Encapsulation of HaCaT Secretome for Enhanced Wound Healing Capacity on Human Dermal Fibroblasts.

In the epidermal and dermal layers of the skin, diverse cell types are reconstituted during the wound healing process. Delays or failures in wound healing are a major issue in skin therapy because they prevent the normal structure and function of wounded tissue from being restored, resulting in ulceration or other skin abnormalities. Human immortalized keratinocytes (HaCAT) cells are a spontaneously immortalized human keratinocyte cell line capable of secreting many bioactive chemicals (a secretome) that stimulate skin cell proliferation, rejuvenation, and regeneration. In this study, the HaCaT secretome was encapsulated with polyesters such as poly (lactic-co-glycolic acid) (PLGA) and cassava starch in an effort to maximize its potential. According to the estimated mechanism of the HaCaT secretome, all treatments were conducted on immortalized dermal fibroblast cell lines, a model of wound healing. Encapsulation of HaCaT secretome and cassava starch enhanced the effectiveness of cell proliferation, migration, and anti-aging. On the other hand, the levels of reactive oxygen species (ROS) were lowered, activating antioxidants in immortalized dermal fibroblast cells. The HaCaT secretome induced in a dose-dependent manner the expression of antioxidant-associated genes, including SOD, CAT, and GPX. Six cytokines, including CCL2 and MCP-1, influenced immunoregulatory and inflammatory processes in cultured HaCAT cells. HaCaT secretome encapsulated in cassava starch can reduce ROS buildup by boosting antioxidant to stimulate wound healing. Hence, the HaCaT secretome may have a new chance in the cosmetics business to develop components for wound prevention and healing.

Development of an animal-free nitrogen source for the liquid surface culture of Cordyceps militaris.

Cordyceps militaris is a medicinal mushroom in Asia in the 21st century, which cordycepin is a significant bioactive compound. This study, investigated the effect of culture conditions and vegetable seed extract powder as a supplementary source of animal-free nitrogen on the production of cordycepin by C. militaris in liquid surface culture. The highest cordycepin production was observed under soybean extract powder (SBEP) conditions, and 80 g L-1 of SBEP supplementation increased cordycepin production to 2.52 g L-1, which was greater than the control (peptone). Quantitative polymerase chain reaction was used to examine the transcription levels, and the results showed that supplementing with SBEP 80 g L-1 significantly increased the expression of genes associated with the carbon metabolic pathway, amino acid metabolism, and two key genes involved in the cordycepin biosynthesis (cns1 and NT5E) compared to peptone-supplemented culture. Under optimal culture conditions, the model predicted a maximum response of cordycepin production of 2.64 g L-1 at a working volume of 147.5 ml, an inoculum size of 8.8% v/v, and a cultivation time of 40.0 days. This optimized culture condition could be used to increase cordycepin production in large-scale bioreactors. Additional research can be conducted to assess the economic viability of this process.

Nanoencapsulated cordyceps extract enhances collagen synthesis and skin cell regeneration through antioxidation and autophagy.

Oxidative stress from reactive oxygen species is the main cause of skin ageing. Cordycepin, a bioactive compound of Cordyceps militaris, contains antioxidant activity. This study examined extracellular matrix, antioxidant effect, autophagy activity, and skin regeneration in human dermal fibroblasts (HDFs) under normal and oxidative stress conditions. Slow disintegration was used to create nano-encapsulated cordyceps extract. HDFs were cultured and treated with 1 M cordycepin, 1 M medium, 0.1 M cordyceps medium loaded nanoparticles (CMP), or 1 mM H2O2. HDFs' senescent phenotypes were assessed, including cell proliferation, ROS scavenging, collagen and elastin synthesis, antioxidant activity, and wound healing. CMP size averaged 184.5 ± 95.2 nm increased cell proliferation and reduced H2O2-induced ROS. Thus, HDFs treated for 48 h increased skin regeneration activity 2.76-fold by expressing extracellular matrix and rescuing H2O2-induced damaged cells. It was significant that this CMP inhibited H2O2-induced oxidative stress and induced autophagy to regenerate HDFs. The developed CMP could be used in cosmetics.

Cordycepin Enhances SIRT1 Expression and Maintains Stemness of Human Mesenchymal Stem Cells.

BACKGROUND/AIM: Mesenchymal stem cells (MSCs) have been employed for therapeutic applications of various degenerative diseases. However, the major concern is MSC aging during the in vitro cultivation. Thus, the approach to delay MSC aging was examined in this research by focusing on the expression of Sirtuin 1 (SIRT1), a key anti-aging marker. MATERIALS AND METHODS: Cordycepin, a bioactive compound derived from Cordyceps militaris, was used to up-regulate SIRT1 and maintain stemness of MSCs. Upon treatment with cordycepin, MSCs were investigated for cell viability, doubling time, key gene/protein expression, galactosidase-associated senescence assay, relative telomere length, and telomerase expression. RESULTS: Cordycepin significantly increased the expression of SIRT1 in MSCs by activating the adenosine monophosphate activated protein kinase (AMPK)-SIRT1 signalling pathway. Moreover, cordycepin maintained the stemness of MSCs by deacetylating SRY-box transcription factor 2 (SOX2) via SIRT1, and cordycepin delayed cellular senescence and aging of MSCs by enhancing autophagy, inhibiting the activity of senescence-associated-galactosidase, maintaining proliferation rate, and increasing telomere activity. CONCLUSION: Cordycepin could be used to increase SIRT1 expression in MSCs for anti-aging applications.

Enhancing Neurological Competence of Nanoencapsulated Cordyceps/Turmeric Extracts in Human Neuroblastoma SH-SY5Y Cells.

Introduction: Neurological diseases, including Alzheimer's, Parkinson's diseases, and brain cancers, are reportedly caused by genetic aberration and cellular malfunction. Herbs with bioactive compounds that have anti-oxidant effects such as cordyceps and turmeric, are of interest to clinical applications due to their minimal adverse effects. The aim of study is to develop the nanoencapsulated cordyceps and turmeric extracts and investigate their capability to enhance the biological activity and improve neuronal function. Methods: Human neuroblastoma SH-SY5Y cells were utilized as a neuronal model to investigate the properties of nanoencapsulated cordyceps or turmeric extracts, called CMP and TEP, respectively. SH-SY5Y cells were treated with either CMP or TEP and examined the biological consequences, including neuronal maturation and neuronal function. Results: The results showed that both CMP and TEP improved cellular uptake efficiency within 6 h by 2.3 and 2.8 times, respectively. Besides, they were able to inhibit cellular proliferation of SH-SY5Y cells up to 153- and 218-fold changes, and increase the expression of mature neuronal markers (TUJ1, PAX6, and NESTIN). Upon the treatment of CMP and TEP, the expression of dopaminergic-specific genes (LMX1B, FOXA2, EN1, and NURR1), and the secretion level of dopamine were significantly improved up to 3.3-fold and 3.0-fold, respectively, while the expression of Alzheimer genes (PSEN1, PSEN2, and APP), and the secretion of amyloid precursor protein were significantly reduced by 32-fold and 108-fold, respectively. Importantly, the autophagy activity was upregulated by CMP and TEP at 6.3- and 5.5-fold changes, respectively. Conclusions: This finding suggested that the nanoencapsulated cordyceps and turmeric extracts accelerated neuronal maturation and alleviated neuronal pathology in human neural cells. This paves the way for nanotechnology-driven drug delivery systems that could potentially be used as an alternative medicine in the future for neurological diseases.

Transgenic Immortalization of Human Dermal Fibroblasts Mediated Through the MicroRNA/SIRT1 Pathway.

BACKGROUND/AIM: Human dermal fibroblasts (HDFs) are widely used as a skin model in cosmetic and pharmaceutical industry due their advantages for the cosmetic industry and medical aspects. Telomeres are key players in controlling cellular aging, in which telomeres and the telomerase enzyme (hTERT) can maintain proliferative capacity and prolong cellular senescence. The primary aim of the study was to elucidate the underlying mechanisms of hTERT/SV40 immortalization of human dermal fibroblasts. MATERIALS AND METHODS: Transgenic expression of hTERT and SV40 large antigen, as well as co-transfection of both factors was performed and their significance evaluated in terms of HDF immortalization efficiency. RESULTS: The results showed that the immortalized fibroblasts of all conditions can be cultured in over 60 passages and maintain their telomere length. Further, key markers of skin cells, such as COL1A1, KRT18 and ELASTIN, were up-regulated in immortalized cells. In addition, p53 expression was enhanced in all immortalized cells, in accordance with activation of the SIRT1 gene upon transgenic immortalization. The significant role of SIRT1 in fibroblast proliferation was assessed by shRNA-knockdown, and it was found that SIRT1 silencing led to loss of Ki67, a proliferation marker. Moreover, miR-93, a SIRT1-targeted miRNA, also had a significantly reduced expression in the co-transfected immortalized cells, highlighting the linkage of the miRNA and SIRT1 pathway in the immortalization of human dermal fibroblasts. CONCLUSION: This evidence from this study could benefit the efficient development of human skin cell lines for use in the cosmetic industry in the future.

Autophagy Promoted Neural Differentiation of Human Placenta-derived Mesenchymal Stem Cells.

BACKGROUND/AIM: Human placenta-derived mesenchymal stem cells (hPMSCs) are multipotent and possess neurogenicity. Numerous studies have shown that Notch inhibition and DNA demethylation promote neural differentiation. Here, we investigated the modulation of autophagy during neural differentiation of hPMSCs, induced by DAPT and 5-Azacytidine. MATERIALS AND METHODS: hPMSCs were treated with DAPT to induce neural differentiation, and the autophagy regulating molecules were used to assess the impact of autophagy on neural differentiation. RESULTS: The hPMSCs presented with typical mesenchymal stem cell phenotypes, in which the majority of cells expressed CD73, CD90 and CD105. hPMSCs were multipotent, capable of differentiating into mesodermal cells. After treatment with DAPT, hPMSCs upregulated the expression of neuronal genes including SOX2, Nestin, and ßIII-tubulin, and the autophagy genes LC3I/II and Beclin. These genes were further increased when 5-Azacytidine was co-supplemented in the culture medium. The inhibition of autophagy by chloroquine impeded the neural differentiation of hPMSCs, marked by the downregulation of ßIII-tubulin, while the activation of autophagy by valproic acid (VPA) instigated the emergence of ßIII-tubulin-positive cells. CONCLUSION: During the differentiation process, autophagy was modulated, implying that autophagy could play a significant role during the differentiation of these cells. The blockage and stimulation of autophagy could either hinder or induce the formation of neural-like cells, respectively. Therefore, the refinement of autophagic activity at an appropriate level might improve the efficiency of stem cell differentiation.

Potential effects and molecular mechanisms of melatonin on the dopaminergic neuronal differentiation of human amniotic fluid mesenchymal stem cells.

Melatonin, a highly lipophilic molecule secreted by the pineal gland in the brain, plays a role in various biological functions. Previous studies reported that melatonin exerts its effect on mesenchymal stem cell (MSC) survival and differentiation into osteogenic- and adipogenic-lineage. However, the effect of melatonin in neurogenic differentiation in amniotic fluid (AF)-MSCs remains to be explored, thus we investigated the potential role of melatonin on dopaminergic neuron differentiation in AF-MSCs. The results showed that various concentrations of melatonin did not affect cell viability and proliferative effects of AF-MSCs. Increases in the levels of neuronal protein marker (ßIII-tubulin) and dopaminergic neuronal markers (tyrosine hydroxylase, TH and NURR1), but decrease in the level of glial fibrillary acidic protein (GFAP), were observed in melatonin-treated AF-MSCs. Melatonin induced alteration in differential expression patterns of mesenchymal stem cell antigens by reducing CD29, CD45, CD73, CD90 and CD105, but no changing CD34 expressing cells. AF-MSCs were sequentially induced in neurobasal medium containing standard inducing cocktails (ST: bFGF, SHH, FGF8, BDNF), 1 µM melatonin, or a combination of ST and melatonin. The levels of TUJ1, TH, MAP2, NURR1 and dopamine transporter (DAT) were significantly increased in all treated groups when compared with control-untreated cells. Pretreated AF-MSCs with non-selective MT1/MT2 receptors antagonist, luzindole and selective MT2 receptor antagonist, 4-P-PDOT diminished melatonin-induced increase in dopaminergic neuronal markers and phosphorylated ERK but did not diminish increase in phosphorylated CaMKII by melatonin. Pretreatment with mitogen-activated protein kinase (MEK) inhibitor, PD98059 and CaMKII inhibitor, KN-93 were able to abolish increase in the levels of dopaminergic markers in melatonin-treated AF-MSCs. These findings suggest that melatonin promotes dopaminergic neuronal differentiation of AF-MSCs possibly via the induction in ERK and CaMKII pathways through melatonin receptor-dependent and -independent mechanisms, respectively.

Methamphetamine toxicity-induced calcineurin activation, nuclear translocation of nuclear factor of activated T-cells and elevation of cyclooxygenase 2 levels are averted by calpastatin overexpression in neuroblastoma SH-SY5Y cells.

Methamphetamine (METH) is an addictive stimulant drug that has many negative consequences, including toxic effects to the brain. Recently, the induction of inflammatory processes has been identified as a potential contributing factor to induce neuronal cell degeneration. It has been demonstrated that the expression of inflammatory agents, such as cyclooxygenase 2 (COX-2), depends on the activation of calcineurin (CaN) and nuclear factor of activated T-cells (NFAT). Moreover, the excessive elevation in cytosolic Ca2+ levels activates the cell death process, including calpain activation in neurons, which was diminished by the overexpression of the calpain inhibitor protein, calpastatin. However, it is unclear whether calpain mediates CaN-NFAT activation in the neurotoxic process. In the present study, we observed that the toxic high dose of METH-treated neuroblastoma SH-SY5Y cells significantly decreased cell viability but increased apoptotic cell death, the active cleaved form of calcineurin, the nuclear translocation of NFAT, and COX-2 levels. Nevertheless, these toxic effects were diminished in METH-treated calpastatin-overexpressing SH-SY5Y cells. These findings might emphasize the role of calpastatin against METH-induced toxicity by a mechanism related to calpain-dependent CaN-NFAT activation-induced COX-2 expression.

Potential role of N-benzylcinnamide in inducing neuronal differentiation from human amniotic fluid mesenchymal stem cells.

Neurodegenerative disorders are characterized by chronic and progressive loss of neurons in structure and function related to aging, such as Alzheimer's disease, the latter characterized by the degeneration of cholinergic neurons in basal forebrain connected to the cerebral cortex and hippocampus. Amniotic fluid mesenchymal stem cells (AF-MSCs) have been proposed as one of the candidates for stem cell therapy of nervous system disorders. This study demonstrates that incubation of AF-MSCs, obtained from 16 to 20 week pregnant women, with 10ng/ml bone morphogenetic protein (BMP)-9 for 48h in conditioned medium resulted in transdifferentiation to cholinergic neuronal-like cells. This phenomenon could also be obtained with N-benzylcinnamide (PT-3). Pre-treatment for 1h with 10nM PT-3 augmented BMP-9 transdifferentiation effect, elevated ßIII-tubulin cell numbers and fluorescence intensity of immunoreactive ChAT, ameliorated BMP-9-related production of reactive oxygen species and enhanced anti-apoptosis status of the neuronal-like cells. The transdiffirentiation process was accompanied by increased p53 but decreased Notch1 and SIRT1 (p53 deacetylase) levels, and activation of p38, ERK1/2 MAPK, and PI3K/Akt pathways, in concert with inactivation of JNK, all of which were accentuated by PT-3 pre-treatment. These findings suggest that N-benzylcinnamide may provide a useful adjuvant in BMP-9-induced transdifferentiation of AFMSCs into ultimately cholinergic neurons.