BACKGROUND: Secreted factors contained in conditioned media (CM) of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) known as secretome, was suspected to have important roles in regulating cells. This study was conducted to investigate the role of CM-hUCB-MSCs-derived secretome in apoptosis and growth of HeLa cells.METHODS: HeLa cells were treated with secretome in various concentrations (0, 0.2, 2 and 20%) for 24 and 48 hours. Trypan blue exclusion assay was performed to detect cell viability. Meanwhile sub-G1 apoptotic assay was performed to detect apoptotic cells. The transition of mitochondrial transmembrane potential (TMP), which occurs in the apoptotic process, was analyzed by mitochondrial membrane potential (ΔΨM) assay. Both sub-G1 and ΔΨM assays were performed using FACSCanto flow cytometer. Statistical analyses were conducted using IBM SPSS Statistics to detect significance level at p<0.05.RESULTS: Secretome significantly induced cell death starting at concentration of 0.2% within a 24-hour period (p<0.05). Secretome significantly induced cell death in concentration and time dependent manner (p<0.05). The cell death was then confirmed as apoptosis through sub-G1 analysis. Due to the underlying apoptotic mechanism, we found distinct decrease of TMP, indicating an increase in mitochondrial membrane permeability of HeLa cells. In addition, we found that HeLa cell growth was inhibited partially by secretome.CONCLUSION: Taken together, we conclude that CMhUCB-MSCs-derived secretome significantly induced apoptosis of HeLa cells in a concentration and time dependent manner through mitochondrial apoptotic pathway. The secretome might also play important role in inhibiting HeLa cell growth.KEYWORDS: umbilical cord blood, mesenchymal stem cell, secretome, apoptosis, growth, cancer
Endothelial progenitor cells (EPCs) clinical applications have been well reported. However, due to low number of EPCs that could be isolated, EPCs expansion study became one of the main focuses. Some optimized mediums to culture EPCs were currently available. However, the proliferation signaling pathway is not clearly disclosed yet. Peripheral blood was collected from eight healthy subjects, followed by mononuclear cells (MNCs) isolation. MNCs were then prepared and cultured for 2 days. After that, non-adherent cells were harvested and further cultured for 3 days. Resulted colony-forming unit (CFU)-Hill colonies were documented and enumerated under an inverted light microscope. To detect membrane markers, immunofluorescence was performed to detect CD34, VEGFR-2, and CD133. Cell documentation was conducted under a fluorescence microscope. To check cell proliferation, XTT Cell Proliferation Assay Kit was used according to kit insert. To detect possible activation of p44/42 MAPK, western blot was performed to detect p44/42 MAPK and phosphorylated p44/42 MAPK. All visualized bands were captured and quantified. Our results showed that EPCs markers (CD34, CD133 and VEGFR-2) were detected in 3 days culture. From XTT cell proliferation assay and CFU enumeration results, we found that EPCs proliferated significantly (p = 0.012) with addition of supplement. Phosphorylated-p42 MAPK expression of EPCs treated with supplement was significantly higher than the one of EPCs without treatment. Significant inhibition of p42 MAPK phosphorylation by U0126 was observed (p = 0.012). By pretreatment of U0126, number of viable cells and CFUs treated with supplement was significantly decreased (p = 0.012). Our results showed that MEK-dependent p42 MAPK pathway might play an important role in EPCs proliferation.
The COVID-19 disease, which is caused by the novel coronavirus, SARS-CoV-2, has affected the world by increasing the mortality rate in 2020. Currently, there is no definite treatment for COVID-19 patients. Several clinical trials have been proposed to overcome this disease and many are still under investigation. In this review, we will be focusing on the potency of mesenchymal stem cells (MSCs) and MSC-derived secretome for treating COVID-19 patients. Fever, cough, headache, dizziness, and fatigue are the common clinical manifestations in COVID-19 patients. In mild and severe cases, cytokines are released hyper-actively which causes a cytokine storm leading to acute respiratory distress syndrome (ARDS). In order to maintain the lung microenvironment in COVID-19 patients, MSCs are used as cell-based therapy approaches as they can act as cell managers which accelerate the immune system to prevent the cytokine storm and promote endogenous repair. Besides, MSCs have shown minimal expression of ACE2 or TMPRSS2, and hence, MSCs are free from SARS-CoV-2 infection. Numerous clinical studies have started worldwide and demonstrated that MSCs have great potential for ARDS treatment in COVID-19 patients. Preliminary data have shown that MSCs and MSC-derived secretome appear to be promising in the treatment of COVID-19. Lay Summary The COVID-19 disease is an infection disease which affects the world in 2020. Currently, there is no definite treatment for COVID-19 patients. However, several clinical trials have been proposed to overcome this disease and one of them is using mesenchymal stem cells (MSCs) and MSC-derived secretome for treating COVID-19 patients. During the infection, cytokines are released hyper-actively which causes a cytokine storm. MSCs play an important role in maintaining the lung microenvironment in COVID-19 patients. They can act as cell managers which accelerate the immune system to prevent the cytokine storm and promote the endogenous repair. Therefore, it is important to explore the clinical trial in the world for treating the COVID-19 disease using MSCs and MSC-derived secretome.
BACKGROUND: A population of circulating Endothelial Progenitor Cells (EPCs) has been reported to play important role in maintaining endothelial function and integrity. Since EPCs culture is crucial and an optimized medium is currently available. Therefore we conducted a study to investigate whether stable angina subjects peripheral blood-derived EPCs could be cultured in this medium. Here, we performed study to detect EPCs characteristics and extracellular signalregulated kinase (Erk)1/2 Mitogen-Activated Protein Kinase (MAPK) pathway as possible underlying pathway for EPCs proliferation.METHODS: Peripheral blood EPCs from 8 stable angina subjects were cultured in an optimized medium with/without addition of supplement for 1 or 3 days. Then, the membrane of cultured EPCs were detected with immunofluorescence method for CD34, Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) and CD133. Colony forming unit (CFU) enumeration was performed. XTT Cell proliferation assay was performed to assess EPCs growth after 1 and 3-days culture. The western blot analysis was performed to detect possible activation of Erk1/2 MAPK.RESULTS: Number of EPCs and CFU cultured for 3 days were significantly higher than the ones cultured for 1 day (p=0.012). EPCs membrane markers from stable angina subjects were detected as well as CFUs were formed. There were significant increase of EPCs number, CFUs number and phosphorylated-Erk2 amount when the groups with and without supplement were compared (p<0.05). Meanwhile U0126, a MAPK Erk1/2 (MEK1/2) inhibitor, significantly inhibited the supplement-induced EPCs number, CFUs number and phosphorylated-Erk2 amount (p<0.05).CONCLUSION: Our results showed that ERK2 MAPK signaling pathway might play an important role in supplement-induced peripheral blood EPCs proliferation in subjects with stable angina.KEYWORDS: endothelial progenitor cell, EPC, p42, Erk2, proliferation
Introduction: Each cell in human body is assigned with a specialized function to perform. Before a cell becomes specialized, it is a stem cell. Stem cell research and therapy is progressing dramatically these days. Stem cell therapy holds enormous treatment potential for many diseases which currently have no or limited therapeutic options. Unfortunately, this potential also comes with side-effects. In this review, the positive and negative effects of regulation of stem cells will be explained.Content: Stem cells are undifferentiated cells that have potential to develop into many different cell types in the body during early life and growth. The type of stem cells are embryonic stem cells, induced pluripotent stem cells, somatic stem cells, foetal stem cells and mesenchymal stem cells. Stem cell transplantation is one form of stem cell therapy, it comes with different sources, and those are autologous and allogenic transplantation stem cells. In an autologous transplant, a patient’s own blood-forming stem cells are collected, meanwhile in an allogeneic transplant, a person’s stem cells are replaced with new stem cells obtained from a donor or from donated umbilical cord blood.Summary: Its abilities to maintain undifferentiated phenotype, self-renewing and differentiate itself into specialized cells, give rise to stem cell as a new innovation for the treatment of various diseases. In the clinical setting, stem cells are being explored in various conditions, such as in tissue repair and regeneration and autoimmune diseases therapy. But along with its benefit, stem cell therapy also holds some harm. It is known that the treatment using stem cell for curing and rehabilitation has the risk in tumor formation.
B ACKGROUND:Dental pulp stem cell (DPSC) and periodontal ligament stem cell (PDLSC) have been suggested as valuable seed cells for bone engineering, suggesting that both stem cells are potential osteogenic sources. Since DPSC and PDLSC seem like to have similar potential in bone formation, we conducted a study to compare morphology, immunophenotype and cell growth of DPSC and PDLSC isolated from the same teeth. METHODS:Human dental pulps and periodontal ligaments were obtained from freshly extracted partial impacted third molar teeth. Collected samples were digested with type I collagenase. Resulted cell suspension was washed and cultured. For biomarker identification, the cells were fixed and bound with anti-fluorescein isothiocyanate (FITC)-cluster of differentiation (CD)117 antibody. For cell growth quantification, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used.Meanwhile for osteogenic differentiation, the cells were cultured in osteogenic medium for 1-3 weeks, fixed and stained with alizarin red. RESULTS:Morphology of dental pulps cell (DPC) and periodontal ligament cell (PDLC) in passage 5 was similar. Clear CD117 green fluorescence of DPC and PDLC in passage 5 was observed. Cell growth rate of PDLC was higher than the one of DPC, 0.3858 and 0.3848 respectively. DPC formed bone nodule on the third week culture in osteogenic medium, while PDLC showed bone nodule formation on the second week culture. CONCLUSION:We suggest that DPC and PDLC are potential seed cells for osteogenic regeneration, since they had cell growth capacity and osteogenic differentiation, particularly PDLC that had faster osteogenic differentiation.
BACKGROUND: Bromelain is a sulfhydryl proteolytic enzyme that can hydrolyze protein, protease or peptide. Bromelain can be found in pineapple stem, fruit and core. Bromelain is composed of 212 amino acid residues with cysteine-25 forming a polypeptide chain that can hydrolyze peptide bonds by H2O. In medicine, bromelain has been developed as antibiotic, cancer drug, anti-inflammatory agent and immunomodulator. In dentistry, bromelain has potential to reduce plaque formation on the teeth and to irrigate root canal.METHODS: Pineapple core was dried for 3 days to get simplicia. Then simplicia was extracted with water solvent for 24 hours. After that, the macerated-pineapple core crude extract-derived bromelain (PCB) was separated by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by Coomassie Brilliant Blue (CBB) staining to ensure the presence of bromelain. In cytotoxic test, NIH-3T3 fibroblast cultures were treated with extracts in various concentrations to for 24 or 48 hours. Number of fibroblasts was calculated using 3-(4,5-dimethylthiazol-2- yl)-2,5-Diphenyltetrazolium bromide (MTT) assay.RESULTS: Pineapple core extraction using maceration method produced relative high yield (concentration: 1.5424 g/mL) of bromelain, which was confirmed by CBB staining results with the molecular weight of 33 kDa. Based on cytotoxic test results of PCB on NIH-3T3 fibroblasts, 24-hours-incubation LD50 was 95.7 g/L, while 48-hours-incubation LD50 was 51.1 g/L.CONCLUSION: PCB has low cytotoxic effect in NIH-3T3 fibroblasts.KEYWORDS: bromelain, pineapple, extract, cytotoxic, MTT
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