to the VEGFR-2/FLK1 promoter. Finally, these modified hADFCs also transduced Wnt signaling. Importantly, on a two-dimensional (2D) gel substrate, a subset of the converted cells formed vascular network-like structures in the presence of VEGF.ConclusionChromatin-modifying agents converted hADFCs to OCT4+ and VEGFR-2+ capillary tube-forming cells in a 2D matrix in VEGF-dependent manner.

Differential decrease in soluble and DNA-bound telomerase in senescent human fibroblasts.

The role of telomere shortening in the induction of replicative cellular senescence (CS) is well known and as a result, the involvement of telomerase and in particular its catalytic subunit, the telomerase reverse transcriptase (TERT) in CS has also been investigated. However, the majority of studies were conducted on cells that generally express high levels of TERT (cancer and immortalized cells) while the role of telomerase in CS in normal cells has been investigated to a much lesser extent. In particular, it was reported that active TERT is expressed in early passages of cultured human keratinocytes but rapidly diminished towards entry to CS, without telomere shortening. With the putative importance of TERT/telomerase in CS and the aging process in mind, we investigated the expression of TERT and telomerase activity in primary cultures of adult human dermal fibroblasts (HDFs) in the in vitro model of replicative CS. We found that (i) HDFs expressed active TERT; (ii) TERT protein levels and telomerase activity were markedly decreased in senescent HDFs; and (iii) the reduction of TERT in the soluble fraction was more pronounced than in the DNA-bound one. The results suggest the importance of the non-canonical (telomere-unrelated) functions of TERT in cellular senescence.

Adult human dermal fibroblasts exposed to nanosecond electrical pulses exhibit genetic biomarkers of mechanical stress

BackgroundExposure of cells to very short (<1µs) electric pulses in the megavolt/meter range have been shown to cause a multitude of effects, both physical and molecular in nature. Physically, nanosecond electrical pulses (nsEP) can cause disruption of the plasma membrane, cellular swelling, shrinking and blebbing. Molecularly, nsEP have been shown to activate signaling pathways, produce oxidative stress, stimulate hormone secretion and induce both apoptotic and necrotic death. We hypothesize that studying the genetic response of primary human dermal fibroblasts exposed to nsEP, will gain insight into the molecular mechanism(s) either activated directly by nsEP, or indirectly through electrophysiology interactions. MethodsMicroarray analysis in conjunction with quantitative real time polymerase chain reaction (qRT-PCR) was used to screen and validate genes selectively upregulated in response to nsEP exposure. ResultsExpression profiles of 486 genes were found to be significantly changed by nsEP exposure. 50% of the top 20 responding genes coded for proteins located in two distinct cellular locations, the plasma membrane and the nucleus. Further analysis of five of the top 20 upregulated genes indicated that the HDFa cells’ response to nsEP exposure included many elements of a mechanical stress response. ConclusionsWe found that several genes, some of which are mechanosensitive, were selectively upregulated due to nsEP exposure. This genetic response appears to be a primary response to the stimuli and not a secondary response to cellular swelling. General significanceThis work provides strong evidence that cells exposed to nsEP interpret the insult as a mechanical stress.

Replicative Senescence in Human Fibroblasts Is Delayed by Hydrogen Sulfide in a NAMPT/SIRT1 Dependent Manner.

Recent evidence suggests that hydrogen sulfide (H2S) has cytoprotective and anti-aging effects. However, the mechanisms for such properties are not fully understood. Here, we show that the expression of the main H2S producing enzyme, CBS, and production of H2S are coordinately diminished in replicative senescent adult human dermal fibroblasts. The reduced production of H2S falls within the same time-frame that the hallmarks of replicative senescence appear including accumulation of SA–β-Gal, enhanced expression of p16, p21, and RRM2B while the expression of RRM2, hTERT, SIRT1, NAMPT, and NAD/NADH ratio all fall. Exogenous H2S increases the expression of hTERT, NAMPT, SIRT1 and NAD/NADH ratio in treated cells. Moreover, H2S safeguards the expression of hTERT in a NAMPT and SIRT1 dependent manner and delays the onset of replicative senescence as evidenced by reduced accumulation of age associated SA–β-Gal and cessation of proliferation. Postponement of loss of cell proliferative capacity without risk of mutagenesis shows implications for use of H2S in delaying the adverse effects of senescence in organisms.

Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts

To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

Human amniotic epithelial cells attenuate TGF-β1-induced human dermal fibroblast transformation to myofibroblasts via TGF-β1/Smad3 pathway

Background aimsKeloids are raised dermal scars that extend beyond the boundaries of the initial injury. To date, there is no treatment to erase scars completely in humans. Growing evidence has shown that the human amniotic epithelial cells have anti-fibrotic properties and can reduce the fibrosis of lung and liver. However, it is unknown whether and how they can influence human keloids. The aim of this study was to investigate whether factors secreted by human amniotic epithelial cells have anti-fibrotic effects on human keloids and to clarify the potential transduction mechanism. MethodsHuman amniotic epithelial cells were isolated and identified both with flow cytometry and immunofluorescent. The α-smooth muscle actin, collagen-I and III gene and protein expression of transforming growth factor (TGF)-β1-treated human adult dermal fibroblasts were partly abolished by human amniotic epithelial cells conditioned medium through stimulating the expression of matrix metalloproteinase (MMP). Furthermore, human amniotic epithelial cells conditioned medium effectively attenuated nuclear import of the Smad2/3 complex. ResultsSoluble human leukocyte antigen G, a human amniotic epithelial cell–derived factor, significantly decreased collagen production in TGF-β1–induced human dermal fibroblasts, although the effect on collagen production was less than that of human amniotic epithelial cell–conditioned medium. ConclusionsThis study demonstrates that human amniotic epithelial cells conditioned medium could down-regulate the expression of fibrosis-related molecules by regulating MMP and tissue inhibitor of metalloproteinase levels, and suppress TGF-β1–induced fibroblast transition, in which the TGF-β1/Smad3 pathway is likely involved. These findings suggest that human amniotic epithelial cells are a potential therapeutic compound for the treatment of keloids.

Quantitative and qualitative toxicological evaluation of thiol-ene "click" chemistry-based polyanhydrides and their degradation products.

Quantitative and qualitative toxicological analyses of crosslinked, surface-eroding polyanhydrides (PAHs) made from thiol-ene "click" polymerizations are reported. The cytotoxicity of these PAHs was investigated against three skin-based cell types; melanoma (A-375), human dermal fibroblast adult (HDFa), and 3T3-J2 (mouse fibroblast) cells, thus providing insight into the potential for these PAHs to be used in dermal drug delivery applications. Apoptosis was evaluated quantitatively and qualitatively using MTT assay and fluorescence microscopic imaging as indication of cytotoxicity. Upon exposure of A-375 and HDFa cells to high concentrations (4000 mg/L) of crosslinked PAH, the respective morphologies remained relatively unchanged compared with nonexposed cells. The 3T3-J2 cell type was more sensitive towards the PAH, exhibiting minimal deformation of cell morphology at 4000 mg/L. The MTT assay and fluorescence imaging revealed that this PAH and its degradation products are highly cytocompatible at high concentrations and cytotoxicity observed is dosage/time dependent. Further, the PAH did not induce inhibition of tested cells' proliferation at high polymer concentration up to 2000 mg/L. The IC50 (concentration of the crosslinked PAH required to inhibit 50% cell viability) for HDFa and A-375 cells was determined to be 4300 ± 70 and 8500 ± 50 mg/L, respectively. The high cytocompatibility of this type of crosslinked PAH, in addition to their degradation products, towards these skin cells (standard and cancer cell types) suggests that the polymer may be viable for dermal-based drug delivery to normal and cancerous diseased tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1936-1945, 2016.

What is really driving cell-surface interactions? Layer-by-layer assembled films may help to answer questions concerning cell attachment and response to biomaterials.

Layer-by-layer (LbL) assembled multicomponent films offer the opportunity to control and to fine-tune cell attachment and behavior on solid surfaces [Layer-by-Layer Films for Biomedical Applications, edited by Picart et al. (Wiley, Weinheim, 2014) and El-Khouri et al., "Multifunctional layer-by-layer architectures for biological applications," in Functional Polymeric Ultrathin Films, edited by Advincula and Knoll (Wiley, Weinheim, 2011), Vol. 1]. At the same time, these films allow for quite detailed physicochemical characterization of static and dynamic surface properties that are typically not available in classic cell culture. In this report, the authors investigate cell adhesion and cytocompatibility of compositionally and morphologically similar thin films composed of oppositely charged synthetic or natural polyelectrolytes in which different physical parameters such as surface charge or water content are varied through chemical composition and deposition conditions. Human adult dermal fibroblasts were chosen as a model because of the need for chemically defined matrix in the field of primary cell amplification. The growth and the stability of the multilayer films in the incubation media were studied dissipation-enhanced quartz crystal micobalance (QCM-D) and ellipsometry. The QCM-D signals observed during the film deposition were analyzed qualitatively to estimate the viscoelastic properties of the films. The authors used contact angle measurements with water to study the contribution of the chemical functionalities to wetting behavior of the films. Most importantly, they also studied the interaction of the films with serum components. Our results underline that cell adhesion is a highly complex process which is not only governed by the functionality of a surface but also by its morphology, its affinity for serum components, and also by changes of surface properties brought about by adsorbing molecules. Of the many LbL-films tested, poly(4-styrenesulfonate)/poly(allyl amine) multilayers were best suited for our fibroblast cultures, which opens a way to avoid gelatin based and similar substrates whose exact chemical composition is unknown.

Placenta Mesenchymal Stem Cell Derived Exosomes Confer Plasticity on Fibroblasts.

Mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) has been reported to enhance wound healing. Exosomes contain nucleic acids, proteins, and lipids, and function as an intercellular communication vehicle for mediating some paracrine effects. However, the function of MSC-derived exosomes (MSC-exo) remains elusive. In this study, we isolated human placenta MSC (PlaMSC)-derived exosomes (PlaMSC-exo) and examined their function in vitro. PlaMSCs were isolated from human term placenta using enzymatic digestion. PlaMSC-exo were prepared from the conditioned medium of PlaMSC (PlaMSC-CM) by ultracentrifugation. The expression of stemness-related genes, such as OCT4 and NANOG, in normal adult human dermal fibroblasts (NHDF) after incubation with PlaMSC-exo was measured by real-time reverse transcriptase PCR analysis (real-time PCR). The effect of PlaMSC-exo on OCT4 transcription activity was assessed using Oct4-EGFP reporter mice-derived dermal fibroblasts. The stimulating effects of PlaMSC-exo on osteoblastic and adipocyte-differentiation of NHDF were evaluated by alkaline phosphatase (ALP), and Alizarin red S- and oil red O-staining, respectively. The expression of osteoblast- and adipocyte-related genes was also assessed by real-time PCR. The treatment of NHDF with PlaMSC-exo significantly upregulated OCT4 and NANOG mRNA expression. PlaMSC-exo also enhanced OCT4 transcription. The NHDF treated with PlaMSC-exo exhibited osteoblastic and adipocyte-differentiation in osteogenic and adipogenic induction media. PlaMSC-exo increase the expression of OCT4 and NANOG mRNA in fibroblasts. As a result, PlaMSC-exo influence the differentiation competence of fibroblasts to both osteoblastic and adipocyte-differentiation. It shows a new feature of MSCs and the possibility of clinical application of MSC-exo. J. Cell. Biochem. 117: 1658-1670, 2016. © 2015 Wiley Periodicals, Inc.

Two Histone Variants TH2A and TH2B Enhance Human Induced Pluripotent Stem Cell Generation.

There are two major methods of reprogramming: generation of induced pluripotent stem cells (iPSCs) by overexpressing embryonic stem cell-specific transcription factors (OCT4, SOX2, KLF4, and c-MYC) and somatic cell nuclear transfer by oocyte-specific factors. Previously, we reported oocyte-enriched histone variants TH2A, TH2B, and the histone chaperone nucleoplasmin (NPM2) enhance the reprogramming by OSKM in mice by inducing open chromatin structure. In this study, we showed that human TH2A, TH2B, and NPM2 enhance the OSKM-induced reprogramming of adult and neonatal human dermal fibroblasts and umbilical vein endothelial cells. Pluripotency of iPSCs generated by coexpressing OSKM, TH2A, TH2B, and NPM2 was shown by in vitro and in vivo differentiation assays. These iPSCs gave rise to highly differentiated teratomas compared to iPSCs induced by OSKM alone. Genome-wide analysis suggests a possibility that TH2A, TH2B, and NPM2 might regulate genes that are involved in naïve stem cell stage. Thus, TH2A, TH2B, and NPM2 enhance reprogramming of human somatic cells and improve the quality of human iPSCs.

Effects of GHK-Cu on MMP and TIMP Expression, Collagen and Elastin Production, and Facial Wrinkle Parameters

Background: Glycyl-L-histidyl-L-lysine-copper (GHK-Cu) is an endogenous tripeptide-copper complex involved in collagen synthesis and is used topically as a skin anti-aging and wound healing agent. However, its biological effects are yet to be fully elucidated. Objectives: To investigate the effects of GHK-Cu on gene expression of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), and on production of collagen and elastin by human adult dermal fibroblasts (HDFa); and to investigate the effectiveness of topical application of GHK-Cu on wrinkle parameters in volunteers. Methods: Cultured HDFa were incubated with GHK-Cu at 0.01, 1 and 100 nM in cell culture medium. Gene expression (mRNA) for MMP1, MMP2, TIMP1 and TIMP2 in treated and control HDFa was measured by RT-PCR. Cellular production of collagen and elastin was measured colourmetrically using commercial assay kits. Correlations between gene expression and collagen and elastin production were determined. A randomised, double-blind clinical trial involving twice daily application of GHK-Cu, encapsulated in lipid-based nano-carrier, to facial skin of female subjects (n= 40, aged 40 to 65) was run over 8 weeks. The formulation vehicle (a serum) and a commercial cosmetic product containing Matrixyl® 3000, a lipophilic GHK derivative, were used as controls. Results: GHK-Cu significantly increased gene expression of MMP1 and MMP2 at the lowest concentration whilst simultaneously increasing the expression of TIMP1 at all the tested concentrations. All examined concentrations of GHK-Cu increased both collagen and elastin production. An increase of the mRNA expression ratio of TIMPs to MMPs was associated with an increase in collagen/elastin production. Application of GHK-Cu in nano-carriers to facial skin of volunteers significantly reduced wrinkle volume (31.6%; p=0.004) compared to Matrixyl® 3000, and significantly reduced wrinkle volume (55.8%; p<0.001) and wrinkle depth (32.8%; p=0.012) compared to control serum. Conclusions: GHK-Cu significantly increased collagen and elastin production by HDFa cells depending on the relative mRNA expression of their TIMP(s) over MMP. Topical application of GHK-Cu with the aid of nano-carriers reduced wrinkle volume to a significantly greater extent than the vehicle alone or a commercial product containing Matrixyl 3000®, a GHK lipophilic derivative.

Human ECM particles produced in vitro are injectable and support angiogenesis and adipogenesis in vivo

Event Abstract Back to Event Human ECM particles produced in vitro are injectable and support angiogenesis and adipogenesis in vivo Nicolas L'Heureux1, 2*, Adam D. Young3*, Nathalie Dusserre1*, Sonya B. Sonnenberg3*, Todd D. Johnson3*, Ricardo L. Rodriguez4*, Todd N. Mcallister1* and Karen L. Christman3* 1 Cytograft Tissue Engineering, United States 2 Université de Bordeaux, U1026 BioTis, France 3 University of California, San Diego, Department of Bioengineering, Sanford Consortium for Regenerative Medicine, United States 4 CosmeticSurg, United States Cell-synthesized ECM assembled by cultured cells has emerged as a promising biological scaffold for tissue engineering[1]. Tissue-engineered blood vessels built using rolled sheets of cell-assembled matrix (CAM) have shown unprecedented long-term patency in humans[2]. In this study, the CAM was mechanically processed into particles to produce an injectable form of the material suitable for many applications including aesthetic treatments. The goal of this study was to assess 1) the composition, 2) the injectability, 3) the stability, and 4) the host’s response to the CAM. Adult human dermal fibroblasts were cultured to promote ECM production and assembly in sheets for 8 weeks as previously described[3]. Dried sheets were milled into particles that passed through a #60 sieve (250µm mesh) and sterilized by gamma irradiation. Particles composition was analyzed using mass spectroscopy, immunolabeling and biochemical testing. Particle suspensions in saline were injected subcutaneously in nude mice along with two commercially available dermal fillers: Juvéderm Ultra XC® (cross-linked hyaluronic acid-based product) and Radiesse® (hydroxyapatite-based product). Each of 12 animals received four injections (upper shoulders and thighs) and was sacrificed after 2, 4, 8 and 12 weeks (4 biopsies per material per time point). Analysis of the ECM revealed that the particles contained numerous components including fibrillar and other collagens, elastic fiber components, fibrogenesis-associated and signaling components as well as glycosaminoglycans. The particle at 175 mg/ml produced a thick solution that could be injected through a 25 Ga needle. Subcutaneous injections were easily performed and, after diffusion of the excess injected fluids (≈24hrs), a palpable and localized bolus was observed. Histologically, the collagen rich particles were easily recognizable and generated a very mild inflammatory reaction with a generally thin fibrous layer around the injection site and only sparse immune cells. Rapid fibroblastic cell migration (2 weeks) was observed between the particles followed by progressive (2-12 week) perfusion by small blood vessels (also between particles). Adipocytes were also observed between particles within the injection bolus as early as 2 weeks but were more abundant as time went by. Cells did not appear to penetrate the particles and no signs of active degradation were observed. On the other hand, Juvéderm® did not form a well-delineated injection bolus and did not elicit an active remodeling process. Radiesse generated a very significant inflammatory response throughout the study period with abundant immune cell migration, giant cell formation, thicker fibrous capsule formation and extensive angiogenesis. These results clearly demonstrate the feasibility of injecting human CAM particles, at a clinically relevant concentration, using a clinically relevant system. Further more, these results support the idea that CAM is not targeted by the non-specific immune system. Finally, CAM particles supported both angiogenesis and adipogenesis. As a whole, these results illustrate the potential of this injectable material as a new tool for long-term tissue reconstruction and rejuvenation. While previous studies have shown great clinical successes using CAM-based tissue engineering[2], this is the first study to provide safety and remodeling data in a controlled setting. Marissa Peck, David Gebhart, Sunny Virk, Casey Mount, and Heinz Scheuenstuhl; Sophia Suarez; Rebecca Braden; Jessica Ungerleider; Dr. Kent Osborn; ASEE/NSF Engineering Innovation Fellows Program; National Science Foundation Graduate Research Fellowship Program