Growth and Osteogenic Differentiation of CD117+ Dental Pulp and Periodontal Ligament Cells

BACKGROUND: 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.KEYWORDS: dental pulp, periodontal ligament, cell, growth, osteogenic differentiatio

Macerated-Pineapple Core Crude Extract-derived Bromelain Has Low Cytotoxic Effect in NIH-3T3 Fibroblast

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, MT

Ischemic Stroke: New Neuron Recovery Approach with Mesenchymal and Neural Stem Cells

Stroke is a leading cause of death and long-term disability. This due to the ischemic event that cause by embolism of blockage blood flow. Thrombolytic agent plasminogen activator (tPA) is the only treatment approved by FDA. However, the used of tPA is limited to the short time window period. Neural stem cells (NSCs) show the potential to repair neuronal damage naturally after stroke. However, isolating NSCs is a challenging process due to the limitations of the method and its invasiveness. Some studies that had used mesenchymal stem cell (MSCs) as the main source of stem cell for therapy show that MSCs have the potency to differentiate into NSCs. in vitro, a differentiation process from MSC to NSC has been developed by combining the supplement or growth factor needed in the culture media.Keywords: stem cells, neuron stem cell, mesenchymal stem cell, stroke, trans-differentiatio

Human umbilical cord blood-mesenchymal stem cell-derived secretome in combination with atorvastatin enhances endothelial progenitor cells proliferation and migration [version 1; peer review: awaiting peer review]

Background: Human umbilical cord blood-mesenchymal stem cell (hUCB-MSC)-derived secretome is known to be able to promote neovascularization and angiogenesis, so it is also thought to have a capability to modulate endothelial progenitor cell (EPC) functions. Atorvastatin is the cornerstone of coronary artery disease (CAD) treatment which can enhance EPCs proliferation and migration. This study aims to analyze the effect of the hUCB-MSC-derived secretome and its combination with atorvastatin toward EPCs proliferation and migration. Methods: EPCs were isolated from a CAD patient’s peripheral blood. Cultured EPCs were divided into a control group and treatment group of 2.5 µM atorvastatin, hUCB-MSC-derived secretome (2%, 10%, and 20% concentration) and its combination. EPCs proliferation was evaluated using an MTT cell proliferation assay, and EPC migration was evaluated using a Transwell migration assay kit. Results: This research showed that hUCB-MSC-derived secretomes significantly increase EPC proliferation and migration in a dose-dependent manner. The high concentration of hUCB-MSC-derived secretome were shown to be superior to atorvastatin in inducing EPC proliferation and migration (p<0.001). A combination of the hUCB-MSC-derived secretome and atorvastatin shown to improve EPCs proliferation and migration compared to hUCB-MSC-derived secretome treatment or atorvastatin alone (p<0.001). Conclusions: This study concluded that the hUCB-MSC-derived secretome work synergistically with atorvastatin treatment in improving EPCs proliferation and migration

Proliferation of Peripheral Blood-derived Endothelial Progenitor Cells from Stable Angina Subjects

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, proliferatio

Investigation on Cell Surface Markers of Dental Pulp Stem Cell Isolated from Impacted Third Molar Based on International Society for Cellular Therapy Proposed Mesenchymal Stem Cell Markers

Background: Recently we have isolated and cultured dental pulp stem cell (DPSC) derived from impacted third molar (DPSC-M3). The DPSC-M3 was suggested as mesenchymal stem cell, however the cell surface markers were not completely clarified. Therefore current study was conducted to investigate the markers.Materials and Methods: Passage 5 DPSC-M3 was cultured, labeled and examined with flow cytometer. All markers were investigated according to the proposed cell surface marker panel for the minimal identification of human mesenchymal stem cell (MSC) by International Society for Cellular Therapy (ISCT). The positive markers were cluster of differentiation (CD)90, CD73, CD105, while the negative markers were CD34, CD45, CD11b, CD19, and Human Leukocyte Antigen (HLA)-DR. Results: Results showed that the size and granularity of DPSC-M3 were ranged from 75 to 230 and 27 to 203, respectively. The cell surface antigens examination showed that CD90, CD105 and CD73 were highly expressed (>95%), meanwhile expressions of CD45, CD34, CD11b, CD19 and HLA-DR were <2%.Conclusion: Since the all markers expression were in accordance to the proposed cell surface marker panel for the minimal identification of human MSC by ISCT, DPSC-M3 could be suggested as an MSC.Keywords: dental pulp, stem cell, dental pulp stem cell, ISCT, flow cytometr

Conditioned Media of Human Umbilical Cord Blood Mesenchymal Stem Cell-derived Secretome Induced Apoptosis and Inhibited Growth of HeLa Cells

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, cance

Is Stem Cell a Curer or an Obstruction?

Stem cell research and therapy are progressing these days dramatically. 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. Stem cells are undifferentiated cells which able to develop into many different cells of types in the body during early life and growth. There are five types of stem cells: embryonic stem cells, induced pluripotent stem cells, somatic stem cells, fetal stem cells and mesenchymal stem cells. Stem cell transplantation is one form of stem cell therapy, it comes with different techniques sourced, and those are autologous and allogeneic transplantation stem cells. In an autologous transplant, a patient's blood-forming stem cells are collected, meanwhile, in an allogeneic transplant, target cells are replaced with new stem cells obtained from a donor or donated umbilical cord blood. Its abilities to maintain the phenotype, self-renewing and differentiate itself into specialized cells, give rise to stem cell as an innovation for the treatment of various diseases. In the clinical setting, stem cells are being explored for different 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 of tumor formation. Keywords: stem cell, therapy, transplantation, tumorigenic, mesenchymal stem cell, allogenei

Positive Correlation between Very Small Embryonic Stem Cell, Hematopoietic Stem Cell, and Endothelial Progenitor Cell in Umbilical Cord Blood Unit

BACKGROUND: Umbilical Cord Blood (UCB) has been widely use as regenerative medicine due to the content of undifferentiated cell which have capability to do self-renewal and differentiation into various type of cell called stem cells. Recent studies show that UCB contains not only hematopoietic stem cell (HSC) but also others stem cell and progenitor cell such as endothelial progenitor cell (EPC) and very small embryonic-like stem cell (VSEL). It is beliefs that HSC and EPC shared the same progenitor. In this study, correlation between the cell number of HSC, EPC and VSEL is analyzed in umbilical cord blood as the source of stem cell for clinical application. METHODS: The cell number of HSC, EPC and VSEL is counted from cryopreserved UCB collected from 22 women delivered via cesarean section which already stored for more than 2 years in this study. Sample were incubated with antibodies such as cluster of differentiation (CD)34-phycoerythrin (PE)/CD45-fluorescein isothiocyanate (FITC), CD133/1 (anti-CD (AC)133)-antigen-presenting cell (APC) and, CD184 (C-X-C chemokine receptor (CXCR)4)-PE.Vio770 to detect the present of HSC, EPC and VSEL in UCB. Sample were analyze using flowcytometer BD FACS Canto II. RESULTS: The cell population of HSC and late-EPC is 0.009% and 0.01% of total cell in UCB. VSEL only represented 0.001% from total cell in UCB, showing the lowest number of cell population in UCB. The correlation between the cell number of HSC and EPC is r=0,483*, p=0.023) and between HSC and VSEL is r=0.510*, p=0.015. CONCLUSION: In this study, both EPC and VSEL have a significant positive correlation with HSC. KEYWORDS: stem cell, umbilical cord blood, endothelial progenitor, flowcytometr

Eleutherine bulbosa bulb extract induces apoptosis and inhibits cell migration by downregulating Sonic hedgehog in human tongue cancer cells: An in vitro study

763-769Eleutherine bulbosa (Mill.) Urb. is a medicinal plant which has long been used to treat cancer. E. bulbosa bulb extract (EBBE) has been reported to show cytotoxicity towards several types of human cancer. However, the cytotoxic effect of EBBE towards tongue cancer cells has not been investigated. The present study aimed to evaluate the effects of EBBE towards the viability, apoptosis, and migratory activities of tongue cancer cells. Human oral squamous cell carcinoma (HSC)-3 cells were treated with various concentrations of EBBE for 24 h. The number of viable and apoptotic HSC-3 cells were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sub-G1 assay, respectively. The migratory activities of HSC-3 cells were assessed using scratch and transwell assay. Sonic hedgehog (SHH) expression was measured using immunoblotting. Upon EBBE treatment, apoptotic HSC-3 cells were significantly higher in a concentration-dependent manner (P <0.05). Meanwhile, viability, migratory activities, and migrated HSC-3 cell number were significantly lower in a concentration-dependent manner (P <0.05). The SHH expression levels in EBBE-treated HSC- 3 cells were also lower in a concentration-dependent manner. EBBE reduces HSC-3 cell viability through apoptosis and inhibits its migratory activities by downregulating SHH expression